人骨髓干细胞在中空纤维滤器中诱导分化构建生物人工肝反应器的可行性研究

目的 探讨以人骨髓干细胞在中空纤维滤器中直接扩增并诱导分化为类肝细胞,从而直接构建成生物人工肝反应器的可行性. 方法 取志愿者骨髓血,人骨髓间充质干细胞分离、扩增至107数量级后种植入中空纤维滤器,继续培养扩增10 d后,以含重组人肝细胞生长因子(rhHGF)、重组人碱性成纤维细胞生长因子4(rhFGF-4)、重组人制瘤素M(rhOSM)等细胞因子的培养液诱导分化,检测培养上清液中白蛋白、甲胎蛋白(AFP)水平;诱导18 d后检测氨代谢、安定代谢及尿素合成等功能.培养结束后以流式细胞仪检测消化滤器内细胞白蛋白表达率.培养全过程监测滤器内培养液葡萄糖浓度变化. 结果 ①扩增培养期人骨髓间充质干细胞在中空纤维滤器内种植后的葡萄糖摄取量呈进行性升高,在诱导分化期保持相对平稳,诱导培养结束时细胞总数达109数量级.②进行诱导培养后6 d,培养上清液中出现AFP,12 d达到峰值;9 d出现白蛋白并持续进行性升高;诱导培养18 d时,滤器内细胞总体氨清除速率为2.0～2.7 mmol/24 h,尿素产生速率为1.8～2.2 mmol/24 h,安定清除速率为3.2～3.8 mg/24 h.③21 d滤器内细胞白蛋白表达率为66.18%～76.91%. 结论 人骨髓干细胞可在中空纤维滤器内扩增,并分化为肝细胞样细胞,成为具有一定功能的生物人工肝反应器.

Construction of a novel bioreactor of bioartificial liver system with human bone marrow mesenchymal stem cells

Objective To design a novel bioreactor of bioartificial liver system by using expanded and differentiated human bone marrow mesenchymal stem cells(hBMMSCs) as active cells. Methods hBMMSCs were isolated from bone marrow of volunteers and grown to 107 population,and then replanted into hollow fiber cartridge to expand continuously for 10 days.They were incubated in differentiation medium containing recombinant human hepatocyte growth factor(rhHGF),recombinant human fibroblast growth factor 4(rhFGF-4),recombinant human oncostatin M(rhOSM),and the cells were induced to differentiate into hepatocyte-like cells for 21 days.The functions of the differentiated cells,such as synthesis of albumin(Alb),alpha fetoprotein(AFP) were determined.Eighteen days later,the functions of metabolism of ammonia and benzodiazepines,and synthesis of urea were monitored.The cellular synthesis rate of Alb was measured with flow cytometer.The glucose levels in the medium were measured during entire culture process. Results ①Glucose-uptake in the cartridge was increased during the culture period,and at the end of culture,the number of cells in the cartridge increased to 109.②After induction,AFP was detected on day 6,reaching the peak level on day 12.Alb was detected on day 9.Eighteen days after being induced,the clearance rate of ammonia and benzodiazepines in the cartridge was 2.02.7 mmol/24 hours and 3.23.8 mg/24 hours,respectively,and urea production rate was 1.82.2 mmol/24 hours.③At the end of the culture,66.18%76.91% of the cells showed positive Alb expression. Conclusion hBMMSCs can be multiply to construct a novel bioreactor of bioartificial liver system in a hollow fiber cartridge.