骨髓间充质干细胞诱导肝细胞耐受肝功能衰竭血清细胞毒性的研究  

Experimental study on bone marrow mesenchymal stem cells-induced tolerance of hepatocytes to the cytotoxicity of sera from the patients with liver failure in vitro

作　　者：顾劲扬[1] 施晓雷[1] 袁献温[1] 张悦[1] 肖江强[1] 丁义涛[1] 

机构地区：[1]南京大学医学院附属鼓楼医院肝胆外科,210008

出　　处：《中华实验外科杂志》2014年第2期308-311,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81170417）;国家自然科学基金青年科学基金资助项目（81300337）;江苏省自然科学基金资助项目（BK2012081）;江苏省卫生厅科技项目（Q201307）;中国博士后基金资助项目（2011M500904）;江苏省博士后科研资助计划项目（1102149C）;南京市卫生青年人才培养工程（qrx11168）;美捷登青年科学家研究基金资助项目（MJR20130001）

摘　　要：目的 探讨与骨髓间充质干细胞（BMSCs）共培养的肝细胞可耐受慢加急性肝功能衰竭患者血清的细胞毒性,以及共培养细胞对肝功能衰竭血清的肝功能支持作用.方法 收集慢性乙型肝炎慢加急性肝功能衰竭患者血清（n=18）及正常志愿者血清（n=18）.建立猪肝细胞与BMSCs最适共培养体系.实验分为4组：肝功能衰竭血清单纯肝细胞培养组（Hep-F）、肝功能衰竭血清共培养组（Co-F）、正常人血清单纯肝细胞培养组（Hep-N）和正常人血清共培养组（Co-N）.血清浓度依次为10％、20％、40％、60％、80％和100％.培养24 h后收集上清液检测白蛋白水平和肝细胞周期确定最合适的肝功能衰竭血清浓度.观察细胞生长状态、超微结构及细胞活力.分别检测60％浓度血清培养组超氧化物歧化酶（SOD）、乳酸脱氢酶（LDH）、谷氨酰胺（GLN）和胆碱酯酶（CHE）水平.更换为含10％胎牛血清的完全培养基继续培养24 h,收集Hep-FB、Co-FB、Hep-NB和Co-NB组上清液,重复检测SOD、LDH、GLN和CHE水平.结果 共培养组肝细胞在60％浓度的肝功能衰竭血清中贴壁数量最多,呈岛状聚集生长.肝细胞表达白蛋白水平最佳,G2～S期细胞比例升至32.08％,细胞内超微结构接近正常,细胞活力较对照组明显改善.Co-F组SOD、GLN和LDH水平与Co-N组之间差异无统计学意义（P＞0.05）,仅CHE水平有所下降,但与Hep-F组比较显著提高.Co-FB组LDH、GLN、CHE活性较Hep-F组有显著改善.结论 与BMSCs共培养的肝细胞可耐受中高浓度慢加急性肝功能衰竭血清的细胞毒性,有效维持肝细胞功能.Objective To clarify the tolerance of hepatocytes co-cultured with bone marrow mesenchymal stem cells （BMSCs） to the cytotoxicity of sera from the patients with liver failure induced,and the supporting effects on liver failure sera by co-culture.Methods Serum samples from 18 patients with acute-on-chronic liver failure due to hepatitis B were collected with informed consent.Normal serum was obtained from 18 normal volunteers.The optimal co-cultivation of BMSCs and primary hepatocytes was generated.Four groups were creased as follows：hepatocyte alone with liver failure serum （Hep-F）,co-culture with liver failure serum （Co-F）,hepatocyte alone with normal serum （Hrp-N）,and co-culture with normal serum （Co-N）.The additional serum concentration was set as 10%,20%,40%,60%,80% and 100%,respectively.The albumin expression and cell cycle of hepatocytes after 24 h culture were examined to determine the optimal liver failure serum concentration.The morphology and viability were also monitored.The indices of hepatic functions in every group were tested.Subsequently,the cells were inoculated in complete medium containing 10% fetal bovine serum for another 24 h,and hepatocyte specific functions of Hep-FB,Co-FB,Hep-NB,and Co-NB were examined,respectively.Results A well attachment and self-organization of islet-like hepatocyte aggregates were encouraged in Co-F group with 60% liver failure serum.It also achieved the best albumin secretion and largest population of hepatocytes in G2-S phase.The cell ultrastructure remained similar to that of hepatocytes in vivo.Meanwhile,hepatocyte viability assay showed a metabolically active,viable cell population in Co-F group instead of quite a few dead cells in Hep-F group.The activities of superoxide dismutase （SOD）,lactate dehydrogenase （LDH）,glutamine （GLN） and CHE in Co-F group were all significantly increased as compared with those in Hep-F group （P 〈 0.05）.The similar trends were observed between Co-FB group and Hep-FB group except SOD.Conclusion

关 键 词：肝细胞 生物人工肝 骨髓间充质干细胞 共培养 

分 类 号：R575.3[医药卫生—消化系统]