Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells.
Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells.
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DOI：
10.3892/or.2015.4241
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发表时间：
2015-11
期刊：
影响因子：
4.2
通讯作者：
Ping Liu;Liang Sun;Hui-Cheng Chen;Shui Sun;Dongsheng Zhou;Bo Pang;Jian Wang
中科院分区：
文献类型：
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作者：
Ping Liu;Liang Sun;Hui-Cheng Chen;Shui Sun;Dongsheng Zhou;Bo Pang;Jian Wang
本研究的目的是为多基因诱导干细胞分化提供理论和实验基础。将携带TGF-β1和IL-10基因的慢病毒载体转染至骨髓来源的间充质干细胞（BMSCs），使其分化为软骨形成。本研究使用2-3个月大的健康新西兰白兔。从每只兔子的髂骨和胫骨干中分离6-8ml骨髓。用percoll 分离介质离心骨髓后吸出BMSC 悬浮液。将BMSCs在体外进行初代培养和传代培养，根据慢病毒载体的不同分为四组：A组，接受转化生长因子β1（TGF-β1）；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BMSCs后12 h，检测荧光表达。 96小时后，MOI=100，转染效率约为70%。 RT-PCR和Western blot分析第7天和第14天A-E组SOX-9聚集蛋白聚糖和Ⅱ型胶原的表达。 A、C组表达的3个基因的表达量均高于B、D、E组的表达量。B组表达的3个基因的表达量高于D组的表达量。A、C组表达的3个基因的表达量无统计学差异。因此，细胞因子在细胞增殖和软骨分化中发挥重要作用。 TGF-β1在分化中具有协同作用。此外，IL-10可能对软骨组织的修复具有保护作用。
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.