联合半肝切除治疗中晚期肝门胆管癌孙谊 吴志宇 郝纯毅 黄信孚 王崑 包全 钱红纲 邢宝才【摘 要 】 目的 探讨联合半肝切除治疗肝门胆管癌的优势以及降低并发症和病死率的对策。方法 回顾性分析1998年-2006年北京大学临床肿瘤学院外科收治并手术治疗的50例肝门胆管癌病例。结果 手术治疗50例中,41例获得切除,切除率56.9% ,R0 根治性切除22例(53.6%)。实施术前PTCD减黄30例。肝门胆管癌联合半肝切除19例,其中R0根治性切除14例(73.6%),手术死亡2例(10.5%)。全组并发症发生率62%,半肝切除组78%。随访半肝切除组1年生存率57.1%,2年生存率27.3 % 。结论 联合半肝切除治疗肝门胆管癌能够提高手术的根治性改善预后,增加术中显露,简化了切除后重建,是肝门胆管癌尤其是BismuthⅢ-Ⅳ型的安全而有效的术式。对于需联合大范围肝切除的肝门胆管癌病例术前减黄是必要的。Clinical analysis of curative resection with hemihepatectomy for advanced hilar cholangiocarcinomaSUN Yi, WU Zhi-yu, HAO Chun-yi, HUANG Xin-fu, WANG Kun, BAO Quan, QIAN Hong-gang, XING Bao-cai. Department of Surgery, Peking University School of Oncology, Beijing Cancer Hospital, Beijing 100036, China[Abstract] Objective To investigate the superiority of curative resection with hemihepatectomy in treatment of hilar cholangiocarcinoma and the strategy to lower the incidence of complications and mortality.Methods The clinical data of 50 patients with hilar cholangiocarcinoma who underwent surgical procedures in Peking University School of Oncology from January 1998 to May 2006 were retrospectively analyzed.Results Forty-one patients (56.9%) underwent surgical resection. Twenty-two (53.6 %) received a radical operation(110).Thirty patients underwent preoperative percutaneous transhepatic cholangio-drainage (PTCD) to reduce the jaundice. Curative resection with hemihepatectomy was performed on 19 cases including 14 cases undergoing 110. Two patients who had undergone hemihepatectomy died of liver failure. The postoperative morbidity rate was 62% in all 50 cases, 78% in the hemihepatectomy group. The 1- and 2-year survival rates of the hemihepatectomy group were 57.1% and 27.3% respectively. Conclusion Curative resection with hemihepatectomy is a safe and effective option for hilar cholangiocarcinoma, especially for Bismuth Ⅲ or Ⅳ type. Preoperative biliary drainage is necessary before a major hepatic resection. 肝门胆管癌(Hilar Cholangiocarcinoma,Klatskin's Tumor)是指原发于肝总管、左右肝管及其汇合部胆管上皮的恶性肿瘤,是最常见的胆道恶性肿瘤(约占50%-70%),其发病率有逐年增高的趋势。目前手术切除仍是惟一被公认的最有效根治性治疗方法。近年来随着影像学及手术技术的进步,强调了手术切除的扩大化及根治性,肝门胆管癌的根治性切除率及预后有了一定程度的提高。我们总结了1998年-2006年北京大学临床肿瘤学院外科联合半肝切除治疗肝门胆管癌的19例的临床资料,现报道如下。对象与方法1. 一般资料:1998年1月-2006年5月北京大学临床肿瘤学院肝胆外科共收治肝门胆管癌72例,其中男44例,女28例,男女比例1.57:1,平均年龄(58.2±10.45)岁(33~74岁),临床表现主要为黄疸(90.1%)、陶土样便(54.9%)及腹痛(27.4%)。手术治疗50例,术前对黄疸严重(T-Bil >171 μmoL/L)的患者常规实施B超引导下经皮径肝穿刺胆管引流(PTCD)减黄治疗,人院时血清总胆红素水平897.3~117.0 μmoL/L,平均(446.9±209.1)μmoL/L;30例施行PTCD减黄(引流左肝7例,引流右肝17例,引流双侧6例),减黄后术前血清总胆红素水平406.2-34.0 μmoL/L,平均(188.0±126.5)μmoL/L;45例(90%)伴有血清CAl9-9不同程度升高。所有病例术前常规行腹部B超,增强CT或MRI检查磁共振胰胆管成像(MRCP)或PTC造影(PTCD减黄病例,经PTCD造影)评估病灶范围及可切除性。2.手术方法及步骤:入腹探查肿瘤的可切除性,远处转移为根治性切除的禁忌,此类病例行内/外引流术;如排除以上情况,首先,于肝十二指肠韧带下端分离显露肝动脉及门静脉,于胰腺上缘水平切断胆总管,送远端切缘冰冻切片;游离胆囊,将其与胆总管牵向上方,自下而上清扫胰头后上方、肝总动脉旁及肝十二指肠韧带内脂肪、淋巴、神经组织,全程显露并骨骼化肝动脉及门静脉,由此可明确其与肿瘤的关系;降低肝门板,进一步探明胆管汇合部及左右肝管受侵情况,Bismuth I型一般不需联合肝切除,Ⅱ型视情况联合切除肝方叶或沿正中裂劈开以使肝门部得到更好的显露,Ⅲ型及部分Ⅳ型则视胆管受侵情况,联合患侧半肝切除。半肝切除前,先行肝外结扎患侧肝动脉及门静脉,游离肝周韧带,显露第2肝门,肝外处理患侧肝静脉;此时,待切除侧(患侧)半肝血流完全阻断,颜色明显变暗,与保留侧分界清晰,切除线划定于分界线略向患侧1 cm,以彭氏多功能手术解剖器(PMOD)刮吸法沿切除线分离肝实质,结扎离断所遇管道,注意保留肝中静脉,以血管闭合器处理第2肝门患侧肝静脉;于肿瘤边缘近侧0.5-1.0 cm离断健侧胆管,胆管切缘标记,送冰冻切片;最后,离断患侧肝动脉、门静脉,移除标本。如肿瘤侵犯肝固有动脉行联合肝固有动脉切除,如肿瘤侵犯门静脉主干则联合切除受侵之门静脉后,行门静脉主干-保留侧门静脉端端吻合。术中视尾状叶胆管受侵情况,联合尾状叶切除。标本移除后,相邻胆管成型后,行肝管空肠Roux-en-Y吻合术。3.病例分组:全部手术病例分为3组,根据所选择术式不同分为半肝切除组、非半肝切除组(未切除肝脏、不规则肝部分切除及肝脏劈开)及未切除组(探查及内/外引流)。结果1.手术完成情况:对72例收治的肝门胆管癌患者,手术治疗50例,完成手术切除41例(切除率56.9%);根治性切除22例(根治性切除率53.6%);联合半肝切除19例,其中左半肝17例,右半肝2例;联合尾状叶切除5例;联合肝固有动脉切除11例(含l例切除后重建),联合门静脉切除吻合1例。根据影像学检查及术中探查情况确定Bismuth分型:I型7例,Ⅱ型5例,Ⅲa型7例,Ⅲb型18例,Ⅳ型13例(表1)。2.病理情况:全部手术切除病例术后病理均证实为肝门胆管癌,包括腺癌40例(高分化6例,中分化18例,低分化16例),腺鳞癌1例;脉管内癌栓6例(14.2%);淋巴结转移11例(26.8%),神经组织受累7例(17.1%),肝脏受侵14例(34.2%)。3.手术方式与根治程度:所有手术切除病例胆管的远近切缘均单独标记送检,根据胆管切缘病理结果及术中情况,将手术根治程度分为:R0切除(病理镜下切缘阴性),R1切除(病理镜下切缘阳性,肉眼未见肿瘤残留),R2切除(肉眼可见肿瘤残留),手术切除病例R0切除22例(R0切除率53.6%),半肝切除组根治切除率73.6%,非半肝切除组根治性切除率40.9%,非半肝切除组中BismuthⅢ-Ⅳ型根治性切除率23.1%。手术根治情况(表2)。4.术中及术后恢复情况:计算各组病例平均手术时间,平均手术出血,平均手术输血,平均术中胆管断端数目,平均胆肠吻合口数目及术后并发症情况。全组病例手术死亡2例,均为联合半肝切除病例,因肝功能衰竭分别死于术后11 d及21 d(表3)。5.随访情况:全组患者以电话、书信及门诊复查方式随访,随访时间2-50个月,随访率88%。未切除组无1年生存;非半肝切除组1年生存率50.0%,2年生存率27.3%;半肝切除组1年生存率57.1%,2年生存率27.3%。讨论 肝门胆管癌发生部位解剖的复杂性及其局部浸润性生长的特点,特别是极易向上侵犯肝脏,致使其手术切除非常困难。近年来,随着肝切除技术及血管外科技术的不断进步,联合肝脏切除及血管切除使得肝门胆管癌的手术根治性切除率明显提高,国外较大宗或综合文献报道平均为34.8%(16%~64%),而手术安全性及术后生存率也有显著进步,国内报道肝门胆管癌根治性切除后5年生存率13.6%~31.0%。回顾我院收治的肝门胆管癌患者,联合半肝切除手术治疗肝门胆管癌有以下明显优势。首先,联合半肝切除能够提高手术切除率,特别是根治性切除率,从而改善预后。肝门胆管癌沿胆管方向黏膜下浸润是其生物学行为的最显著的特点,甚至在早期肝门胆管癌未出现周围组织浸润、未发生梗阻之前,肿瘤常常已沿胆管方向黏膜下扩散,而且向肝内方向浸润范围明显大于十二指肠方向。这正是肝门胆管癌根治性切除率低、预后差的最根本原因,尤其是对于BismuthⅢ型或Ⅳ型的病例来讲,此类患者患侧肝内二级胆管往往已被肿瘤侵犯,不联合切除受累的肝叶的切除很难达到5 mm无癌这一根治性切除的标准。本组病例半肝切除组根治切除率73.6%(14/19)明显高于非半肝切除组根治性切除率40.9%(9/22),差异有统计学意义(P=0.035),而排除非半肝切除组中Bismuth I型和Ⅱ型病例的影响,对于BismuthⅢ型或Ⅳ型的病例来讲,两组根治性切除率分别为73.6%(14/19)及23.1%(3/13),两者差异更有统计学意义(P=0.005)。国内外亦有联合肝叶切除提高根治性切除率的文献报道口。对周围组织的直接侵犯是肝门胆管癌的另一生长特点,BismuthⅢ型或Ⅳ型病变达左右肝管进而侵犯紧邻的肝组织比较常见。本组手术切除病例中,病理证实肝侵犯者14例(34.2%);赵建勋等报道肝门胆管癌切除后病理报告肝组织受累高达54.3%,并且认为,根治性切除应该是整块标本全周无癌的立体概念,而不仅仅是胆管切缘的无癌,采用半肝切除的根治效果是肯定的。另外,肝门血管尤其是门静脉受侵是制约肝门胆管癌切除根治性的关键因素,而采取半肝切除术式可以将患侧受侵血管一并切除,一定程度上提高了根治性切除率。临床实践证明,肝门胆管癌手术切缘无癌是根治性手术切除要点,是改善患者术后生存率的关键因素,联合半肝切除能够提高肝门胆管癌的根治性切除率,从而改善患者预后,提高术后生存率。本组病例半肝切除组较非半肝切除组生存率无明显优势,可能与病例数较少,随访时间较短有关。对于BismuthⅢ~Ⅳ型的病例来讲,根治性切除后,待吻合的胆管往往已达肝内二级甚至更高级的胆管,胆管断端数目多,管壁薄以及临近的血管及肝组织又阻碍了相邻胆管成型,从而给胆肠吻合带来很大困难,工作量大,需要手术医师极大地细致与耐心;梁力建等认为肝门胆管癌术后胆瘘多发生在胆肠吻合口,手术的扩大化增加了胆瘘的发生,复杂的成型及繁多的吻合口是胆瘘发生的根本原因。联合半肝切除后,胆管残端数目明显减少,简化了胆肠吻合的手术操作,吻合口数目的减少可能减少胆瘘等并发症的发生。但本组病例半肝切除组在平均手术时间、术后住院时间及术后并发症方面较非半肝切除组并无优势,分析其原因:半肝切除简化了胆肠吻合的手术操作可能被半肝切除的操作所抵消,而吻合口数目减少可能减少胆瘘的优势会被半肝切除后肝创面的胆瘘所抵消,使得半肝切除平均手术时间长于非半肝切除组,而术后并发症(包括胆瘘)亦较多。 另外,半肝切除后,肝门部得以良好的显露,方便探查及手术操作。肝门胆管癌肿瘤的浸润常常使肝十二指肠韧带结构向上收缩;而胆道梗阻引起的黄疸常常使肝脏肿大且质地变硬,这就使得原本不易显露的病灶更加伸人肝内,给探查及手术操作带来了很大的困难。不少文献中报道了诸如:分离肝门板、肝方叶切除及肝脏劈开等增加显露的方法,半肝切除可以更好的暴露保留侧的肝内二级胆管,方便了手术探查,并且为进一步的胆管成型和胆肠吻合等手术操作提供了充分的显露和良好的空间。合并严重黄疸且持续时间较长的患者实施大范围肝切除,无疑会增加术后合并症及死亡率,如何提高手术安全性,尽量减少并发症和死亡率?我们认为:术前进行必要的评估与准备,过硬的手术技术、足够的细致与耐心以及围手术期积极正确的处理是降低手术合并症和死亡率的关键。对黄疸比较严重(T-Bil>171μmoL/L)且肝内胆管扩张超过5 mm的患者实施B超引导下PTCD外引流。有效的实施术PTCD减黄,可以解除胆道梗阻,减轻黄疸,改善肝功能,减少大范围肝切除术后合并症及死亡率,尤其是肝功能衰竭的发生。Kawasaki等及Seyama等副报道,肝门胆管癌扩大切除前减黄治疗,手术死亡率为0-1.3%。黄志强认为,胆道引流会增加胆道感染的机会,但对于联合广泛肝叶切除尤其是扩大的右半肝切除者,术前胆道引流减轻黄疸有益于肝功能恢复。PTCD引流后到胆红素水平正常或接近正常、肝细胞功能恢复往往需要2-4周甚至更长时间,是否延期手术会影响预后呢?seyama等报道,延期手术并不影响远期效果。留置PTCD的另一优势是通过观察引流胆汁的性状可以判断肝功能情况。我们的经验是:引流胆汁稀薄、量大,虽然血清胆红素可以明显下降,提示肝功能往往较差,术后易出现肝功能不全等合并症,故对于此类患者一方面进一步加强减黄保肝治疗尽可能延长引流时间以期肝功能好转,另一方面手术应慎选大范围肝切除和/或肝固有动脉的联合切除。经PTCD行胆管造影能够更经济且更清晰直观的反映病变范围、判断分型,对指导手术切除有很重要的意义。 综上所述,联合半肝切除治疗肝门胆管癌能够提高手术的根治性,改善预后,但远期的疗效观察有待病例数的进一步扩大及随访时间的进一步延长。参考文献[1]周宁新.肝门部胆管癌扩大根治术及疗效评价.中国普外基础与临床杂志,2005,12:323-324.[2]肖梅,周宁新,黄志强,等。联合肝时切除治疗肝门部胆管癌(附74例报告).中国实用外科杂志,2006,26:42-44.[3]蔡秀军,虞洪,梁霄,等.腹腔镜刮吸法在肝切除术的临床应用.中华医学杂志,2005,85:16l-163.[4]易滨,张柏和,张永杰,等.手术方式与肝门部胆管癌预后的关系分析.中华外科杂志,2005,43:842-845.[5]董家鸿.胆管癌根治性手术的适应证、术式选择及远期疗效.中国实用外科杂志,2001,2l:461-464.[6]程庆保,张柏和,张永杰,等.肝门部胆管癌预后因素分析.肿瘤,2005,25:166-169.[7]Sakomoto E,Nimura M,carboni F,et a1.The Pattem of infiltration at the proximal border of hilar bile duct carcinoma:a histologic analysis of 62 resected cases. 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提高对结直肠癌肝转移外科切除的认识邢宝才 结直肠癌是常见的恶性肿瘤之一,有60%-70%结直肠癌病人会发生肝脏转移,其中同时性肝转移大约25%,异时性肝转移病人占35%~45%。未经治疗的病人中位生存时间6-18个月。但是,最近10年,随着化疗药物(Oxaliplatin和Irinotecan)及靶向药物(Cetuximab和Bevacizumab)在临床的广泛应用,肝转移灶外科切除技术的推广及围手术期综合治疗模式的提出和应用,结直肠癌肝转移病人的治疗效果和生存有了明显的改善。但是,在诸多的治疗手段中,结直肠癌肝转移灶的手术切除是目前最有效且可能达到根治的治疗方法。结直肠癌肝转移的外科切除是目前常见肿瘤研究的热点。 1.外科切除在结直肠癌肝转移中的作用:对于结直肠癌肝转移,大约20%的病人在发现肝转移时有手术切除的机会,法国等国家的10年前的多项研究已经显示手术切除后,结直肠肝转移病人的5年生存率可达到35%-40%。而采用任何非手术治疗方法的病人,5年生存率一般<5%。影响病人手术疗效的因素包括肝转移灶的数目,大小,同时性/异时性,是否是根治性的切除,肝外有无转移及肿瘤标记物的高低等因素。手术切除与其他治疗手段相比有明显的生存优势,手术切除肝转移灶是能够手术切除病人获得长期生存的最佳治疗方式。有手术切除机会的病人还是很有限的。在选择手术切除的病人上,应遵循两个原则(1)手术的安全性:手术切除肝脏转移病灶后,残余肝脏是否能够维持正常的肝脏功能,一般认为残余肝脏应>30%,对于手术前进行过化疗的病人残余肝脏应保留更高的比例,以避免手术后出现肝功能不全。最新的研究报告,结直肠癌肝转移手术切除后死亡率接近为0%。(2)手术的彻底性:手术应能够切除肝脏内外的所有转移病灶,以达到根治性的切除。因为只有根治性的切除病人才能从手术中获得生存时间的延长。所以,我们应学习国外在结直肠癌肝转移治疗方面的经验,改变对结直肠癌肝转移在分期与选择治疗方面的认识,提高肝转移癌的手术切除率,进而延长肝转移癌病人的生存时间。 2.肝转移癌不能手术切除的外科策略:尽管肝转移灶的切除所取得的疗效已经得到众多研究所证实,但是,毕竟在发现肝转移时还是仅仅20%的病人有手术切除的机会。大部分的病人会是由于存在左右两叶多发的病变,或是手术后残余肝脏体积过小,在发现肝脏转移的时候,由于不具备彻底性与安全性两条原则,而没有手术切除的机会。肝转移癌不能手术切除的最主要的原因是残余肝体积不足,若采用手术切除的方法,病人有可能发生手术后的肝功能衰竭。如何使诊断时不具备手术机会的病人,获得手术机会。目前,国际上主要采用门静脉栓塞和二期切除的方法1.门静脉栓塞是通过结扎或栓塞门脉,产生肝右叶的萎缩和肝左叶的肥大,增加残余肝的体积。通常是手术切除肝左叶的病灶及结扎门脉右支,4周左右进行第二次手术——右半肝切除。通过上述的外科策略,可以使部分由于残余肝不足,没有手术机会的病人获得手术切除的机会,降低了手术后的危险。门脉结扎和二期切除这一新的外科策略,可以使10%左右的病人,获得手术切除的机会,使这些病人得到生存的延长。 3.新辅助化疗提高肝转移癌的切除率:尽管外科切除在结直肠癌的治疗上有明显的优势,但是,仅仅有20%的病人可能达到根治性的切除。近10年,随着新的化疗药物与靶向药物在临床上的应用,不断增加的研究结果证实,新辅助化疗可以使一部分肝转移癌病人肿瘤体积缩小,增加手术的切除率。Folfox或Folfiri的化疗,对40%~50%的肝转移病人有效。巴黎Paul Brousse医院对1988--2003年进行新辅助化疗的2047例病人进行分析,有14%的病人通过新辅助治疗由不能手术切除,获得了手术切除的机会。2008年ASCO和ESMO上报告的Crystal和Opus研究显示,爱必妥联合化疗可以使新辅助治疗的疗效提高到77%。爱必妥的疗效与结直肠癌原发病变k-ras是否有突变相关。在选择应用爱必妥之前,检测k-ras是必要的。 随着结直肠癌病人的增多,结直肠癌肝转移也在不断增加。结直肠癌肝转移有着其自身的生物学特点,对能够达到根治性手术切除的病人,应积极手术切除。对没有机会手术切除的病人,通过围手术期的综合治疗,争取获得手术切除的机会。我们应该不断学习该领域的最新进展,提高对结直肠癌的肝转移的认识,造福结直肠癌肝转移的病人。参考文献[1]Adam R,Miller R。Pitombo M,et eL Two—stage hepatectomy approach for initially unrcsectable colorectal hepatic metastases.Surg Oneol Clin N Am,2007.16:525-536.[2]Nordlinger B,Guiguet M,Vailhm Jc,et a1.Surgical resection of colorectal carcinoma metastases to the liver.A prognostic scoring system to improvecase selection,basedon 1568 patients.Cancer,1996.77:1253-1262.[3]Jaeck D,Oussoultzoglou E,Rasso E,et aL A two-stage hepateetomy procedure combined with portal vein embolization to achieve curative resection for initially unresectable multiple and bilobar colorectal liver metastases. Ann surg,2004,240:1037-105l.[4]Jaeck D.Pessaux P.Bilobar colorectal liver metastases:treatment options. Surg Oncol Clin N Am,2008,17:553-568.[5]kichman L The role of chemotherapy in the curative treatment of patients with liver metastases from colorectalcancer. Surg oncol Clin N Am.2007.16:537-556.
Proteome Analysis of Hepatocellular Carcinoma by Two-dimensional Difference Gel ElectrophoresisWei Sun, Baocai Xing, Yi Sun, Xiaojuan Du**, Min Lu, Chunyi Hao, Zhuang Lu,Wei Mi, Songfeng Wu, Handong Wei, Xue Gao, Yunping Zhu, Ying Jiang,Xiaohong Qian, and Fuchu He§§Hepatocellular carcinoma (HCC) is a highly malignant tumor,and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio > 2, p < 0.01) in tumor samples, whereas 158 spots were down-regulated (ratio < 2, p < 0.01). Seventy one gene products were identified among these spots.Members of the heat shock protein 70 and 90 families were simultaneously up-regulated, whereas metabolism associated proteins were decreased in HCC samples. The down-regulation of mitochondrial and peroxisomal proteins in these results suggested loss of special organelle functions during HCC carcinogenesis. Four metabolic enzymes involved in the methylation cycle in the liver were down-regulated in HCC tissues, indicating S-adenosylmethionine deficiency in HCC. Two gene products,glyceraldehyde-3-phosphate dehydrogenase andformimidoyltransferase-cyclodeaminase, were identified from inversely altered spots, suggesting that differentisoforms or post-translational modifications of these two proteins might play different roles in HCC. For the first time, the overexpression of Hcp70/Hsp90-organizing protein and heterogeneous nuclear ribonucleoproteinsC1/C2 in HCC tissues was confirmed by Western blot and then by immunohistochemistry staining in 70 HCC samples,suggesting their potential as protein tumor markers. In summary, we profiled proteome alterations in HCC tissues, and these results may provide useful insights for understanding the mechanism involved in the process of HCC carcinogenesis. Molecular & Cellular Proteomics 6:1798–1808, 2007.Proteomics analysis is currently considered to be a powerful tool for global evaluation of protein expression, and proteomics has been widely applied in analysis of diseases, especially in fields of cancer research. Quantitative protein expression profiling is a crucial part of proteomics, and such profiling requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. Two-dimensional electrophoresis (2DE)1 was a technique that was widely usedfor proteomics research. However, intergel variation and excessive time/labor costs have been common problems with standard 2DE. Two-dimensional (2D) DIGE might therefore be considered as one of the most significant advances in quantitative proteomics. Using the 2D DIGE approach, different samples prelabeled with mass- and charge-matched fluorescent cyanine dyes are co-separated in the same 2D gel, and an internal standard is used in every gel that has negated the problem of intergel variation (1). Moreover with the great sensitivity and dynamic range that is afforded by these dyes, 2D DIGE can give greater accuracy of quantitation than silver staining (2). It has been reported that the correlation between quantitation by 2D DIGE and metabolic stable isotope labeling is exceptionally good (3). In addition, this method reduces the number of gels needed for one experiment. With these advantages over traditional 2DE, 2D DIGE gives more a accuratequalitative and quantitative analysis (4) and has thus been applied to proteomics studies in several human cancers, such as colon cancer (5), prostate cancer (6), and pancreatic cancer (7).Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is responsible for approximately one million deaths each year (8). HCC is especially frequent in Asia due to a high prevalence of chronic HBV and HCV infections. In China, HCC has been ranked as the second most frequent fatal cancer since the 1990s (9), and the majorityof HCCs in China are caused by HBV infection.So far, most of the research has focused on HBV/HCV associated HCC. Studies have been done in different laboratories in Asia, Europe, and America. Several laboratories in Korea (10, 11), China (12, 13), and Singapore (14) have conducted research on HBV-associated HCC. Also there have been other studies on HCC with various or unclearly described viral origins in laboratories in Korea (15–17), Hong Kong (18), Taiwan (19), and Germany (20). Some of their results could be coincident, but the diversity is still distinct due to the differences in sampling and techniques used. In view of both the large number of patients affected together with the limitations of diagnostic methods and effective therapy for HCC, there is an urgent need to find key carcinogenesis-associated molecules for HBV-associated HCC diagnosis. To accurately quantitate differences between samples and achieve statistical significance, we used 2D DIGE to analyze paired clinical tissue samples of HCC from China with HBV infection background. All tumor samples were neoplasms of intermediate differentiation (Edmondson grade II or III) representativeof the majority of clinical HCC cases. The differentially expressed spots were identified, and the proteins ofinterest were further validated by Western blot and immunohistochemistry staining.EXPERIMENTAL PROCEDURESReagents—Cy2, Cy3, and Cy5 were purchased from GE Healthcare.dimethylformamide was purchased from Aldrich. DTT, urea,agarose, glycerol, bromphenol blue, CHAPS, mineral oil, acrylamide,Bis, Tris base, glycine, SDS, iodoacetamide, ammonium persulfate,TEMED, Immobiline DryStrip gels (24 cm, pH 3–10), and Bio-Lyte solutions (pH 3–10) were purchased from Bio-Rad. Thiourea was purchased from Fluka (Buchs, Switzerland). Protease inhibitor mixture was purchased from Roche Applied Science. ACN and methanol were purchased from Fisher. TFA was purchased from Merck. Trypsin (sequencing grade) was purchased from Promega (Madison, WI). All buffers were prepared with Milli-Q water (Millipore, Bedford, MA). Tissue Collection and Sample Preparation—HCC tissues and adjacent nontumorous liver tissue counterparts used for 2D-DIGE were collected from 12 HBV-associated HCC patients who underwent hepatectomy at Beijing Cancer Hospital (Table I). None of these patients received antineoplastic therapy prior to surgery. After resection, specimens were rinsed thoroughly in ice-cold normal saline and snap frozen in liquid nitrogen. Necrotic tissue was excluded, and nontumor liver tissues were confirmed to contain no tumor cells by histopathologic evaluation. Access to human tissues complied with the guidelines of the Ethics Committee.For each sample, 0.2 g of tissue was grinded into powder in liquid nitrogen with a precooled mortar and pestle. Samples were then homogenized on ice in 1 ml of lysis buffer (7 M urea, 2 M thiourea, 4%CHAPS, 30 mM Tris-Cl, pH 8.5, protease inhibitor mixture) using a glass homogenizer. After sonication on ice for 10 s using an ultrasonic processor, the samples were centrifuged for 30 min at 20,627 g (12,000 rpm) to remove particulate materials. Protein concentrations were determined in duplicate by the Bradford method (Bio-Rad) and confirmed by SDS-PAGE. 2D DIGE and Imaging—The pH of the protein was adjusted to 8.5by 50 mM NaOH, and the concentration was adjusted to 5 mg/ml with lysis buffer. Equal amounts of proteins from the 12 pairs of samples were pooled together as the internal standard. Tumor and nontumor counterparts of each patient were randomly labeled with Cy3 or Cy5, whereas internal standards were labeled with Cy2 using 400 pmol of fluorochrome/50 g of protein. Labeling was performed for 30 min on ice in the dark. Reactions were then quenched by the addition of 1 of lysine (10 mM) for 10 min on ice in the dark. Fifty-microgram Cy3- and Cy5-labeled samples from each patient were combined before mixing with 50 g of Cy2-labeled internal standard. Then an equal volume of 2 sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% Bio-Lyte, pH 3–10, 20 mg/ml DTT) was added to the sample, and the total volume was made up to 410 with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Bio-Lyte, 10 mg/ml DTT). Samples were actively rehydrated into 24-cm pH 3–10 IPG strips (Bio-Rad) at 17 °C for 12 h using a Protean IEF cell (Bio-Rad). Isoelectric focusing was performed for a total of 80 kV-h (ramped to 250 V in 30 min, held at 1000 V for 1 h, ramped to 10,000 V in 5 h, and held at 10,000 V for 60 kV-h). The IPG strips were equilibrated in equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-Cl, pH 8.8, 30% glycerol) supplemented with 0.5% DTT for 15 min at room temperature followed by 4.5% iodoacetamide in equilibration buffer for another 15-min incubation at room temperature. IPG strips were placed on the top of 12% homogeneous polyacrylamidegels that had been precast with low fluorescence glass plates using an Ettan DALTtwelve gel caster. The second dimension SDS-PAGE was carried out using the Protean Plus system (Bio-Rad). After 2DE, gels were scanned on the Typhoon 9410 scanner with Ettan DALT gel alignment guides using excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). The intensity was adjusted to ensure that the maximum volume of each image was within 60,000–90,000. Data Analysis—Analysis of 2D DIGE was done using DeCyder 5.0 software (GE Healthcare) according to the manufacturer’s recommendations. Briefly the DeCyder biological variation analysis module was used to detect spots (the estimated number of spots was 2500) and simultaneously match all 36 protein spot maps from 12 gels. Allmatches were also confirmed manually. The paired t test was used for statistical analysis of the data. Protein spots that were differentially expressed in tumor and nontumor groups were marked. Only spots altered consistently in at least five of the 12 patients were selected for identification. In-gel Digestion—Spot picking was carried out with preparative gels. Two-dimensional electrophoresis was performed as described under “2D DIGE and Imaging” except that the IPG strips were loaded with 500–1000 μg of protein, and gels were stained with Coomassie Brilliant Blue. Protein spots of interest were excised and destained with 25 mM ammonium bicarbonate, 50% ACN. Gels were then dried completely by centrifugal lyophilization. In-gel digestion was performed with 0.01 g/l trypsin (Promega) in 25 mM ammonium bicarbonate for 15 h at 37 °C. The supernatants were collected, and the tryptic peptides were extracted from the gel sequentially with 5% TFA at 40 °C for 1 h and with 2.5% TFA, 50% ACN at 30 °C for 1 h. The extracts were pooled and dried completely by centrifugal lyophilization. Protein Identification—Peptide mixtures were redissolved in 0.5% TFA, and 1 l of peptide solution was mixed with 1 l of matrix (4-hydroxy--cyanocinnamic acid in 30% ACN, 0.1% TFA) before spotting on the target plate. MALDI-TOF mass spectrometry and tandem TOF/TOF mass spectrometry were carried out on a 4700 Proteomics Analyzer (Applied Biosystems). Peptide mass maps were acquired in positive reflection mode, averaging 1500 laser shots per MALDI-TOF spectrum and 3000 shots per TOF/TOF spectrum (the resolution was 20,000). The 4700 calibration mixtures (Applied Biosystems) were used to calibrate the spectrum to a mass tolerance within 0.1 Da. Parent mass peaks with a mass range of 600–4000 Da and minimum signal to noise ratio of 15 were picked out for tandem TOF/TOF analysis. Combined mass and mass/mass spectra were used to interrogate human sequences in the Swiss-Prot database (UniProt_SP sprot_84 (230,133 sequences; 84,471,903 residues)) using the MASCOT database search algorithms (version 1.9). Searches were performed to allow for carbamidomethylation, oxidation, and amaximum of one missed trypsin cleavage. Peptide tolerance and MS/MS tolerance were both 0.2 Da. All of the automatic data analysis and database searching were fulfilled by the GPS ExplorerTM software (version 3.6, Applied Biosystems). Known contaminant ions (keratin) were excluded. The confident identification had a statistically significant (p 0.05) protein score (based on combined mass and mass/mass spectra) and best ion score (based on mass/mass spectra).Redundancy of proteins that appeared in the database under differentnames and accession numbers was eliminated. If more than one protein was identified in one spot, the single protein member with the highest protein score (top rank) was singled out from the multiprotein family. The molecular weight and pI values of most proteins were consistent with the gel regions from which the spots were excised. Western Blot—Proteins from the 12 paired tumor and nontumor tissues were separated on 12% polyacrylamide gels and transferred to PVDF membranes (Amersham Biosciences). These blots were incubated for 2 h at room temperature in Tris-buffered-saline with Tween (20 mM Tris-Cl, 140 mM NaCl, pH 7.5, 0.05% Tween 20) containing 5% skim milk. Primary antibodies used were anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (diluted 1:1000, Kangcheng Biotechnology), anti-Hcp70/Hsp90-organizing protein (HOP) monoclonal antibody (diluted 1:1000, Stressgen), and anti-heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) monoclonal antibody (diluted 1:1000, Abcam).Blots were incubated with primary antibodies overnight at 4 °C. After washing three times in Tris-buffered-saline with Tween, blots wereincubated with horseradish peroxidase-conjugated secondary antibody(diluted 1:10,000, Santa Cruz Biotechnology) for 1 h at room temperature. Immunoreactive complexes were visualized using ECL reagents (Santa Cruz Biotechnology). Immunohistochemistry Staining—The samples used for immunohistochemistry staining were formalin-fixed, paraffin-embedded tissues, which included paired HCC tumor and nontumor liver from each patient. Seventy HCC samples (54 male; 16 female; age, 51 13 years) were used for HOP immunocytochemistry staining, including seven samples analyzed by 2D DIGE. Seventy HCC samples (56 male; 14 female; age, 52 12 years) were used for hnRNP C1/C2 immunocytochemistry staining, including eight samples analyzed by 2D DIGE. There were 63 samples (49 male; 14 female; age, 52 12 years) in which both HOP and hnRNP C1/C2 were detected. Paraffin sections (5 m) were deparaffinized and rehydrated in PBS. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS for 10 min. Antigen retrieval was performed by heating the sections in 0.01 M citrate buffer in a microwave oven. Nonspecific binding was blocked by incubating the tissue sectionswith 10% BSA (Sigma) in PBS for 60 min. Slides were then incubated overnight at 4 °C with either polyclonal hnRNP C1/C2 antibody (Santa Cruz Biotechnology) at a dilution of 1:800 or monoclonal HOP antibody (GeneTex) at a dilution of 1:50. Controls without primary antibodies were also included. After three washes with PBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Immunocomplexes were detected with 3,3-diaminobenzidine (Sigma) as a chromogen resulting in deposition of a brown reaction product. After counterstaining with Gill hematoxylin, sections were dehydrated and mounted for microscopic viewing. Specimens with 0, 25, 50, 75, and 100% positively stained tumor or nonneoplastic hepatocytes were scored as respectively. RESULTSAnalysis of Differentially Expressed Proteins—After 2D DIGE, the Cy2, Cy3, and Cy5 channels of each gel were individually imaged, and the images were analyzed using DeCyder 5.0 software. Among 2338 matched protein spots, 61 were significantly up-regulated in the tumor group (ratiotumor/nontumor 2, p 0.01; spots shown in Fig. 1A), whereas 158 spots were down-regulated (rationontumor/tumor 2, p 0.01; spots shown in Fig. 1B). Identification of Differentially Expressed Proteins-One hundred and twenty-two differentially expressed spots were identified, and their detailed information are listed in Supplemental Table S1. Among the 61 significantly up-regulated spots in the tumor group, 34 spots corresponding to 23 different gene products were identified. Seven of them were heat shock proteins or chaperones: 78-kDa glucose-regulated protein precursor (GRP 78), heat shock 70-kDa protein 1 (HSP 70.1), and heat shock 70-kDa protein 4 (HSP 70RY) are members of heat shock protein (HSP) 70 family, HSP 90 (HSP 84 and HSP 90) and GRP 94 are members of HSP 90 family, and HOP and T-complex protein 1 are chaperones. Other up-regulated proteins were those associated with transcription and translation, cytoskeleton, and DNA replication.The 23 up-regulated proteins were located in the cytoplasm (43%), nucleus (26%), and endoplasmic reticulum (13%).Some previously reported HCC-associated proteins, such as proliferating cell nuclear antigen (21, 22) and hepatomaderived growth factor (23) were also found to be up-regulated in this study. Furthermore there were two up-regulated proteins that correlate with telomerase activity, including hnRNP C1/C2 and nm23-H2.Among the 158 significantly down-regulated spots in the tumor group, 88 spots were identified to represent 48 different gene products. They are involved in biotransformation (31%), oxidoreduction (17%), amino acid metabolism (15%), lipid metabolism (12%), protein binding and transport (10%), cytoskeleton (4%). and other functions (shown in Fig. 2A; based on Swiss-Prot database annotation). Interestingly four metabolicenzymes involved in the methylation cycle in the liver were down-regulated in HCC tissues, including S-adenosylmethionine synthetase (or methionine adenosyltransferase (MAT)), glycine N-methyltransferase (GNMT), betaine-homocysteine S-methyltransferase (BHMT), and catechol O-methyltransferase(COMT). The subcellular locations of these down-regulated proteins are shown in Fig. 2B. More than halfof these proteins are located in the cytoplasm (35%) or mitochondria (27%), and 8% are located in peroxisomes.As reported in other gel-based researches, in the results of this study, different spots were identified to be products of the same gene. It is of particular interest that differentially expressed protein spots were found to be products from the same gene. For example, we identified seven spots as GAPDH of which six of higher molecular mass (41 kDa) were up-regulated, whereas one of lower molecular mass (36 kDa)was down-regulated. The DIGE gel maps of this 36-kDa spot are displayed in Fig. 3A and showed its down-regulation in tumor tissues of samples 1–11. Similarly one up-regulated spot and a second down-regulated spot were both identified to be formimidoyltransferase-cyclodeaminase (FTCD) as shown in Fig. 4. Protein Validation by Western Blot—To verify DIGE results,the 12 pairs of HCC samples were further analyzed by Western blot. The specificity of the antibodies against GAPDH, HOP, and hnRNP C1/C2 was verified by 2DE Western blot (Fig. 5). In DIGE results, we identified seven spots as GAPDH of which six of high abundance with similar molecular mass (41 kDa) and different pI values were up-regulated, whereas one of low abundance and lower molecular mass (36 kDa) was down-regulated in HCC tissues. As shown in Fig. 3B, theup-regulation of a 41-kDa GAPDH was observed in all 12 tumor tissues as compared with the nontumor counterparts, whereas a 36-kDa GAPDH was detected in nine nontumor tissues (failure of detection in samples 5 and 6 might be due to very low abundance) and one tumor tissue. These results were consistent with the DIGE results as shown in Fig. 3A. We found in the DIGE results that the HOP level was increasedin HCC tissues (as shown in Fig. 3C). The increased HOP levels were confirmed in all 12 tumor tissues by Western blotting (Fig. 3D), consistent with the DIGE result. Our DIGE results also showed that the intensity of an hnRNP C1/C2-containing spot was increased in HCC tissues (data not shown). To investigate the alteration of hnRNP C1/C2 in HCC, Western blot was performed, and two bands were detectedby anti-hnRNP C1/C2 monoclonal antibody (Fig. 3E). The 42-kDa band was up-regulated in 10 of 12 tumor tissues, and the 67-kDa band was detected in all tumor tissues but was weak or non-detectable in nontumor samples. Immunohistochemistry Staining—Immunohistochemistry staining of hnRNP C1/C2 and HOP was performed on paraffin sections that contained both tumor tissue and adjacent nontumor liver tissue from 70 HCC patients. Results showed that the positive staining rate of hnRNP C1/C2 was 100% in tumor tissues and 7% (5 of 70) in nontumor tissues (shown in Table II). Meanwhile HOP was expressed in 100% (70 of 70) tumor tissues and 6% (4 of 70) nontumor tissues (Table III). More interestingly, we found that in nontumor tissues only a minor portion of hepatocytes were positively stained (), whereas many more hepatocytes in tumor tissues (mainly , , and ) expressed hnRNP C1/C2 and HOP. Both hnRNP C1/C2 and HOP were stained in the nucleus of hepatocytes as shown in Fig. 6. DISCUSSIONThere have been several reports on proteomics analysis of HCC using cell lines (22, 24–27) and animal models (28 –30), but further validation of results in human clinical samples seemed to be indicated (31) as carcinogenesis in human beings may have significant differences from in vitro or rodent neoplasm. HCC is one of the most frequent malignant tumor types worldwide with a very high morbidity and mortality, andearly diagnosis is very important. Human serum is a type of feasible sample for clinical detection. Much attention has been paid to comparisons of sera among populations with sequential progression of liver diseases, such as different stages of chronic liver disease and HCC (32–37), and new research aspect such as glycosylation has also been applied (38). However, the high abundance proteins and the wide protein dynamic range remain as technical challenges to serum or plasma proteome analysis. Moreover the diseaserelatedproteins in serum or plasma may be significantly diluted (5 liters plasma in an adult) or intermixed withunrelated proteins from other organs of the human body. In contrast, those disease-related proteins have the highest concentrations in tissue. Those proteins will have clinical application potential if they could be released into the blood, therefore tissue is a material that adapts to both carcinogenesis mechanism research and biomarker discovery. However, most of these previous proteomics studies lacked further validation in larger populations. We analyzed the proteome of paired tumor and nontumor liver tissues from 12 HCC patients using the 2D DIGE technique. Among the differentially expressed protein spots, 73 gene products wereidentified. This result was also consistent with gene expression profiling studies of HCC (39). In conjunction with Westernblot results, which are of recognized consistency, our data have yielded accurate information about the proteomic alterations in HCC tissues, which in turn may help shed light on understanding the mechanism of HCC carcinogenesis. Furthermore two up-regulated proteins, HOP and hnRNP C1/C2,with no report about the relationship between them and HCC so far, were identified as potential protein markers of HCC by immunohistochemistry staining in 70 paired HCC tissues. Members of Heat Shock Protein 70 and 90 Families Were Both Up-regulated in HCC Tissues—In this study, we identified five heat shock proteins that were up-regulated in HCC tissues, including three members of the HSP 70 family (GRP 78, HSP 70.1, and HSP 70RY) and two members of the HSP 90 family (HSP 90 and GRP 94). Takashima et al. (40) found four members of the HSP 70 family overexpressed in HCVassociated HCC tissues, including GRP 78, heat shock cognate 71-kDa protein (HSC 70), GRP 75, and HSP 70.1. In the present study, two of these four proteins, GRP 78 and HSP 70.1, were found to be up-regulated in HBV-associated HCC tissues, suggesting these two proteins may not be specific for either HCV or HBV infection-associated HCC. In addition, there were two HSP 90 family members up-regulated in HBVassociated HCC. One of them, HSP 90, is known as anessential component of several signal transduction pathways and has been identified as an essential host factor for HBV replication (41). We also identified and validated the overexpression of HOP, which mediates the association of the molecular chaperones HSC 70 (ratiotumor/nontumor 1.6 in our results) and HSP 90 (42). This simultaneous overexpression of members of HSP 70 and 90 families in human HBV-associated HCC was also found in the research of a Korean group (10). However, only members of the HSP 70 family were reported to be up-regulated in HCV-associated HCC. This may reflect a difference in pathogenesis between HBV and HCV infection, and together with the up-regulation of HOP, these results suggested that members of HSP 70 and 90 families may be involvedin HBV-related carcinogenesis. Mitochondrial and Peroxisomal Proteins Were Downregulated— A finding of note was that 27% of the downregulated proteins in our research were located in mitochondria. In contrast, in the human proteome, the proportion of proteins assigned to the mitochondria is only 4% (43). This suggested that mitochondria are altered significantly duringthe HCC carcinogenesis, consistent with previous cancer biological research (44–46), and confirmed the conclusion of Chignard and Beretta (47) that by means of 2DE/MS mitochondrial proteins made up the second largest proportion (19%) of the dysregulated proteins identified in HCC. In our results, 8% of the down-regulated proteins were peroxisomal proteins, including the peroxisomal marker protein catalase. It has been demonstrated that human hepatocellular tumor cells contain fewer peroxisomes than extrafocal hepatocytes (48). Neoplastic transformation may affect the biogenesis of this organelle and might thus be responsible forsome of the metabolic derangements observed in the disease processes of HCC. The down-regulation of mitochondrial and peroxisomal proteins in this study was consistent with cancer biological research and indicated that detailed subcellular (e.g. mitochondria and peroxisome) proteome analysis may be a method for the demonstration of organelle alterations in carcinogenesis (49). Down-regulation of Methylation-related Enzymes—In this study, four metabolic enzymes involved in the methylation cycle in the liver, MAT, GNMT, BHMT, and COMT, were down-regulated in HCC tissues. MAT is an enzyme essential for the formation of S-adenosylmethionine (AdoMet) in the methylation cycle. The down-regulation of MAT, GNMT, BHMT, and COMT reflected the decrease of AdoMet in the liver. Chronic deficiency in AdoMet results in spontaneousdevelopment of steatohepatitis and HCC, but the mechanism remains unknown (50). A study in knock-out mice showed that the deficiency of AdoMet impairs mitochondrial function and generates oxidative stress in the liver (51). Our result is consistent with earlier studies showing that the mRNA levels of MAT, BHMT, and GNMT are markedly reduced in human cirrhosis and HCC (52, 53). However, proteome data regardingthe alterations of these enzymes in human HCC tissues are discrepant. Liang et al. (14) found the down-regulation of MAT, BHMT, and GNMT in poorly differentiated HCC and of BHMT in well differentiated HCC, whereas Lee et al. (19) reported the up-regulation of MAT in HCC tissues. The samples they used were seven pairs of HBV-associated HCC oreight pairs of HCC of heterogeneous pathogenic backgrounds (two HBV- and HCV-infected, three HBV-infected, one HCV-infected, and two without HBV or HCV infection). Sample differences might be the reason for result discrepancy. Our results strongly supported the Liang et al. (14) data, which demonstrated similar alterations of these three enzymes in HBV-associated HCC tissues and confirmed the relationship between AdoMet deficiency and carcinogenesis at least in HBV-associated HCC. Another enzyme involved in the methylation cycle, COMT, was also found to be downregulated in human HCC tissues. In a previous report, the low activity COMT (L) alleles were considered as a high risk genotype(54), and our research is the first report about its expression level alteration in human HCC tissues.Different Isoforms or Modifications of GAPDH and FTCD May Play Different Roles in HCC Carcinogenesis—Two gene products, GAPDH and FTCD, were identified from inversely altered spots. We identified seven spots as GAPDH of which six spots were up-regulated, whereas one spot was downregulated. Besides the up-regulated spots consistent with results in previous cancer researches (10, 55–57), we found another 36-kDa spot of GAPDH down-regulated in HCC tissues. This down-regulation was further validated by Westernblot. Similarly two spots were shown to correspond to FTCD with one up-regulated and a second down-regulated. FTCD is a folate-dependent enzyme and also a liver-specific autoantigen in patients with autoimmune hepatitis (58). FTCD was also listed among the down-regulated proteins in two HCCproteome analysis reports (12, 14), but its relationship with HCC is not clear. It is possible that the two inversely changed,spots correspond to different isoforms or modifications. For the first time, we present the co-existence of conversely changed spots corresponding to GAPDH and FTCD, which may have different isoforms or modifications playing different roles in HCC. HnRNP C1/C2 and HOP May Be Potential Markers in HCC Tissues—Two identified up-regulated proteins, hnRNP C1/C2 and nm23-H2, both correlate to telomerase activity. nm23-H2 has been shown to promote the expression of the c-myc gene and might be associated positively with telomerase activity in HCC (59), whereas there are no reports about the relationshipbetween hnRNP C1/C2 and HCC. hnRNP C1/C2 is one of the hnRNPs bound to telomerase (60), and the application of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA (61). Knockdown of hnRNP C1/C2 by small interfering RNA inhibited the growth of HeLa cells (62). In lung epithelial cells, hnRNP C was found to interact with and regulate the stability of urokinase receptor mRNA (63). Other members of the hnRNP family were reported to be related to HBV or HCC. For example, hnRNP K (64), hnRNP H/H (65), and Hcc-1 (with sequence matches to hnRNP but localized to chromosome 7q22.1, which is different than hnRNP C1/C2) (66). But there has been no report about the up-regulation of hnRNP C1/C2 in HCC. Because telomeraseactivity is up-regulated in most HCCs, we supposed thathnRNP C1/C2 could be a new candidate marker for HCC and chose it for further validation. HOP is a molecular chaperone. It was reported that HOP regulates the processes of proliferation and myogenesis in cardiac development (67, 68). But there has been no report about the relationship between HOPand HCC. The mass spectra of hnRNP C1/C2 and HOP are shown in Figs. 7 and 8. In the present study, we validated the up-regulation of hnRNP C1/C2 and HOP in HCC tissues by Western blot in 12 pairs of HCC tissues. In addition, immunohistochemistry was also performed with anti-hnRNP C1/C2 and anti-HOP antibodies in 70 HCC samples. The results showed that the positive staining rates of hnRNP C1/C2 were 100% in tumor tissues and 7% in nontumor tissues, whereas HOP was expressed in 100% of tumor tissues and 6% of nontumor tissues.This overexpression of hnRNP C1/C2 and HOP in HCC tissues was significant.Up to this point, there has been no report about the relationship between hnRNP C1/C2 or HOP and HCC. We validated the overexpression of hnRNP C1/C2 and HOP in HCC tissues by Western blot and immunohistochemistry for the first time, but their functions in carcinogenesis need to be further investigated. 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王崑 黄信孚 孙谊 邢宝才【摘要】目的 探讨胰体尾肿瘤整块切除联合腹腔干切除(Appleby手术)的安全性及可行性。方法 对6例接受Appleby手术的侵犯腹腔干的胰腺肿瘤患者的临床资料进行分析。结果 6例患者中,5例为原发性胰体尾癌,1例为胰体尾转移瘤(腹膜后神经鞘瘤术后),行胰体尾肿瘤整块切除联合腹腔于切除术,均未行血管重建。6例手术均获得成功。手术时间4~6 h,术中出血400~1200 Inl,术后肝功能一过性升高,经保肝治疗,2周内全部恢复正常,肝脏和胆囊均有正常动脉血供,无手术死亡,无严重并发症发生。结论 胰体尾肿瘤整块切除联合腹腔干切除手术是安全可行的,它可以提高肿瘤的切除率并可缓解疼痛。 胰体尾恶性肿瘤发病隐匿,一经发现往往侵犯腹腔干,因而切除率很低,预后极差。国外报道,胰体尾癌的切除率为10%~22%,1年生存率为8%-9%,自2005年1月,我院肝胆胰腺外科针对6例侵犯腹腔干的肿瘤患者施行Appleby手术,均未行动脉重建,未出现严重并发症和手术死亡,现报告如下。临床资料一、一般资料 本组6例患者中5例为原发胰体尾癌,1例为胰体尾转移瘤(腹膜后神经鞘瘤术后)。男性4例,女性2例,年龄43~78岁,中位年龄64.3岁。肿瘤平均体积:4.5 cm3。所有患者术前行腹部B超和CT检查显示肿瘤均位于胰体尾部,均侵犯腹腔干(图1),而肠系膜上动静脉、门静脉未受侵犯。二、手术方法手术步骤如下:术中探查确认肿瘤位于胰体尾,局部浸润严重,均包绕腹腔干。解剖肝十二指肠韧带,沿肝固有动脉分离至肝总动脉,靠近胃十二指肠动脉起始部,结扎肝总动脉,确认肝固有动脉搏动良好。沿腹主动脉向下分离,确认肠系膜上动脉未受侵,横断胰颈部,沿肠系膜上动脉表面向上分离,在肠系膜上动脉发出的上方可以看到腹腔干的起始部,在此切断之,连同标本一并移出(图2)。切除的范围包括:胰体尾,脾,腹腔干,肝总动脉,胃左动脉,腹腔神经丛,腹膜后脂肪组织及腹主动脉旁淋巴结。本组每例患者由于肿瘤侵犯的周围器官有所不同,因此,联合切除的其他器官也不相同,其中近端胃切除2例,左肾切除1例,左肾上腺切除2例,结肠部分切除1例,1例患者肝脏单发转移灶行左肝部分切除。5例手术达到R0切除。三、结果6例患者的手术过程均较顺利,无手术死亡。6例患者均未施行动脉重建。手术时间4~6 h,出血400~1200 m1。1例患者术后由于低血糖出现低血糖性昏迷,抢救无效死亡。无胰瘘,无腹腔感染,肝脏和胆囊未出现缺血和坏死,部分患者术后出现肝功能一过性增高,经保肝治疗后,术后2周肝功能均恢复正常(图3)。6例患者术后均未出现胃黏膜缺血等病变。术后患者行彩色多普勒超声检查:肝脏和胆囊均可见正常动脉血流。2例患者出现腹泻,药物治疗后好转。伴有腰背部疼痛的患者症状均有所减轻。随访结果:1例术后死于低血糖昏迷(术前有顽固性低血糖病史),1例术后3个月肿瘤复发后死亡。4例仍存活,最长18个月,其余3例分别为11个月,6个月和4个月,仍在继续随访中。讨论 1953年Appleby等在给进展期胃癌的患者施行胃癌根治术时,为了更彻底的切除肿瘤和淋巴结清扫而施行胰体尾联合腹腔于切除,Appleby手术由此得名。随着腹腔干结扎在创伤外科手术中的应用以及腹腔干切除在腹腔干动脉瘤中的相继开展,根治性胰体尾切除联合腹腔干切除也在国外逐渐开展。1976年Nimura等将此术式首先用于胰体尾的扩大根治术。施行Appleby手术可提高手术切除率特别是R0切除率。Kondo等报告:13例胰体尾肿瘤患者行Appleby手术R0切除率达到67%,无手术死亡,无严重并发症的产生。本组6例肿瘤均侵犯腹腔干,不切除腹腔干均无法达到R0切除,实行Appleby手术后,R0切除率达到83%,高于常规胰体尾切除术。胰体尾肿瘤患者术前常合并顽固性腰背疼痛,由于术中切除腹腔神经丛和神经节可以获得较好的止痛效果,不用再长期服用镇痛药物,明显改善了生活质量。Appleby手术的理论基础为:肝总动脉分出肝固有动脉和胃十二指肠动脉,当结扎肝总动脉后,在保证肠系膜上动脉的维持正常的血流的前提下,部分血流可通过胰十二指肠上、下动脉的血管弓经胃十二指肠动脉逆向进入肝固有动脉从而保证肝脏血流。Appl曲y手术后常见的特异的并发症有:1.由于切除腹腔干导致的缺血性并发症包括胃黏膜缺血伴溃疡,肝脏缺血导致肝功能受损,肝脓肿,胆囊缺血坏死等;2.切除腹腔神经丛导致的顽固性腹泻。以上症状经药物及保守治疗均可缓解好转。由于担心术后出现严重的肝脏功能损害和胆囊缺血坏死以及胃黏膜缺血等并发症的发生,文献报道的此种手术多数病例进行了动脉重建。本组6例患者Appleby术中动脉均未重建,对可能出现的并发症进行了严密的监测后发现,所有患者自术前施行胃肠减压直至术后胃肠功能恢复,未出现胃痛、出血及溃疡等胃黏膜损害的表现。所有术后患者均监测肝功能,部分患者出现转氨酶一过性升高,2周后全部恢复正常,没有患者出现肝功能不全或肝功能衰竭表现。所有患者术后1个月复查腹部彩色多普勒超声显示肝脏动脉血供良好,胆囊亦有良好血供、无缺血坏死表现,亦无急慢性胆囊炎表现。2例患者出现腹泻,药物治疗后好转。本组6例患者经Appleby手术提高了手术切除率,特别是R0切除率。无手术死亡,也无严重手术并发症的产生,肿瘤侵犯神经产生的疼痛均获得很好控制,提高了生活质量,证明此种手术是安全和有效的。通过本组病例,我们认为选择Appleby手术的适应证为:1.局部进展期胰体尾肿瘤;2.腹腔干受侵犯,而肠系膜上动脉未受侵;3.无远处广泛转移。尽管有个案报道Appleby术后存活5年和13年,但是术后平均生存期仍少于1年(6.6-11.2个月)。由于缺乏长期随访和大宗病例的循证医学证据,Appleby手术能否延长生存期尚有待进一步研究。参考文献[1] Nordbck IH,HnhRH,Btnoll JK,et a1.carcinoma of the body and tail of the pancreas.Am J Surg,1992,164:26-31.[2] Johnson CD,SchwaU G,F1echtenmacher J,et a1.Resection for adenocarcinoma of the body and tail of the pancreas. Br J Surg,1993.80:l177-1179.[3] Brennan MF, Moccia RD, Klimstra D. Management of adenocarcinoma of the body and tail of the pancreas.Ann Surg,1996,223:506-511.[4] Appleby LH.The coeliac axis is in the expansion of the operation for gastric carcinoma.Cancer,1953,6:704-707.[5] Kavic sM,Atweh N,Ivy ME,et a1.celiac axis 1igation after gumshot wound to the abdomen:case repoort and literature review.J trauma Injury Infect Crit Care,2001,50:738-739.[6] Bret PM,Partensky c,Bretagno1le M.obstmctive jaundice by a dissecting aneurysm of celiac axis and hapatic artery.Dig Dis Sci.1987.32:1431-1434.[7] Nimura Y,Hattori T,Miura K,et a1.Experience of Appleby’s operation for advanced carcinoma of the paIncreatic body and tail.Shuiutsu,1976,30:885-889.[8] Kondo S,Katoh H,Himno S,et a1.Results of radical distal pancreatectomy with en bloc resection of the celiac anery for locally advanced cancer of the pancreatic body.Langenbecks Arch Surg,2003,388:10l-106.[9] Mayumi T,Nimum Y,Kamiya J,et a1.Distal pancreatectomy with en b1oc resection of the celiac artery for carcinoma of the body and tail of the pancreas.Int J Pancreatol,1997,22:15-21.[10] Ozaki H,Kinoshita T,Kosuge T,et a1.An aggressive therapeutic approach to carcinoma of the body and tail of the pancreas.Cancer.1996.77:2240-2245.
专家论坛合理规范地治疗胆囊癌邢宝才, 王宏伟作者单位:100036北京,北京大学临床肿瘤学院、北京肿瘤医院肝胆外科,恶性肿瘤发病机制及转化研究教育部重点实验室第一作者:邢宝才(1963一),男,医学博士,教授,博士研究生导师,主任医师 胆囊癌是胆道系统最常见的恶性肿瘤。我国胆囊癌发病率占同期胆道疾病构成比的0.4%-3.8%,平均为1.53%。胆囊癌发病率虽然较低,但恶性程度高,预后非常差,5年总生存率仅为5%。 胆囊癌的肿瘤分期对其治疗方式的选择及预后判断起重要作用。胆囊癌有多个分期系统,包括Nevin分期、TNM分期及日本胆道外科协会分期。Nevin分期根据肿瘤侵犯胆囊壁的深度和转移情况将胆囊癌分为5期:I期,黏膜层内原位癌;II期,侵入黏膜和肌层;Ⅲ期,侵犯胆囊壁全层;IV期,侵犯胆囊壁全层和胆囊淋巴结;V期,侵犯或转移至肝及其他部位。以前大多数学者使用Nevin分期系统,但随着对胆囊癌认识加深及治疗理念的改变,Nevin分期已逐渐被TNM分期所取代。TNM分期根据肿瘤侵犯深度、淋巴结转移情况及有无远处转移把胆囊癌分为4期。最新的第六版TNM分期中原发肿瘤浸润深度:Tx为原发肿瘤无法评估;T0为无原发肿瘤证据;Tis为原位癌;T1为肿瘤侵及固有层或肌层,T1a为肿瘤侵及固有层,Tlb为肿瘤侵及肌层;T2为肿瘤侵及肌层周围结缔组织,尚未侵透浆膜或侵入肝脏;T3为肿瘤侵透浆膜和(或)直接侵及肝脏和(或)1个其他邻近器官或组织;T4为肿瘤直接侵及门静脉或肝动脉主干或侵及2个或更多的肝外器官或组织。淋巴结转移:Nx为区域淋巴结转移无法评估;N0为无区域淋巴结转移;NI为有区域淋巴结转移。远处转移:Mx为远处转移无法评估;M0为无远处转移;M1为有远处转移。第六版TNM分期较第五版改动较大,改动后鼓励外科医师积极手术治疗胆囊癌及更加积极地清扫淋巴结。1 规范的胆囊癌手术方法 外科手术仍然是治愈胆囊癌的唯一办法。其术式包括单纯胆囊切除、胆囊癌根治手术及扩大根治术等。手术方式应根据TNM分期及肿瘤侵犯的范围来选择,并应确保达到R0切除。对T分期为Tis及T1a的患者行单纯胆囊切除术即可达到根治效果。而Tlb患者,由于淋巴结转移几率升高,单纯胆囊切除术可能达不到R0切除,越来越多的学者建议行胆囊癌根治术。T2患者应至少行胆囊癌根治性手术,即切除完整的胆囊,胆囊床所在的肝Ⅳ段、V段次全切和区域淋巴结清扫,同时可根据肿瘤的位置及切缘来决定是否行扩大切除(如右半肝切除及胆管切除)。T3、T4期患者常需行扩大根治术,其切除范围可包括右半肝、肝中叶甚至右三叶切除,肝外胆管、胰十二指肠切除,胰周淋巴结、肝门淋巴结清扫,腹腔干、下腔静脉及腹主动脉旁淋巴结清扫等。但扩大根治术尚存在争议,手术前,应综合考虑患者的全身情况,判断其对手术的耐受能力,尽量减少或避免与手术相关的死亡。此外,在开腹手术前也可行腹腔镜探查以确定疾病分期,避免不必要的开腹手术。2 胆囊肿物的合理处理 临床中,如果胆囊肿物的影像学表现提示高度怀疑胆囊癌或无法鉴别肿物良恶性时,应积极开腹手术探查,术中送冰冻病理检查,根据肿瘤浸润深度来决定治疗的方式。为了预防胆汁外漏、癌细胞播散,活检时应遵循整块切除原则,不可切开胆囊行切取活检。如肿物与周围组织关系密切,应部分切除周围受累组织,以明确病理分期。另外,对于术前怀疑胆囊癌的患者,应让患者在有条件进行胆囊癌根治手术的肝胆外科中心接受治疗,避免轻易行单纯胆囊切除手术。单纯胆囊切除术后往往有疾病残留,术后周围组织如结肠肝曲常与胆囊床粘连,在二次手术时需行扩大切除术。这些患者的预后可能较术中获得诊断立即行根治手术的患者差。3 意外胆囊癌的处理 意外胆囊癌是指因胆囊良性疾病行胆囊切除的术中或术后病理发现的胆囊癌。国内报道的腹腔镜意外胆囊癌发生率为0.12%-3.86%,开腹胆囊切除术的发生率约为1.7%-2.3%。大部分的意外胆囊癌为T1、T2期患者。意外胆囊癌的手术方式取决于病理分期。在腹腔镜术中发现意外胆囊癌时应立即中转开腹。对于术后病理检查时发现的胆囊癌,如病理分期为pTis、pTla,没有发生术中胆漏且切缘阴性的患者,无须行二次手术;如病理分期为pTlb以上则应尽早接受二次手术,手术原则同前。腹腔镜Trocar创口的切除尚存在争议。为避免术后才发现胆囊癌的被动局面,建议在胆囊切除术后常规剖检胆囊,见疑似病变时应送冰冻病理检查。在良性疾病的手术探查过程中发现意外胆囊癌时,如果手术医师没有做好肝切除的准备,应停止手术,并将患者转院到有经验的肝胆外科中心进行二次手术。这些未行胆囊切除手术的患者在接受二次手术后其预后并未受到影响。4 进展期胆囊癌的治疗对临床及影像学表现符合进展期胆囊癌的患者,可行针吸活检以明确诊断。此类患者预后非常差,有黄疸、上消化道梗阻的患者可行姑息手术或放置支架治疗。无黄疸等症状的胆囊癌患者可考虑放化疗。胆囊癌是恶性程度非常高的肿瘤,手术切除是唯一治愈胆囊癌的方法。大量循证医学证据表明,合理规范的手术能够显著改善患者的预后。同时,胆囊癌的手术创伤较大,死亡率及并发症发生率较高,患者应尽量在经验丰富的肝胆外科中心接受治疗。参考文献1.邹声泉,张林.全国胆囊癌临床流行病学调查报告[J].中国实用外科杂志,2000,20(1):43-46.2.Wakai T,Shirai Y,Yokoyama N,et al.Early gallbladder carcinoma does not warrant radical resection[J].Br J Surg,2001,88(5):675-678.3.Principe A,Del Gaudio M,Ercolani G,et al.Radical surgery for gallbladder carcinoma:possibilities of survival[J].Hepatogastroenterology,2006,53(71):660-664.4.Ito H,Matros E,Brooks DC,et al.Treatment outcomes associated with surgery for gallbladder cancer:a 20-year experience[J].J Gastrointest Surg,2004,8(2):183-190.5.Lai EC,Lau WY.Aggressive surgical resection for carcinoma of gallbladder[J].ANZ J Surg,2005,75(6):441-444.6.Steinert R,Nestler G,Sagynaliev E,et al.Laparoscopic cholecystectomy and gallbladder cancer[J].J Surg Oncol,2006,93(8):682-689.7.Fong Y,Jarnagin W,Blumgart LH.Gallbladder cancer:comparison of patients presenting initially for definitive operation with those presenting after prior noncurative intervention[J].Ann Surg,2000,232(4):557-569.
保留脾脏和脾血管的胰体尾切除手术邢宝才 孙谊 包全 钱红纲 郝纯毅 黄信孚 王怡 顾晋 季加孚 【摘要】 目的 探讨胰腺体尾部肿瘤进行保留脾脏和脾血管的胰体尾切除手术的可行性与安全性。方法 对2003年12月至2005年1月收治的4例胰腺体尾部实性假乳头状肿瘤患者,采用了保留脾脏与脾血管的方法进行胰腺远端的切除。在肿瘤的右侧横断胰腺,显露脾动静脉,自胰腺的体部向脾脏方向分离脾动静脉血管与胰腺。不需离断脾动静脉和胃短血管,不需游离脾脏。结果 4例患者均成功完成保留脾脏和脾血管的胰体尾切除手术。全部病例的平均手术时间为208 min ±52 min;手术中的平均出血为475 ml ±96 ml;手术均未需要输血;手术后平均住院时间为18 d ±13 d。手术后1例患者出现胰瘘,经保守治疗治愈。无其他合并症。随访无复发与转移。结论 保留脾脏和脾血管的胰腺远端切除是安全可行的,对于胰腺良性与交界性肿瘤的切除是最佳的选择。 胰腺体尾部与脾动静脉和脾脏的位置毗邻关系密切。当胰腺体尾部发生肿瘤,传统的治疗方法是行胰腺的远端及脾脏的联合切除。近年来,随着对脾脏解剖学及其功能的认识不断深入,脾脏的抗感染、抗肿瘤的免疫功能已经被确认。保留脾脏的胰腺体尾部切除的手术逐渐引起人们的关注[ 1 ] 。对于位于胰腺体尾部的胰腺癌,由于肿瘤在发生的早期就会出现胰腺周围的浸润,为达到手术的根治性,一般主张应行胰腺远端及脾脏的联合切除。但是,于胰腺体尾部的良性肿瘤和交界性肿瘤,在完全切除胰腺肿瘤的同时保留患者脾脏的功能,应是此类患者最佳的、最合理的治疗方式[ 2 ] 。我们对4例胰腺体尾部实性假乳头状肿瘤患者的手术治疗的过程进行了分析研究。对象与方法1. 对象: 2003年12月至2005年1月,北京肿瘤医院外科共进行了4例保留脾脏和脾血管的胰腺体部切除手术。其中男1 例,女3 例。年龄17 ~37岁,中位年龄30岁。所有的病例均无背部疼痛,消瘦等临床症状,偶然发现腹部包块或体检发现胰腺占位。入院后检查肿瘤标记物癌胚抗原(CEA) 、CA199均在正常范围。B 型超声、CT或磁共振成像(MR I)检查显示4例患者的肿瘤均位于胰腺的体尾部,肠系膜血管的左侧,边界清楚,影像学检查显示肿瘤与与脾血管有清楚的界限。4例患者中,肿瘤最大的6.0 cm ×4.5 cm, 最小的4.5 cm ×3.2 cm。综合各项检查结果临床诊断为胰腺的良性或交接性肿瘤。因此,本研究选择了切除胰腺肿瘤同时保留脾脏和脾血管的胰腺体尾部切除的手术方式。2. 手术方式:手术经上腹部正中切口入腹,分离胃结肠韧带至脾脏的下极,将胃翻向上方充分显露胰腺及肿瘤。分离胰腺下缘,显露结肠中血管及肠系膜上静脉;分离胰腺上缘与肝总动脉。自肠系膜上静脉前方,胰腺后向上分离,于胰腺颈部横断胰腺,胰头侧胰管结扎,胰腺近断端间断褥式缝合。清楚显露门静脉2肠系膜上静脉,脾静脉,脾动脉。提起胰腺远断端,沿脾动静脉的表面向左分离,脾动静脉与胰腺体尾部间的穿支仔细分离,结扎切断,直至脾门,全部切除胰腺的体尾部及其肿瘤。脾动静脉主干及其脾门的分支、脾脏完整保留。手术过程未处理脾胃韧带以及脾结肠韧带,未游离脾脏。结果所有4个病例均成功完成保留脾脏和脾血管的胰腺体尾部切除, 手术后脾脏血运完全正常(图1) 。全部病例的平均手术时间为208 min ±52 min;手术中的平均出血为475 ml ±96 ml;手术均未需要输血;手术后平均住院时间为18 d ±13 d。手术后1例患者出现胰瘘,经保守治疗治愈;由于胰瘘造成手术后住院时间的延长,手术后住院37 d;其余病例手术后恢复顺利,无手术后的合并症。病理结果:全部病例手术后的病理结果均为胰腺实性—假乳头状肿瘤。病例随访观察时间为7~19个月,中位随访时间1313个月。全部病例手术后血糖在正常的范围,手术后患者均无发热的表现;复查B型超声,脾脏血供正常,无脾脏脓肿的表现。随访期间,患者均无复发与转移的征象。讨论 对于胰腺体尾部肿瘤,在行胰腺远端切除的过程中通常会一并切除脾脏。这是因为一方面联合脾脏切除可以降低手术难度,简化手术,缩短手术时间;另一方面,人们既往对于脾脏的功能认识不够,认为切除与保留脾脏对于人体的功能影响不大。近些年来,随着脾脏外科基础与临床研究的不断深入,研究显示脾脏是人体重要的功能器官,在维持人体正常活动方面发挥着重要的作用,尤其是脾脏的抗感染与抗肿瘤功能已被确认。因此,对于胰腺体尾部肿瘤是否一定进行联合脾脏的胰腺远端切除手术,是否应该尽可能的保留脾脏,引起了人们广泛的兴趣。 对于胰腺的恶性肿瘤如胰腺癌,由于其生物学行为的特殊性,在肿瘤的早期,大部分胰腺癌的患者就会出现胰腺周围尤其是肿瘤后方的脂肪神经组织的浸润与转移,手术后复发率较高,所以,为了达到手术切除的彻底性,对于胰腺体尾部的胰腺癌,手术治疗时应在切除胰腺远端的同时联合脾脏血管与脾脏切除[ 3, 4 ] 。但是,对于胰腺远端的良性肿瘤或交接性肿瘤,肿瘤的生物学行为较好,没有肿瘤周围的浸润与转移,同时肿瘤与脾动静脉间有解剖间隙可以进行分离,因此,在行胰腺远端切除时,保留脾脏的术式是可行的。在切除胰腺肿瘤的同时,还能达到保留脾脏功能的目的。1988年Warshaw[ 5 ]首先报道了保留脾脏的胰腺远端切除手术。其方法是保留胃短与胃左的动静脉血管,以保证脾脏的血供,但不保留脾脏的动静脉[ 6 ] 。此术式的缺点是有可能引起脾脏的缺血,造成脾脏的梗塞和脓肿形成。1996年Kimura等[ 6 ]报道了保留脾脏保留脾血管的胰腺远端切除术,在切除胰腺远端的同时保留脾脏的动静脉和胃短与胃左血管。此术式的优点是保证了脾脏的血供,避免了脾脏的缺血;但是,此术式技术难度大,手术技巧要求高。在胰腺体尾部肿瘤与脾脏血管能够分离的情况下,保留脾脏同时保留脾脏血管是理想的保脾性手术方法。姜洪池等在我国率先开展了保脾手术[ 1 ] 。但是,相关文献报道不多。本研究采用了保脾与保留脾血管的方法进行胰腺远端切除。手术过程自胰腺的断端向脾门分离,仔细处理脾动静脉向胰腺的分支,仅切除胰腺的远端,保留了脾脏的动静脉和胃左与胃短血管,没有游离脾脏。本组研究结果显示, 平均手术时间208 min, 手术平均出血475 ml,患者的平均术后住院时间为18 d。手术后患者没有脾脏缺血造成的脾脓肿发生,说明保留脾脏保留脾血管的胰腺远端手术是安全可行的。此外,本组4例患者均选择的是胰腺的实性假乳头状肿瘤。WHO将胰腺实性假乳头状肿瘤划分为交界性胰腺肿瘤。其特点是女性最为多见,发病年龄一般为10~40岁,肿瘤一般呈圆形,边界清楚,有完整的包膜,瘤细胞大小不一,呈实性片状,形成假乳头。根据此类肿瘤的生物学行为较好,有完整的包膜,能够与脾脏血管分离的特点,我们选择了进行保留脾脏和脾血管的胰腺远端切除手术。在切除肿瘤的同时保留了脾脏。在手术后的随访过程中,所有病例尽管是胰腺的交界性肿瘤,但是,没有发现复发与转移,说明对于胰腺体尾部的良性与交界性肿瘤,保留脾脏与脾血管的胰腺远端切除可能是最佳的手术方式[ 7, 8 ] 。参考文献1 代文杰,姜洪池. 保留脾脏的胰体尾切除. 中华肝胆外科杂志,2001, 7: 333-334.2 Benoist S, Dugue L, SauvanetA, et al. Is there a role of the spleen in distal pancreatectomy. J Am Coll Surg, 1999, 188: 2552260.3 倪泉兴,张群华,曹国海,等. 分阶段综合治疗胰头癌16 例临床分析. 中华医学杂志, 2000, 80: 252-254.4 母德清, 彭淑牖,王国凤. 胰头癌切除术后复发因素的探讨. 中华医学杂志, 2003, 83: 1657-1660.5 Warshaw AL. Conervasion of the sp leen with distal pancreatectomy.Arch Surg, 1988, 123: 550-553.6 Kimura W, Inoue T, Futakawa N, et al. Spleen preserving distal pancreatectomy with conservation of the sp lenic artery and vein.Surgery, 1996, 120: 885-890.7 Lukish JR, Rothstein JH, Petruzziello M, et al. Spleen preserving pancreatectomy for cystic pancreatic neop lasms. Am Surg, 1999, 65:596-599.8 Kimura W, Fuse A, Hirai I, et al. Spleen preserving distal pancreatectomy for intraductal papillary mucinous tumor.Hepatogastroenterology, 2004, 51: 86-90.