Fu Weili, Chen Gang, Tang Xin, Li Qi, Ll Jian
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2015 Apr;29(4):472-6.
To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells.
Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR.
GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000).
Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.
研究重组腺病毒-骨形态发生蛋白12（Ad-BMP-12）转染对兔外周血间充质干细胞（MSCs）向肌腱/韧带细胞分化的影响。
取3-4月龄新西兰兔，分离外周血MSCs并体外培养至第3代。采用AdEasy系统制备重组腺病毒载体系统，转染第3代MSCs（转染组）；未转染的MSCs作为对照（未转染组）。倒置相差显微镜下观察转染细胞的形态特征及生长情况。采用流式细胞术（FCM）及荧光显微镜检测转染效率及绿色荧光蛋白（GFP）表达。体外培养14 d后，采用免疫组织化学及实时荧光定量PCR检测肌腱/韧带特异性标志物的表达。
转染后8 h可见外周血MSCs表达GFP。转染后24 h，倒置相差显微镜下细胞形态清晰，生长缓慢，荧光显微镜下同一视野几乎所有细胞均表达GFP。FCM分析显示，转染组转染效率为99.57%，未转染组为2.46%。免疫组织化学显示，Ⅰ型胶原表达随体外培养时间逐渐增加。实时荧光定量PCR结果显示，转染组肌腱/韧带特异性基因（肌腱调节蛋白、腱生蛋白-C、核心蛋白聚糖）的mRNA表达明显高于未转染组（0.061±0.013比0.004±0.002，t=-7.700，P=0.031；0.029±0.008比0.003±0.001，t=-5.741，P=0.020；0.679±0.067比0.142±0.024，t=-12.998，P=0.000）。
Ad-BMP-12可显著促进外周血MSCs向肌腱/韧带成纤维细胞分化，增强肌腱/韧带特异性表型分化的表达，为外周血MSCs应用于肌腱/韧带再生提供依据。
