乙型肝炎患者骨髓间充质干细胞培养方法的优化和生物学特性

目的比较乙型肝炎患者骨髓间充质干细胞（MSCs）多种体外培养方法的效果，确定乙型肝炎患者MSCs的最佳体外培养方案；并对其生物学特性进行初步研究。方法比较2种接种方案和5种不同成分培养液的乙型肝炎患者MSCs体外培养成功率。比较5种不同成分培养液和4种换液方案的乙型肝炎患者MSCs生长曲线。进行乙型肝炎患者MSCs的形态观察和表面分子鉴定，传代培养以初步了解其生长特性。结果密度梯度离心接种法的体外培养成功率（88％）高于全骨髓接种法（76％），但差异无统计学意义。5种培养液的体外培养成功率差异有统计学意义（P〈0．01），其中患者自体血清培养液的培养成功率最高（100％），3种FBS培养液的培养成功率次之，健康成人血清培养液的最低（56％）。患者自体血清培养液的MSCs生长曲线最高，健康成人血清培养液的生长曲线最低，3种FBS培养液的MSCs生长曲线集中位于上述两者之间。以2d或者3d一次全量换液的MSCs生长曲线较高。乙型肝炎患者MSCs与正常人MSCs形态相同，表面分子表达一致，生长特性近似。结论乙型肝炎患者MSCs的体外培养方案以密度梯度离心法分离后，自体血清培养液进行培养，2～3d全量换液为佳，可以较快获得纯度较高的MSCs。乙型肝炎患者MSCs的生物学特性和正常人的近似。

Optimization of cultivation methods of MSCs from hepatitis B patients and in investigation of their biological characteristics

Objective To optimize cultivation methods of bone marrow mesenchymal stem cells (MSCs)from hepatitis B patients and to investigate their biological characteristics.Methods Growth curves of hepatitis B patients'MSCs cultivated with five culture media and two inoculation methods were compared; the shapes,appearances,surface markers and bionomics of the cultivated MSCs were studied.Results In- oculating the cells obtained directly from bone marrow aspirations was not as successful as using the marrow cells after their density gradient centrifugations(76% vs 88%),but the differences in the results were not statistically significant(P>0.05).The successful cultivation rates using five culture media were different and the differences were statistically significant(P<0.01).The autoserum medium was most successful,fatal bovine serum(FBS)medium was next successful and the non-patient serum medium was the least successful. The growth curves of the cultivations using the different media were parallel to this.Changing the whole culture media every 2 or 3 days was better than changing half of the media.The shapes,appearances,surface markers and the growth characteristics of MSCs from the hepatitis B patients were almost the same as MSCs from the normal adult.Conclusion The best cultivation method of MSCs from hepatitis B patients is: separating marrow cells using density gradient centrifugal separation,cultivating them using an autoserum culture medium,and completely changing the medium every 2-3 days.The biological characteristics of MSCs from the hepatitis B patients using the above methods are almost the same as those from normal adults.