基于骨髓干细胞与肝细胞共培养体系的左归丸血清药理学研究

Studies of Zuogui Wan medicated serum pharmacology based on the co-culture system of bone marrow stem cells and hepatocytes

背景：课题组前期研究表明,左归丸含药血清可促进骨髓干细胞转化肝细胞,虽能排除骨髓干细胞与肝细胞的融合,但无法模拟体内骨髓干细胞与肝细胞的相互作用。目的：观察左归丸含药血清对骨髓干细胞转化肝细胞的影响。方法：利用透过性支持物（聚酯Transwell透明嵌套）建立骨髓干细胞与肝组织细胞共培养体系,实验分为空白血清组和左归丸含药血清组,前者在培养体系中加入10%正常大鼠血清,后者在培养体系中加入10%左归丸含药大鼠血清,骨髓干细胞与肝组织细胞共培养7,14,21,28,35d时分别取透过性支持物中的骨髓干细胞爬片用PAS法检测糖元,免疫细胞化学法检测肝细胞标志物甲胎蛋白、CK18、ALB的表达。结果与结论：10%含药血清组与空白血清组相比,在相同时间点糖原阳性细胞率增高,差异有显著性意义（P〈0.01）;共培养7d时,甲胎蛋白阳性细胞率显著增高（P〈0.01）;共培养14,21,28,35d时,甲胎蛋白阳性细胞率显著下降（P〈0.05~0.01）;相同时间点CK18、ALB阳性细胞率显著增高（P〈0.05）。提示在骨髓干细胞与肝细胞共培养体系中使用左归丸含药血清是促进骨髓干细胞转化为肝细胞、并维持其肝细胞功能的较好培养条件。

BACKGROUND：Previous studies have shown that Zuogui Wan medicated serum can promote transformation of bone marrow stem cells into hepatocytes.The confluence of bone marrow stem cells and hepatocytes can be eliminated,but interaction of bone marrow stem cells and hepatocytes in vivo cannot be simulated.OBJECTIVE：To observe the effect of Zuogui Wan medicated serum on the transformation of bone marrow cells into hepatocytes.METHODS：We established the co-culture system of bone marrow stem cells and hepatic tissue cells by using permeable supports（Transwell PTFE membrane insert） and established 10% Zuogui Wan medicated serum group and blank serum group.Medicated serum group added 10% Zuogui Wan medicated serum in the medium,whereas blank serum group added 10% normal rat serum in the medium.Bone marrow stem cells and hepatocytes were cocultured for 7,14,21,28 and 35 days.Bone marrow cell slides in the permeable supports were gathered and glycogen was detected by PAS staining method.Hepatocyte markers（α-fetoprotein,CK18,albumin） were measured by immunocytochemistry method.RESULTS AND CONCLUSION：Compared with blank serum group,the glycogen positive cell rate was obviously increased at the same time point（P 0.01）,and the α fetoprotein positive cell rate was significantly increased at 7 days after co-culture（P 0.01）.At 14,21,28 and 35 days after co-culture,the α fetoprotein positive cell rate had a remarkable decrease（P 0.05-0.01）,and the positive cell rate of CK18 and albumin was apparently raised（P 0.05）.Using Zuogui Wan medicated serum in the co-culture system of bone marrow stem cells and hepatocytes was a better culture condition to promote bone marrow stem cell forming hepatocyte and maintain hepatocyte function.