转人AT-Ⅲ猪骨髓间充质干细胞抑制异种移植凝血紊乱的体外研究

Genetically modified bone marrow mesenehymal stem cells can regulate the coagulation in an in vitro model of xenotransplantation clotting disorders

目的观察转染人抗凝血酶-3（hAT-Ⅲ）的猪骨髓间充质干细胞（MSC）在体外异种移植凝血模型中的抗凝血作用。方法提取a-1，3-半乳糖转移酶基因敲除小型猪的MSC，利用流式细胞术鉴定其表型。构建含hAT-Ⅲ的慢病毒载体，转染MSC，使其表达hAT-Ⅲ，分别应用逆转录聚合酶链反应、蛋白质印迹法和免疫荧光技术检测hAT_Ⅲ的表达情况。在体外人血浆和猪内皮细胞共培养体系中，分别加入转染空质粒MSC（空转染组）、转染hAT-ⅢMSC（转染组）、重组hAT-ⅢMSC（重组hAT-Ⅲ组）和经鼠抗hAT_Ⅲ阻断性抗体处理的转染hAT-Ⅲ MSC（抗hAT-Ⅲ组），并提取上清液检测凝血酶一抗凝血酶复合物（TAT）的含量，并进行复钙实验，检测凝集时间。结果提取的细胞具有MSC表型，转染后成功表达hAT-Ⅲ基因，实验组相对表达量为2．80±0．51。空转染组、转染组、重组hAT-Ⅲ组和抗hAT-Ⅲ组TAT含量为（4．33±1．20）ng／ml、（15．00±2．89）ng／ml、（22．67±1．45）ng／ml、（3．02±1．16）ng／ml；在凝血模型中转染组TAT含量高于空转染组和抗hAT-Ⅲ组（P〈0．05）。空转染组、转染组、重组hAT-Ⅲ组和抗hAT-Ⅲ组的凝集时间为（150．0±4．58）S、（172．0±5．51）s、（181．3±4．06）s和（132．7±6．12）S；转染组凝集时间少于空转染组和抗hAT-Ⅲ组（P〈0．05）。结论基因修饰的MSC能够表达hAT-Ⅲ，并可以有效抑制体外异种移植凝血模型中人血浆中的凝血酶。

Objective We aimed to identify the anticoagulation effect of genetically modified bone marrow mesenchymal stem ceils （MSCs） in a blood coagulation model of xenotransplantation in vitro. Method Isolated MSCs from α1,3-galactosyltransferase gene-knockout （GTKO） pig, and identified the phenotype of MSCs by flow cytometry. Transfected the human antithrombin UI （hAT-Ill） gene into MSCs using lentivirus. Reverse transcription polymerase chain reaction （RT- PIER）, Western blot and immunofluorescence were used to detect the hAT-Ill gene expression. Enzyme-linked immunosorbent assay （ELISA） was used to detect the thrombin-antithrombin complex of the supernatant in the coagulation model in vitro. Result RT-PCR showed positive expression of hAT-Ⅲ in the experimental groups （2. 80 ± 0. 51）, while the hAT-III was scarcely found in the control group（1.00 ± 0. 11）. There was statistically difference between two groups （P〈0. 01）. Western blot and IF showed positive expression of hAT-Ⅲ in the experimental group. Higher thrombin AT-Ⅲ complex （TAT） concentration were observed in the transfection group than other control groups （P〈 0. 05）,and the coltting time were shorter than other control groups （P〈0. 05）. Conclusion MSCs transfected with hAT-Ⅲ can effectively alleviate the coagulation disorders in a in vitro model.