人骨髓干细胞异体内诱导分化为肝细胞的实验(英文)

Xenogenic differentiation of human bone marrow stem cells into hepatocytes

背景:如何获取来源丰富的高质量人源性肝细胞是生物人工肝和肝细胞移植共同面临的问题。骨髓干细胞在适当条件下可分化为肝细胞,为获取肝细胞提供了新的思路。目的:探讨人骨髓干细胞在大鼠体内诱导分化为肝细胞的方法,为解决临床肝脏移植和生物人工肝来源提供新的思路。设计:随机对照实验。单位:南方医科大学珠江医院普通外科。材料:实验于2004-05/2005-02在南方医科大学珠江医院中心实验室完成。选取雄性清洁级SD大鼠40只,随机分为5组:单纯模型组、造模+人骨髓干细胞移植14d组、造模+人骨髓干细胞移植28d组、造模+CL-1细胞(人肝细胞系)移植14d组、造模+CL-1细胞(人肝细胞系)移植28d组,8只/组。方法:①5组均建立2-乙酰氨基芴+四氯化碳+环磷酰胺肝损伤模型。②利用治疗性肝再生策略,将人骨髓干细胞移植到大鼠肝脏内诱导分化为肝细胞。造模+人骨髓干细胞移植14d组移植人骨髓干细胞后观察14d,造模+人骨髓干细胞移植28d组移植人骨髓干细胞后观察28d,造模+CL-1细胞移植14d组移植CL-1细胞后观察14d,造模+CL-1细胞移植28d组移植CL-1细胞后观察28d,单纯模型组未进行细胞移植。③各组在正常状态下以及移植前后进行血清标本的采集与肝功能检测。应用免疫组化、聚合酶链反应法、实时荧光定量反转录聚合酶链反应技术检测各组大鼠肝脏内人白蛋白的mRNA的表达。主要观察指标:①人骨髓干细胞移植对大鼠肝功能转氨酶及总胆红素含量的影响。②各组大鼠病理切片肝细胞形态检查结果。③各组大鼠肝脏中人主要组织相容性抗原-Ⅰ类阳性率。④各组大鼠肝脏中大鼠Sox11序列、人Alu-sx序列、人白蛋白mRNA基因的表达。结果:实验选用40只大鼠,全部进入结果分析。①人骨髓干细胞移植对大鼠肝功能转氨酶及总胆红素含量的影响:各细胞移植组在正常状态以及细胞移植前与单纯模型组基本相似(P>0.05);与正常状态比较,细胞移植前各组均显著增高(P<0.01);与细胞移植前比较,各细胞移植组在细胞移植后均显著降低(P<0.01),但仍高于正常状态下的转氨酶、总胆红素含量(P<0.01)。②各组大鼠病理切片肝细胞形态检查结果:正常情况下肝脏病理切片显示肝细胞排列成条索状,围绕中央静脉成放射状排列,汇管区无炎性细胞浸润;单纯模型组呈大片状坏死表现;人骨髓干细胞移植组可见一些坏死后增生性改变;CL-1细胞移植组可见卵圆细胞以及小胆管增生。③各组大鼠肝脏中人主要组织相容性抗原-Ⅰ类阳性率:单纯模型组为0,造模+人骨髓干细胞移植14d组为(13.03±0.18)%,造模+人骨髓干细胞移植28d组为(9.47±0.46)%,造模+CL-1细胞移植14d组为(10.27±0.50)%,造模+CL-1细胞移植28d组为(9.84±0.23)%。④各组大鼠肝脏中基因序列聚合酶链反应检测结果:单纯模型组大鼠肝脏中只检测到大鼠Sox11序列;而人骨髓干细胞移植、CL-1细胞移植各时间组均可检测到人Alu-sx序列和大鼠Sox11序列。⑤各组大鼠肝脏组织实时荧光定量反转录聚合酶链反应检测结果:单纯模型组大鼠肝脏组织中未检测到人白蛋白mRNA基因表达,人骨髓干细胞移植、CL-1细胞移植各时间组以及阳性对照均可检测到人白蛋白mRNA基因表达。结论:人骨髓干细胞移植能促进肝损伤大鼠的肝功能恢复,人源性细胞在肝损伤大鼠肝脏中的替代率约10%,提示人骨髓干细胞在异体内均可转化为肝细胞并可实现部分替代。

BACKGROUND： How to obtain human-derived hepatocyte of high quality is the key problem for both bioartificial liver and hepatocyte transplantation. Bone marrow stem cells （BMSCs） can differentiate hepatocyte under proper condition, which provides a new think for obtaining hepatocyte. OBJECTIVE： To investigate the methods of the trans-differentiation of human BMSCs into hepatocyte in rats so as to provide a new think for clinical transplantation of liver and source of bioartificial liver. DESIGN： Randomized controlled study. SETTING： General Surgery of Zhujiang Hospital of Southern Medical University. MATERIALS： The experiment was conducted at Central Laboratory of Zhujiang Hospital of Southern Medical University from May 2004 to February 2005. Totally 40 male SD rats of clean grade were divided randomly into five groups： model group, modeling ＋ 14-day transplantation group of human BMSCs, modeling ＋ 28-day transplantation group of human BMSCs, modeling ＋ 14-day transplantation group of CL-1 cell （human hepatocyte family）, and modeling ＋ 28-day transplantation group of CL-1 cell （human hepatocyte family） with 8 in each group. METHODS： ① Injured models of human hepatocytes induced by 2- acetaminofluorene ＋ carbon tetrachloride ＋ cyclophosphamide were established in all 5 groups. ②Human BMSCs were transplanted into liver and differentiated into bepatocyte with remedial liver regeneration. Human BMSCs were observed for 14 days in modeling ＋ 14-day transplantation group of human BMSCs, for 28 days in modeling ＋ 28-day transplantation group of human BMSCs, for 14 days in modeling ＋ 14-day transplantation group of CL-1 cell and for 28 days in modeling ＋ 28- day transplantation group of CL-1 cell. However, cells in model group were not transplanted. ③ The serum samples were selected and hepatic function of rats was measured at normal state, before and after transplantation. The expressions of human albumin mRNA in liver were detected by immunohistocbemistry staining, polymerase chain reaction （PCR） and real time reverse transcription polymerase chain reaction （RT-PCR） respectively. MAIN OUTCOME MEASURES：①Effect of transplantation of human BMSCson hepatic function and content of total bilirubin;② Morphological results of pathological section of hepatic cells; ③ Positive rate of main his- tocompatibility antigen in liver;, ④ Ranks of Soxl 1, Alu-sx and expression of human albumin mRNA in liver. RESULTS： Totally 40 rats entered the final analysis.① Effect of transplantation of human BMSCs on hepatic function and content of total bilirubin： Hepatic function and content of total bilirubin in each transplantation group were similar to those in model group at normal state and before transplantation （P 〉 0.05）; values in each group were obviously increased before transplantation as compared with those at normal state （P 〈 0.01） and were obviously decreased after transplantation as compared with those before transplantation （P 〈 0.01） but were higher than those at normal state （P 〈 0.01）. ② Morphological results of pathological section of hepatic cells： At normal state, pathological section of hepatic cells showed that hepatic cells lined in strip-chorda shape and radian shape around central vein; and inflammatory cells were not infiltrated in crossed-channel area. Necrosis was observed in model group. Proliferated changes were observed in transplantation groups of human BMSCs after a few of necrosis, and ovale-reund cells and small bile duct proliferation were observed in transplantation groups of CL-1 cells. ③Positive rate of main histocompatibility antigen-1 in liver： Positive rate was 0 in model group; （13.03±0.18）% in modeling ＋ 14-day transplantation group of human BMSCs; （9.47±0.46）% modeling ＋ 28-day transplantation group of human BMSCs; （10.27±0.50）% in modeling ＋ 14-day transplantation group of CL-1 cell; and （9.84±0.23）% in modeling ＋ 28-day transplantation group of CL-1 cell. ④ Results of gene ranks with PCR： Soxll was detected in model group, but Soxll and Alu-sx were detected in both transplantation groups of human BMSCs and CL-1 cells at various time points respectively. ⑤ Results of real time fluorescent quantitative RT-PCR： Expression of human albumin mRNA was not observed in model group, but expression of that was observed in transplantation groups of human BMSCs and CL-1 cell as well as positive controls at various time points respectively. CONCLUSION： Human BMSCs can promote recovery of hepatic function. Replaceable rate of human-derived cells is 10% in liver of rats, which suggests that human BMSCs can converse into hepatocyte in xenoma and replace partly.