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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 observe the feasibility of differentiating into fibroblastic cartilage-like cells in vitro by synovial-derived MSCs (SMSCs) transfected with adenovirus-mediated BMP-2/7 co-expression vector. Methods Six New Zealand white rabbits (3 months old, weighing 2.1 ± 0.3 kg) were randomly divided into 3 groups. The SMSCs were isolated and purified from synovial tissue. The morphological observation and immunofluorescence staining, flow cytometry and cell cycle identification , And tested its adipogenic, osteogenic, multi-directional differentiation into cartilage potential. At the same time, the pAdTrack-BMP-2 internal ribosome entry site (IRES) -BMP-7 adenoviral vector was packaged and the proliferation kinetics, karyotype analysis and flow cytometry were used to detect the cellular DNA Content, tumorigenicity analysis and other safety identification. In vitro cultured in incomplete cartilage medium to observe the differentiation status, parallel RT-PCR detection, immunofluorescence staining and toluidine blue staining. Results SMSCs isolated from synovial tissue of rabbits showed the characteristics of MSCs, such as cell morphology, surface markers and multidirectional differentiation potential. The transfection rate of pAdTrack-BMP-2-IRES-BMP-7 transfected SMSCs was about 70%. The safety identification results showed that the doubled time, chromosome number and DNA content of the transfected SMSCs were normal, and no tumorigenicity. After induced by in vitro incomplete cartilage medium for 21 days, the expression of type I and type II collagen in SMSCs was significantly increased by RT-PCR, which belonged to type II collagen. The expression of RhoA and Sox-9 in chondrocytes was induced Also significantly increased. Immunofluorescence staining and toluidine blue staining also showed that the transfected SMSCs differentiated into fibrochondrocytes. Conclusion The adenovirus vector is safe and effective, and can induce rabbit SMSCs to differentiate into fibrochondrocytes in vitro.