摘要
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。
The purpose of this study was to investigate the expression of human Factor Ⅸ （hF Ⅸ） in retrovirus-transfected human umbilical cord tissue derived mesenchymal stem cells （hUCT-MSCs）. The pLEGFP-N1-hFⅨ vector was generated by cloning a 3.0 kb Bgl Ⅱ - BamH Ⅰ fragment from the pIRES2-EGFP-hFⅨ plasmid containing the hFⅨ cDNA and part of intron 1 of hFIX in pLEGFP-N1 vector. The retroviral supematants were produced from the Phoenix packaging cell line and then infected the hUCT-MSCs. After selection with G418 for 10 day, the expression of FⅨ was detected by ELISA and Western blot. The biological activity of FⅨ was determined by the clotting assay employing human Factor Ⅸ-deficient plasma. The results showed that compared with the activity of pooled human normal plasma （100%）, transduced cells produced biologically active hFIX with 100 - 130% activity in two-day culture supernatant and expressed hFⅨ at levels of 2.68±0.36 μg/106 cells/24 hours after G418 selection for 10 days. The secretion of hFⅨ into culture supernatant was also confirmed by Western blot analysis. It is concluded that genetically modified hUCT-MSCs can express biologically active hFⅨ and thus serve as an efficient drug delivery vehicle carrying hFⅨ used as a way of somatic gene therapy for hemophilia B.
出处
《中国实验血液学杂志》
CAS
CSCD
2009年第1期184-187,共4页
Journal of Experimental Hematology
基金
中国科学与技术部863资助项目(2006AA02A110)
国家自然科学基金(30570357和30600238)
