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为了研究逆转录病毒(pLEGFP-N1)转染的人凝血因子Ⅸ(hFⅨ)基因在人脐带间充质干细胞中的表达,应用DNA重组技术将hFⅨcDNA构建入pLEGFP-N1载体,转导入包装细胞系Pheonix细胞,应用病毒上清感染人脐带组织源间充质干细胞(hUCT-MSCs),经G418筛选10天后获得全部的转染阳性细胞,从蛋白质水平和其功能活性上检测hFⅨ的表达。结果显示:配养上清液中可检测到hFⅨ的表达,每24小时分泌量达2.68±0.36μg/106细胞。Western blot检测表明,转导hFⅨ的hUCT-MSCs能分泌预期分子大小的hFⅨ入上清。功能性凝集测定实验表明了转导FⅨ的hUCT-MSCs2天培养上清中hFⅨ的活性为100%-130%。结论:pLEGFP-N1-hFⅨ能有效地转导hUCT-MSCs,并在其子代细胞中表达具有凝血活性的hFⅨ,这为hUCT-MSCs成为血友病B基因治疗的细胞载体研究奠定了基础。
HFIX cDNA was constructed into pLEGFP-N1 vector and transfected into packaging cell lines in order to study the expression of human coagulation factor Ⅸ (hFIX) gene transfected with human retroviral vector (pLEGFP-N1) in human umbilical cord mesenchymal stem cells. Pheonix cells. Human umbilical cord tissue-derived mesenchymal stem cells (hUCT-MSCs) were infected with virus supernatants. All transfection positive cells were obtained after screening by G418 for 10 days. The expression of hFⅨ was detected from the protein level and its functional activity. The results showed that the expression of hFⅨ was detected in the supernatant of the culture, and the secretion of hFIX reached 2.68 ± 0.36 μg / 106 cells every 24 hours. Western blot analysis showed that hUCT-MSCs transduced with hFIX secreted the desired molecular size hFIX into the supernatant. Functional agglutination assay experiments showed that the activity of hFIX in hUCT-2 MSCs transfected with FIX was 100% -130%. CONCLUSION: pLEGFP-N1-hFⅨ transduces hUCT-MSCs efficiently and expresses clotting activity of hFIX in its offspring cells, which lays the foundation for hUCT-MSCs to be the carrier of hemophilia B gene therapy.