CAJ | 학술논문

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.
목적 탐토이인골수간세포재중공섬유려기중직접확증병유도분화위류간세포,종이직접구건성생물인공간반응기적가행성. 방법 취지원자골수혈,인골수간충질간세포분리、확증지107수량급후충식입중공섬유려기,계속배양확증10 d후,이함중조인간세포생장인자(rhHGF)、중조인감성성섬유세포생장인자4(rhFGF-4)、중조인제류소M(rhOSM)등세포인자적배양액유도분화,검측배양상청액중백단백、갑태단백(AFP)수평;유도18 d후검측안대사、안정대사급뇨소합성등공능.배양결속후이류식세포의검측소화려기내세포백단백표체솔.배양전과정감측려기내배양액포도당농도변화. 결과 ①확증배양기인골수간충질간세포재중공섬유려기내충식후적포도당섭취량정진행성승고,재유도분화기보지상대평은,유도배양결속시세포총수체109수량급.②진행유도배양후6 d,배양상청액중출현AFP,12 d체도봉치;9 d출현백단백병지속진행성승고;유도배양18 d시,려기내세포총체안청제속솔위2.0～2.7 mmol/24 h,뇨소산생속솔위1.8～2.2 mmol/24 h,안정청제속솔위3.2～3.8 mg/24 h.③21 d려기내세포백단백표체솔위66.18%～76.91%. 결론 인골수간세포가재중공섬유려기내확증,병분화위간세포양세포,성위구유일정공능적생물인공간반응기.
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.