TGF-β1诱导马鹿茸MSCs软骨分化及c-myc基因的表达

doi:10.13560/j.cnki.biotech.bull.1985.2016.03.018

摘要/Abstract

摘要： 鹿茸间充质干细胞(MSCs)是维持茸再生与骨化的重要组织, 旨在研究鹿茸MSCs的软骨分化及原癌基因c-myc对该过程的调控作用。利用成年塔里木马鹿生长60 d的鹿茸第2代间充质干细胞(MSCs, P2), 通过TGF-β1(10 ng/mL浓度)刺激, 诱导塔里木马鹿茸间充质干细胞向软骨分化, 采用免疫组化和阿利新蓝染色鉴定诱导结果, 并通过qPCR方法检测软骨分化过程中c-myc基因的表达变化。结果显示, MSCs在诱导后的第9天开始出现细胞形态变化, 由梭形向多角形转变, 原来菊花状的分布逐渐向铺路石状变化, 至14 d可观察到软骨陷窝, 21 d软骨细胞基质明显, 并开始出现细胞凋亡。非诱导组28 d细胞出现凋亡, 细胞内发现空泡。35 d两组细胞凋亡明显, 细胞折光性变差, 间隙变大。阿利新蓝染色鉴定, 诱导至第14天细胞基质中开始出现大量阳性染色。免疫组化实验检测, 诱导至21 d的细胞基质中出现棕色Col II阳性反应物, 随培养时间增加颜色加深, 并集中分布在细胞及其周围基质中。在软骨分化进程中, 第7、14、21和28天, 诱导组c-myc基因表达与非诱导组相比显著下调(P<0.05), 但诱导至35 d, 诱导组c-myc表达与非诱导组相比无显著差异(P>0.05)。在TGF-β1刺激下, 塔里木马鹿茸MSCs可以分化成软骨, 原癌基因c-myc下调表达诱导鹿茸MSCs进入凋亡状态并分化为软骨细胞。

关键词: 马鹿茸, 间充质干细胞, 软骨分化, 转化生长因子β1, c-myc基因

Abstract: Antler mesenchymal stem cells(MSCs)play a pivotal role on the antler regeneration and ossification. To investigate the chondrogenic differentiation of antler MSCs and the regulation role of the proto-oncogene c-myc in this process, the second passage cells(MSCs, P2)of 60 d antler from adult Tarim wapiti were induced to chondrogenesis by the stimulation of transforming growth factor TGF-β1(10 ng/mL)in vitro. The inducing effects were identified by Alcian blue staining and immunohistochemics, and the expressions of gene c-myc during this process were detected by qPCR. The results demonstrated that, on the day 9 after induction, some MSCs begun to change from spindle-shaped to rounded or polygon, and the chrysanthemums-shaped pattern of the original MSCs gradually changed to paving stone like；the cartilage capsules were observed on the day 14, and the cartilage extracellular matrix was obvious and cell apoptosis appeared on the day 21. While the cells in the control group were observed to have apoptosis on the day 28, and the cavitations were discovered in the cells. On the day 35, massive cell apoptosis of both groups were observed, and the cell refraction became weak and the gaps between cells became larger. The identification by Alcian blue staining revealed that heavy positive staining emerged in the cellular matrix from the day 14 after stimulating. The detection by immunohistochemistry demonstrated that positive brown Col II reactant was in the cellular matrix from the day 21 after stimulating, and the color became darker along with the culture time, mainly distributed in the cells and their surround matrix. In the cartilage differentiation process from the day 7 to 28, the expressions of gene c-myc in the cells of the induced group were significantly lower than that of control group(P<0.05), while there was no significant difference after the day 35(P>0.05). In conclusion, Tarim wapiti antler MSCs differentiated into cartilage under the stimulation of TGF-β1. The down-regulated expression of proto-oncogene c-myc induced the apoptosis of antler MSCs and then differentiation to chondrogenic cells.

Key words: wapiti antler, mesenchymal stem cells, differentiation, transforming growth factor β1, c-myc gene

韩春梅,王姗姗,高庆华,郑永富,马梦婷,张勤. TGF-β1诱导马鹿茸MSCs软骨分化及c-myc基因的表达[J]. 生物技术通报, 2016, 32(3): 109-114.

HAN Chun-mei, WANG Shan-shan, GAO Qing-hua, ZHENG Yong-fu, MA Meng-ting, ZHANG Qin. TGF-β1-induced Differentiation of Wapiti Antler Mesenchymal Stem Cells to Cartilage and Expression Profile of Gene c-myc[J]. Biotechnology Bulletin, 2016, 32(3): 109-114.

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