摘要
背景:骨髓间充质干细胞具有取材方便、多向分化及低免疫原性等优点,是转基因细胞移植镇痛领域中的理想载体细胞,但由于骨髓中间充质干细胞含量有限、体外培养的复制性衰老等问题,制约其在镇痛研究中的应用。目的:构建永生化大鼠骨髓间充质干细胞株,为转基因细胞移植镇痛提供载体细胞来源。方法:构建携带人端粒酶反转录酶基因的慢病毒pL V-Puro-EF1α-h TERT并转染第3代骨髓间充质干细胞后进行嘌呤霉素筛选,获得阳性细胞克隆并扩大培养,分别采用RT-PCR和TRAP法检测转染细胞人端粒酶反转录酶的表达和端粒酶活性,同时观察细胞形态、增殖和诱导分化能力、表面分子以及Nestin、MHC-Ⅰ和MHC-Ⅱ的表达,检测细胞染色体核型和致瘤性。结果与结论:人端粒酶反转录酶基因修饰的骨髓间充质干细胞可以体外连续培养超过30代。与未转染的细胞和对照病毒转染的细胞相比,慢病毒转染的骨髓间充质干细胞人端粒酶反转录酶mR NA表达、端粒酶活性以及细胞增殖能力均提高,细胞周期主要处于G2/M以及S期,增殖指数明显升高;细胞表面分子CD29、CD44、CD90表达阳性率大于70%,而CD34、CD45表达阳性率不足5%,胞浆Nestin的表达阳性,而MHC-Ⅰ和MHC-Ⅱ未见表达,保留了干细胞成骨、成脂、成神经的分化能力,细胞形态、染色体核型均正常,裸鼠接种无致瘤性。以上结果提示成功构建了人端粒酶反转录酶基因修饰的永生化大鼠骨髓间充质干细胞株,为转基因细胞移植用于镇痛研究提供了生物学性状均一、稳定的安全载体细胞。
BACKGROUND： Because of convenient source, multi-lineage differentiation and low immunogenicity, bone marrow mesenchymal stem cells are the ideal cell type to serve as vectors of transgenic cells in pain managemen However, the replicative senescence and small amount of cells obtained from the bone marrow restrict the application of bone marrow mesenchymal stem cells in pain research.OBJECTIVE： To construct human telomerase reverse transcriptase（hT ERT）-immortalized rat bone marrow mesenchymal stem cells as transgenic cellular vectors for pain therapy.METHODS： Bone marrow mesenchymal stem cells were obtained from whole rat bone marrow, and then transfected with a lentivirus containing the hT ERT（pL V-Puro-EF1α-hT ERT） followed by puromycin selection.hT ERT expression and telomerase activity in these transfected cells were determined by RT-PCR and TRAP.Morphological changes, capacity of cell growth and multi-lineage differentiation, chromosome karyotype andtumorigenicity were observed in vitro. Moreover, the expression of cell surface molecule, Nestin, MHC-I and MHC-II in transfected cells were also detected by flow cytometry and immunocytochemistry.RESULTS AND CONCLUSION： The bone marrow mesenchymal stem cells genetically modified by hT ERT could be cultured and passaged through 30 generations in vitro. Compared to the primary and negative transfected cells, the h TERT-modified bone marrow mesenchymal stem cells showed higher expression of h TERT m RNA, telomerase activity and cell proliferation. Most of transfected cells stayed at G2/M and S stages. The proliferation index of the transfected cells were increased dramatically. The positive rates of CD29, CD44 and CD90 were over 70%, but the positive rates of CD34 and CD45 were less than 5%. Transfected cells were positive for Nestin in the cytoplasm, but negative for MHC-1 and MHC-11. In addition, this cell line continued to exhibit the characteristics of fibroblastic bone marrow mesenchymal stem cells, including phenotype, differentiation into ost
出处
《中国组织工程研究》
CAS
北大核心
2015年第23期3621-3627,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金面上项目(81171468)
教育部高等学校博士学科点专项科研基金(200805581112)~~
