α-1抗胰蛋白酶缺乏症是一种常染色体共显性遗传性疾病,以血液中低水平的α-1抗胰蛋白酶为标志,临床上主要表现为年轻患者的肺气肿和急/慢性肝损伤甚至肝癌等,治疗方法包括对症治疗和α-1抗胰蛋白酶的补充,但无法阻止肝脏纤维化的进展。目前国内已有10余例该疾病的报道,但是对该疾病的认识仍然不足,生物治疗药物空白。本文现就肝细胞移植治疗该疾病的研究进展进行介绍。
α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normalfunction of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease thatdegrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutantα1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitoractivity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increaseshepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might havea competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing humanAAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation wasaccelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulationwith engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liverinjury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types ofhost hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest thatwild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternativeto protein replacement for treating emphysema in AAT-ZZ individuals.
Background and Aims: Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permitsextensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated withradiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIRto a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liverlobes by regional HIR should be sufficient to correct some inherited metabolic disorders.Methods: Hepatocytes (107) isolated from wildtype F344 rats or Wistar-RHA rats were engrafted into the livers of congeneicdipeptidylpeptidase IV deficient (DPPIV2) rats or uridinediphosphoglucuronateglucuronosyltransferase-1A1-deficientjaundiced Gunn rats respectively by intrasplenic injection 24 hr after HIR (50 Gy) targeted to the median lobe, or medianplus left liver lobes. An adenovector expressing hepatocyte growth factor (1011 particles) was injected intravenously 24 hrafter transplantation.Results: Three months after hepatocyte transplantation in DPPIV2 rats, 30–60% of the recipient hepatocytes were replacedby donor cells in the irradiated lobe, but not in the nonirradiated lobes. In Gunn rats receiving median lobe HIR, serumbilirubin declined from pretreatment levels of 5.1760.78 mg/dl to 0.9660.30 mg/dl in 8 weeks and remained at this levelthroughout the 16 week observation period. A similar effect was observed in the group, receiving median plus left lobeirradiation.Conclusions: As little as 20% repopulation of 30% of the liver volume was sufficient to correct hyperbilirubinemia in Gunnrats, highlighting the potential of regiospecific HIR in hepatocyte transplantation-based therapy of inherited metabolic liverdiseases.Citation.
Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) andthen differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelialcells shed in human urine and transplanting them into animal livers has not been described systematically. We reportreprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by adetailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by deliveringthe pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration,such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses.After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHepsexpressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involvedin cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleledthat of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant humanSERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps thatcould be potentially useful for disease modeling, pharmacological development, and regenerative medicine.
目的 制备人α-1 抗胰蛋白酶缺乏症 (α-1 Antitrypsin Deficiency)转基因小鼠。 方法 克隆人的α-1抗胰蛋白酶基因SERPINA1及“Z”点突变,采用该基因的肝细胞表达天然启动子,采用DNA原核显微注射法,将含目的片段的质粒线性化后注射至C57BL/6N小鼠受精卵雄原核,使其随机整合到宿主基因组中,将整合后的受精卵植入到该品系假孕雌鼠输卵管内,小鼠出生后取鼠尾提取染色体DNA进行PCR和DNA序列分析,筛选出携带“Z”突变α-1 抗胰蛋白酶基因的阳性小鼠,各组织提取RNA并进行RT-PCR检测α-1 抗胰蛋白酶基因的转录;肝脏活检病理切片进行淀粉酶/PAS染色和α- 1抗胰蛋白酶免疫荧光染色检查。结果 PCR和DNA序列分析证实了α-1抗胰蛋白酶基因及“Z”突变,基因分型PCR阳性产物为425bp。RT-PCR显示人α-1 抗胰蛋白酶基因仅在肝组织阳性,而在心、肺、肝、肾、脑和血液等其它组织显示阴性。肝脏活检切片淀粉酶/PAS染色显示肝细胞内大小不等阳性红色颗粒,即抵抗淀粉酶的变异的α-1 抗胰蛋白酶。α- 1抗胰蛋白酶免疫荧光染色肝细胞内可见大小不等特异性的红色颗粒。结论 成功建立α-1 抗胰蛋白酶缺乏症转基因小鼠模型。