定量RT—PCR检测肝脏损伤与移植干细胞定居量的关系

Determination of hepatic injury and localization of transplanted hepatocytes by quantitative RT-PCR

目的探索经不同途径移植到同种异体大鼠的大鼠骨髓间质干细胞（MSCs），在受体大鼠肝脏的定居情况。方法以绿色荧光蛋白标记从大鼠骨髓中分离培养的MSCs，体外扩增后，分别从不同途径移植到同种异体正常和肝脏损伤大鼠体内，于移植后的第3、7天，通过荧光定量PCR检测移植的MSCs在大鼠体肝内的表达情况。结果不同途径移植的同种异体MSCs，均可在受体大鼠肝脏内定居，定居于肝脏受损大鼠的细胞量大于非肝脏受损大鼠，差异显著（P〈0.05）。在肝脏受损大鼠，移植途径与MSCs在肝脏的定居量无明显的相关性（P〉0.05）。非肝脏受损大鼠，移植途径与MSCs在肝脏的定居量明显相关（P〈0.05），并与移植后时间有关。结论标记的MSCs可以定居于受体大鼠肝脏，其定居时间与细胞数量，与肝脏是否受损伤关系密切，与移植途径关系不密切；标记的MSCs可定居于肝脏未受损伤的大鼠肝脏，定植的细胞量与移植途径和移植后时间有关。

Objective To study the capacity of allograft of rat mesenchymal stem cells （MSCs） settled in receptor＇s livers transplanted by different ways. Methods MSCs were obtained from rat bone marrow and cultured in vitro in conditional DMEM medium with 10% of fetal bovine serum. Then they were identified by their monoclonal antibodies CD29, CD44, CD34, CD45, CDg0, SH-2 and SH-3. The cells were transduced with a greenfluorescent protein （GFP） retroviral vector. Ex vivo expanded MSCs transduced with the GFP retroviral vector were subsequently infused into tail arteries or portal vein of rats. Some of the rats were treated with carbon tetrachloride to induce centrolobular liver necrosis and others were healthy. The livers of rats were recovered from each recipient and evaluated for the presence of the GFP transgene in purified genomic DNA by sensitive RT-PCR on the 3rd and 7th d following infusion of MSCs. Results All the rats receiving allografting of GFP-transduced MSCs were alive until the study was over. The GFP transgene in purified DNA was found in all livers of rats infused with GFP-transduced MSCs by sensitive RT-PCR. DNA copy of GFP was significantly higher in the rats treated with carbon tetrachloride than in those without the treatment （P〈0. 05）. In the rats treated with carbon tetrachloride, the transplanting way was not correlated with number of the MSCs in the liver （P〉0.05）. However, the transplanting way was correlated to the number of MSCs in the liver in the rats without the treatment （P〈0. 05）. Conclusions MSCs appear to distribute in a disparate fashion following infusion in tail arteries or portal vein in rats treated with carbon tetrachloride to induce centrolobular liver necrosis. However, the timing and number of MSCs of homing to the liver following infusion may to some degree be influenced by liver injury.