胚胎干细胞源性肝样细胞移植治疗急性肝功能衰竭模型小鼠(英文)

Embryonic stem cell-derived hepatocyte-like cell transplantation for acute liver failure

背景:采用胚胎干细胞源性肝样细胞进行移植治疗时,诱导分化的肝样细胞致瘤性及其对肝脏生化代谢的影响有待观察。目的:评价胚胎干细胞源性肝样细胞移植对急性肝功能衰竭模型小鼠的治疗效果。设计:随机对照观察。单位:中山大学附属第一医院。材料:实验于2005-01/2006-02在中山大学附属第一医院中心实验室完成。选用转染绿色荧光蛋白基因的小鼠D3-ES细胞(129系小鼠分离建系),由中山大学眼科中心黄冰教授馈赠。选用40只清洁级6周龄D3-129系小鼠,雌雄不限,由中山大学实验动物中心提供,许可证号码为SCXK(粤)2004-0011,实验过程中对动物的处置符合动物伦理学标准。转化生长因子、碱性成纤维细胞生长因子、肝细胞生长因子为美国GibcoBRL公司产品。方法:①利用转化生长因子、成纤维细胞生长因子和肝细胞生长因子联合诱导小鼠D3-ES细胞分化为肝样细胞,将细胞悬液以2.0×106/只注入20只小鼠肝包膜下,其余20只小鼠作为对照组,仅给予生理盐水注射。②注射后24h,采用5μL/20g四氯化碳腹腔注射,诱发两组小鼠急性肝功能衰竭,观察小鼠生存质量及平均生存时间;急性肝功能衰竭24h,取小鼠后腔静脉血进行总胆红素、谷丙转氨酶、白蛋白、血糖、凝血酶原时间等肝功能指标检测;小鼠死亡后取肝脏标本,观察有无肿瘤生成,应用苏木精-伊红染色和免疫组化观察移植细胞生长和白蛋白表达情况。主要观察指标:①小鼠生存质量及平均生存时间。②肝功能指标检测结果。③移植细胞体内生长及肿瘤形成情况。结果:①小鼠生存质量及平均生存时间:诱发急性急性肝功能衰竭后,对照组先出现共济失调等中枢神经系统症状,对照组14只及移植组8只出现腹水。对照组平均存活时间短于移植组,差异有统计学意义(23,62h,P<0.05)。②小鼠肝功能指标检测结果:对照组及移植组总胆红素及谷丙转氨酶较造模前升高,白蛋白及血糖较造模前低,凝血酶原时间较造模前延长,差异有显著性意义(P<0.01),移植组总胆红素及谷丙转氨酶较对照组下降,血糖较对照组高,凝血酶原时间较对照组短,差异有显著性意义(P<0.05)。③移植细胞体内生长及肿瘤形成情况:移植组小鼠死亡后取肝脏标本做病理切片观察,发现肝脏组织结构无明显改变,无肿瘤形成,移植细胞与小鼠肝样细胞形成很好的排列连接并表达白蛋白。结论:胚胎干细胞源性肝样细胞移植能改善急性肝功能衰竭模型小鼠生活质量,延长小鼠生存时间,移植细胞对肝脏生化代谢起到了较好的支持作用。

BACKGROUND： Effects of embryonic stem cell-derived hepatocyte-like cell transplantation on oncogenicity of differentiated hepatocyte-like cells and biochemical metabolism of liver should be further studied. OBJECTIVE： To evaluate the therapeutic efficacy of embryonic stem cell-derived hepatocyte-like cell transplantation on the acute liver failure. DESIGN： Randomized controlled study. SETTING： the First Affiliated Hospital of Sun Yat-sen University. MATERIALS： This study was performed at the Central Laboratory, the First Affiliated Hospital of Sun Yat-sen University from January 2005 to February 2006. D3-ES cells extracted from the mice which underwent transfection of green fluorescent protein were graciously presented by professor Huang, Ophthalmology Center of Sun Yat-sen University. Forty 6-week-old D3-129 mice of clean grade and irrespective of gender were provided by Experimental Animal Center of Sun Yat-sen University [certification： SCXK （yue） 2004-0011]. The experimental animals were disposed according to ethical criteria. Transforming growth factor, basic fibroblast growth factor, and hepatocyte growth factor were provided by Gibco BRL Company, USA. METHODS： Transforming growth factor, basic fibroblast growth factor, and hepatocyte growth factor were combined to differentiate D3-ES cells into hepatic cells. Cell suspension was poured into liver capsule of 20 mice with 2.0×106 cells per mouse. Another 20 mice that determined as the controls were injected with saline. Twenty-four hours later, intraperitoneal injection of 5 μL/20 g carbon tetrachloride was used to induce acute liver failure and to observe quality of life and mean survival time. Twenty-four hours after acute liver failure, vena cava posterior blood was drawn to detect total bilirubin, glutamate-pyruvate transaminase, albumin, blood glucose, pro-time prothrombin time, and other hepatic functional parameters. By scarification, hepatic samples were obtained to evaluate oncogenesis condition, and then HE staining and immunohistochemistry were adopted to detect growth of transplanted cells and albumin expression. MAIN OUTCOME MEASURES： Quality of life, average survival time, hepatic functional parameters, growth of transplanted cells, and oncogenesis condition. RESULTS： Quality of life and average survival time： After the onset of acute liver failure, mice in the control group had incoordination and other symptoms of central nervous system. In addition, 14 mice in the control group and 8 in the transplantation group had abdominal dropsy. Average survival time in the control group was significantly shorter than that in the transplantation group （23, 62 hours, P 〈 0.05）. Hepatic functional parameters： Levels of total bilirubin and glutamate-pyruvate transaminase in the control and transplantation groups were higher than those before modeling; levels of albumin and blood glucose were lower than those before modeling; pro-time prothrombin time was significantly longer than that before modeling （P 〈 0.01）. Furthermore, levels of total bilirubin and glutamate-pyruvate transaminase in the transplantation group were lower than those in the control group; blood glucose in the transplantation group was higher than that in the control group, and pro-time prothrombin time in the transplantation group was significantly shorter than that in the control group （P 〈 0.05）. Growth of transplanted cells and oncogenesis condition： Pathological section demonstrated that structure of liver tissue was not changed remarkably, and tumor was not formed. Moreover, transplanted cells and hepatocyte-like cell were well arranged and combined to express albumin. CONCLUSION： Embryonic stem cell-derived hepatocyte-like cell transplantation can improve quality of life, prolong survival time of model mice with acute liver failure; additionally, transplanted cells may well support biochemical metabolism of liver tissue.