肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究  

Hepatocyte growth factor induces differentiation of adult rat mesenchymal stem cells into a hepatocyte lineage in vitro

作　　者：张刚庆[1] 方驰华[1] 池达智[2] 

机构地区：[1]南方医科大学珠江医院肝胆外科,广州510028 [2]南方医科大学珠江医院急诊外科,广州510028

出　　处：《中华外科杂志》2005年第11期716-720,共5页Chinese Journal of Surgery

基　　金：广东省自然科学基金资助项目(2001;010593;2002;020097)

摘　　要：目的探讨成年大鼠骨髓间充质干细胞(MSCs)在体外是否可定向诱导分化为肝细胞及其方法。方法采用梯度离心法,分离纯化SD大鼠的MSCs,流式细胞仪检测和碱性磷酸酶染色鉴定细胞类型。根据培养基中肝细胞生长因子(HGF)浓度不同,将MSCs分为4组进行诱导分化:A组0ng/ml,B组10ng/ml,C组20ng/ml,D组40ng/ml。倒置显微镜连续观察细胞分化过程的形态学变化。于培养的1,3,7,14,21,28d,分别以逆转录聚合酶链反应(RTPCR)和细胞免疫组化检测各组细胞甲胎蛋白(AFP)、细胞角蛋白18(CK18)和白蛋白的基因和蛋白表达。结果分离纯化的SD大鼠MSCs的表面标志为CD29+,CD44+,CD34-,CD45-和CD90+,细胞的碱性磷酸酶染色阴性,细胞纯度达98%以上。C组与D组的MSCs,于培养第7天出现AFP基因表达,第14天表达增强,第28天表达减弱;第14天始出现白蛋白、CK18基因表达,而后持续。B组和A组在培养过程中,未出现AFP、CK18和白蛋白的表达。C组与D组MSCs的AFP细胞免疫组化染色培养第7天即出现阳性,第14天白蛋白和CK18免疫组化染色阳性。A组和B组的MSCs的AFP、白蛋白和CK18的免疫组化染色均阴性。结论成年大鼠MSCs在较高浓度的HGF的诱导下,可分化为肝细胞。Objective To explore whether the mesenchymal stem cells (MSCs) of rats can be induced into hepatocytes and the condition of differentiation in vitro. Methods Mesenchymal stem cells were collected from the femora of SD rats by density gradient centrifugation and identified by flow cytometric analysis and alkaline phosphatase (AKP) staining. MSCs were divided into 4 groups to induce differentiation with the different concentration of hepatocyte growth factor (HGF) in culture medium. The concentration of each group was group A 0 ng/ml, group B 10 ng/ml, group C 20 ng/ml and group D 40 ng/ml, respectively. The morphological changes of MSCs were observed by phase-contrast microscope. On day 1,3,7,14,21 and 28, mRNA of albumin, AFP and CK18 of MSCs of each group were examined by reverse transcription polymerase chain reaction (RT-PCR), and the expressions of them were also detected with immunohistochemistry technique. Results Mesenchymal stem cells collected from the femora of SD rats expressed antigens of CD29, CD44 and CD90, but not CD34 and CD45. AKP staining was negative for all of MSCs. On day 7, AFP mRNA of MSCs in group C and D could be detected by RT-PCR, and increased on day 14, and then directed on day 21. Albumin and CK18 mRNA of MSCs in group C and D could also be detected from day 14 to day 28 by RT-PCR. On the contrary, mRNA of AFP, CK18 and albumin was not detected in group A and B of culture. Immunocytochemical analysis for CK18, albumin and AFP showed positive staining reaction for AFP on day 7, for CK18 and albumin on day 14 in group C and D, and negative staining reaction both in group A and B of culture. Conclusion MSCs of adult rats cultured in high concentration of HGF can differentiate into hepatocytes.

关 键 词：骨髓间充质干细胞 肝细胞生长因子 实验研究 细胞分化 逆转录聚合酶链反应 免疫组化染色 MSCs 流式细胞仪检测 碱性磷酸酶染色 CK18 定向诱导分化 免疫组化检测 CD29^+ CD44^+ CD34^- 成年大鼠 SD大鼠 分离纯化 基因表达 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R692[医药卫生—基础医学]