逆转录病毒载体介导Fn-TPO基因修饰人骨髓间充质干细胞  

Retroviral vector mediated fibronectin-thrombopoietin gene modification to human bone marrow mesenchymal stem cells

作　　者：于洁[1] 张磊[1] 莫姝[1] 杨光[1] 李欣[1] 宪莹[1] 

机构地区：[1]重庆医科大学儿童医院血液肿瘤科,重庆400014

出　　处：《第三军医大学学报》2006年第24期2396-2399,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30100095)~~

摘　　要：目的应用逆转录病毒载体介导纤维连接蛋白-血小板生成素(Fn-TPO)基因修饰人骨髓间充质干细胞(mesenchymalstemcells,MSCs),观察Fn-TPO基因修饰对于骨髓MSCs的影响。方法构建携带Fn-TPO基因的重组逆转录病毒载体,并以其对骨髓MSCs进行体外基因修饰;台盼蓝拒染法计数活细胞数检测基因修饰后骨髓MSCs的体外增殖能力,RT-PCR检测Fn-TPO基因在骨髓MSCs中的表达情况,MTT法检测基因修饰后骨髓MSCs黏附造血细胞的能力,ELISA检测基因修饰后骨髓MSCs分泌至细胞培养液中的TPO浓度。结果成功构建携Fn-TPO基因的重组逆转录病毒载体且以该逆转录病毒载体对骨髓MSCs进行体外基因修饰;Fn-TPO基因在骨髓MSCs内能够正常转录;基因修饰后的骨髓MSCs体外增殖能力无明显改变(P>0.05);黏附造血细胞能力增强(P=0.01),分泌TPO的能力增强且不随培养时间延长而显著减弱(P<0.01),但受细胞生长状态影响。结论成功应用携带Fn-TPO基因的逆转录病毒载体对人骨髓MSCs进行基因修饰,使基因修饰后骨髓MSCs黏附造血细胞分泌TPO能力增强。Objective To modify human bone morrow mesenchymal stem cells （MSCs） with fibronectinthrombopoietin （Fn-TPO） gene carried retroviral vector and investigate the influence of Fn-TPO gene modification on human bone marrow MSCs. Methods The retroviral vector containing Fn-TPO gene was constructed and transfected into bone marrow MSCs. The proliferation capacity of bone marrow MSCs after gene modification was assayed by trypan blue exclusion assay. The transcription of Fn-TPO gene was assayed by RT-PCR. The capacity of bone marrow MSCs to adhere hematopoietic cells was assayed by MTT and the concentration of TPO in the bone marrow MSCs culture supernatant was assayed by ELISA. Results Retroviral vector containing Fn-TPO gene was successfully constructed and modified bone morrow MSCs. Fn-TPO gene was expressed by bone morrow MSCs after gene modification. The viability of MSCs had no significant difference between pregene-modified and post-gene-modified （ P 〉 0. 05 ）. The adhering ability of gene modified bone marrow MSCs to hematopoietic cells was reinforced （P =0. 01 ）. The concentration of TPO in the bone morrow MSCs culture supernatant rose from （5.58 ±0.37） ng/ml to （7.46 ±0.59） ng/ml （P 〈0. 01 ） and would not significantly decline in short time period, but was influenced by MSCs growth status. Conclusion Human bone morrow MSCs was successfully modified by Fn-TPO gene carried retroviral vector and the status of gene modified MSCs was assayed. This would facilitate further study of fibronectin and thrombopoietin genes on hematopoietic regulation and hematological system diseases.

关 键 词：骨髓间充质干细胞 纤维连接蛋白 血小板生成素 逆转录病毒载体 基因修饰 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R373[医药卫生—基础医学]