缝隙连接细胞间通讯在大鼠骨髓基质细胞成骨分化中的作用

发布时间：2019-03-25 15:56

【摘要】： 目的:探讨缝隙连接细胞间通讯在大鼠骨髓基质细胞成骨分化中的作用。方法:采取全骨髓贴壁分离筛选法从雄性Sprague-Dawley大鼠中的股骨、胫骨中提取骨髓基质细胞并在L-DMEM中进行培养。细胞传代培养至第3代时,分成对照组、诱导组、实验组共3组,选用地塞米松+β-甘油磷酸钠+维生素C为成骨诱导剂、18α-甘草次酸为缝隙连接通讯阻滞剂,3组细胞分别加入培养液、培养液+成骨诱导剂、培养液+成骨诱导剂+18α-甘草次酸进行培养。培养过程中在不同时间点检测各组细胞的增殖、碱性磷酸酶相对活性,同时在第7天应用细胞免疫荧光技术定位缝隙连接蛋白43,RT-PCR法检测缝隙连接蛋白43、骨钙素、骨涎蛋白的mRNA水平。在第10天应用划痕标记染料示踪法评估缝隙连接细胞间通讯功能,在第21天茜素红S染色法检验基质中钙结节形成能力。结果:在第7、9天的增殖能力,实验组诱导组对照组。第9、11天的碱性磷酸酶相对活性,对照组实验组诱导组。第7天,发现缝隙连接蛋白43定位于胞浆、胞膜,缝隙连接蛋白43的mRNA水平诱导组和实验组间无显著差别,但均高于对照组;骨钙素、骨涎蛋白的mRNA对照组实验组诱导组。第10天,显示缝隙连接通讯功能对照组实验组诱导组(均以P0.05为有显著统计学意义)。第21天,钙结节形成能力方面,肉眼观察显示实验组诱导组,对照组无钙结节形成。结论:18α-甘草次酸能通过阻滞由缝隙连接蛋白43介导的缝隙连接细胞间通讯能力,从而减弱成骨诱导剂对大鼠基质细胞的成骨分化作用。
[Abstract]:Aim: to investigate the role of gap junctional intercellular communication in osteoblast differentiation of rat bone marrow stromal cells (BMSCs). Methods: bone marrow stromal cells were isolated from femur and tibia of male Sprague-Dawley rats and cultured in L-DMEM. When the cells were subcultured to the third generation, they were divided into control group, induction group and experimental group. Dexamethasone 尾-sodium glycerophosphate vitamin C was used as osteogenic inducer and 18 伪-glycyrrhizinic acid was used as gap junctional communication blocker. The cells of the three groups were cultured with 18 伪-glycyrrhizinic acid, the osteoblast inducer. Cell proliferation and the relative activity of alkaline phosphatase (ALP) were measured at different time points in the culture process. At the same time, gap junction protein 43 and osteocalcin were detected by RT-PCR and immunofluorescence technique on the 7th day, respectively, and the expression of gap junctional protein 43 and osteocalcin were detected by RT-PCR. MRNA level of bone sialoprotein. The intercellular communication function of gap junctions was evaluated by scratch-labeled dye tracer method on the 10th day, and the ability of calcium nodule formation in matrix was examined by 21-day alizarin red S staining. Results: on the 7th and 9th day, the proliferation ability of the experimental group was induced in the control group. On the 9th and 11th day, the relative activity of alkaline phosphatase (ALP) was induced in the experimental group of the control group. On the 7th day, it was found that there was no significant difference in the mRNA level of gap junctional protein 43 in cytoplasm, cell membrane and gap junctional protein 43 between the two groups, but it was higher than that in the control group, while osteocalcin and osteocalcin in the mRNA control group were significantly higher than those in the control group. On the 10th day, the gap junctional communication function was shown in the experimental group (P0.05 was statistically significant). On the 21st day, the ability of calcium nodule formation was observed by naked eye, and no calcium nodule was observed in the experimental group and the control group. Conclusion: 18 伪-glycyrrhizinic acid can inhibit the osteoblastic differentiation of rat stromal cells by blocking the gap junctional intercellular communication mediated by gap junctional protein 43 (gap junctional protein 43).
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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