Liu Ping, Sun Liang, Chen Hui, Sun Shui, Zhou Dongsheng, Pang Bo, Wang Jian
Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Oncol Rep. 2015 Nov;34(5):2618-26. doi: 10.3892/or.2015.4241. Epub 2015 Sep 1.
The aim of the present study was to provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3 months of age were used in the present study. A 6-8 ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured in vitro, then divided into four groups according to the difference of lentivirus vectors: group A, receiving transforming growth factor β1 (TGF‑β1); group B, receiving TGF-β1 and Interleukin-10 (IL-10); group C, empty vector transfection; and group D, receiving no cell growth factor. Fluorescence expression was detected 12 h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96 h. Expression of SOX-9 aggrecan and Type Ⅱ collagen in groups A-E on day 7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groups A and C were higher compared to the expression in groups B, D and E. The expression level of the three genes expressed in group B was higher compared to the expression in group D. The expression level of three genes expressed in group A and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue.
本研究的目的是通过多个基因的诱导为干细胞分化提供理论和实验基础。携带转化生长因子β1（TGF-β1）和白细胞介素10（IL-10）基因的慢病毒载体被转染到向软骨生成分化的骨髓间充质干细胞（BMSCs）中。本研究使用了2至3月龄的健康新西兰白兔。从每只兔子的髂骨和胫骨干中分离出6至8毫升骨髓。通过percoll分离介质对骨髓进行离心后吸出BMSCs悬液。BMSCs在体外进行原代培养和传代培养，然后根据慢病毒载体的差异分为四组：A组，接受转化生长因子β1（TGF-β1）；B组，接受TGF-β1和白细胞介素10（IL-10）；C组，空载体转染；D组，不接受细胞生长因子。将携带TGF-β1和IL-10基因的慢病毒载体转染到BMSCs后12小时检测荧光表达。96小时后，当感染复数（MOI）=100时，转染效率约为70%。通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析检测A-E组在第7天和第14天SOX-9、聚集蛋白聚糖和Ⅱ型胶原蛋白的表达。与B、D和E组相比，A组和C组中三个基因的表达水平较高。与D组相比，B组中三个基因的表达水平较高。A组和C组中三个基因的表达水平无统计学差异。因此，细胞因子在细胞增殖和软骨分化中起重要作用。TGF-β1在分化中具有协同作用。此外，IL-10可能在软骨组织修复中具有保护作用。
