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

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

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。

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 Inoculating 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 MSCsfrom the hepatitis B patients using the above methods are almost the same as those from normal adults.