腺病毒介导BMP-2/7共表达转染兔滑膜MSCs体外向纤维软骨样细胞分化的研究  

IN VITRO DIFFERENTIATION OF SYNOVIAL-DERIVED MESENCHYMAL STEM CELLS INFECTED BY ADENOVIRUS VECTOR MEDIATED BY BONE MORPHOGENETIC PROTEIN 2/7 GENES INTO FIBROCARTILAGE CELLS IN RABBITS

作　　者：符培亮[1] 张雷 吴海山[1] 丛锐军[1] 陈松[1] 丁喆如[1] 胡凯猛[2] 

机构地区：[1]上海长征医院关节外科,上海200003 [2]第二军医大学组织胚胎学教研室

出　　处：《中国修复重建外科杂志》2013年第3期345-352,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金青年科学基金资助项目(81000798)~~

摘　　要：目的通过使用腺病毒介导BMP-2/7共表达载体转染兔滑膜MSCs(synovial-derived MSCs,SMSCs),观察其在体外向纤维软骨样细胞分化的可行性。方法取3月龄新西兰大白兔6只,雌雄不限,体重(2.1±0.3)kg;取滑膜组织分离纯化SMSCs后,进行形态学观察及免疫细胞荧光染色、流式细胞仪、细胞周期鉴定,并检测其成脂、成骨、成软骨多向分化潜能。同时包装pAdTrack-BMP-2-内部核糖体进入位点(internal ribosome entry site,IRES)-BMP-7腺病毒载体,转染SMSCs后进行增殖动力学、核型分析、流式细胞仪检测细胞DNA含量、致瘤性分析等安全性鉴定。体外在不完全成软骨培养基中培养,观察其分化状态,并行RT-PCR检测、免疫荧光染色及甲苯胺蓝染色检测。结果从兔滑膜组织中分离出的SMSCs,其细胞形态、表面标记、多向分化潜能等均符合MSCs的特点。pAdTrack-BMP-2-IRES-BMP-7转染SMSCs的转染率约为70%。安全性鉴定结果显示转染后的SMSCs倍增时间、染色体数目及DNA含量均正常,且无致瘤性。体外不完全成软骨培养基诱导培养21 d后,RT-PCR检测示SMSCs的Ⅰ、Ⅱ型胶原表达明显增加,以Ⅱ型胶原为主;软骨分化信号分子RhoA与Sox-9的表达经诱导后也明显增加。免疫荧光染色及甲苯胺蓝染色结果亦显示转染后的SMSCs向纤维软骨样细胞分化。结论使用腺病毒载体安全有效,可在体外定向诱导兔SMSCs向纤维软骨样细胞分化。Objective To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells （SMSCs） differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 （BMP-2/7） genes in vitro. Methods SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing （2.1 ＋ 0.3） kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site （IRES）-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT- PCR, immunofluorescent staining, and toluidine blue staining. Results SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrackBMP-2- IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showeddifferentiation of SMSCs into fibrocartilage cells. Conclusion It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

关 键 词：腺病毒载体 BMP-2 7 滑膜MSCs 纤维软骨样细胞 兔 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]