【摘要】： 目的:探討縫隙連接細(xì)胞間通訊在大鼠骨髓基質(zhì)細(xì)胞成骨分化中的作用。 方法:采取全骨髓貼壁分離篩選法從雄性Sprague-Dawley大鼠中的股骨、脛骨中提取骨髓基質(zhì)細(xì)胞并在L-DMEM中進(jìn)行培養(yǎng)。細(xì)胞傳代培養(yǎng)至第3代時(shí),分成對(duì)照組、誘導(dǎo)組、實(shí)驗(yàn)組共3組,選用地塞米松+β-甘油磷酸鈉+維生素C為成骨誘導(dǎo)劑、18α-甘草次酸為縫隙連接通訊阻滯劑,3組細(xì)胞分別加入培養(yǎng)液、培養(yǎng)液+成骨誘導(dǎo)劑、培養(yǎng)液+成骨誘導(dǎo)劑+18α-甘草次酸進(jìn)行培養(yǎng)。培養(yǎng)過程中在不同時(shí)間點(diǎn)檢測(cè)各組細(xì)胞的增殖、堿性磷酸酶相對(duì)活性,同時(shí)在第7天應(yīng)用細(xì)胞免疫熒光技術(shù)定位縫隙連接蛋白43,RT-PCR法檢測(cè)縫隙連接蛋白43、骨鈣素、骨涎蛋白的mRNA水平。在第10天應(yīng)用劃痕標(biāo)記染料示蹤法評(píng)估縫隙連接細(xì)胞間通訊功能,在第21天茜素紅S染色法檢驗(yàn)基質(zhì)中鈣結(jié)節(jié)形成能力。 結(jié)果:在第7、9天的增殖能力,實(shí)驗(yàn)組誘導(dǎo)組對(duì)照組。第9、11天的堿性磷酸酶相對(duì)活性,對(duì)照組實(shí)驗(yàn)組誘導(dǎo)組。第7天,發(fā)現(xiàn)縫隙連接蛋白43定位于胞漿、胞膜,縫隙連接蛋白43的mRNA水平誘導(dǎo)組和實(shí)驗(yàn)組間無顯著差別,但均高于對(duì)照組;骨鈣素、骨涎蛋白的mRNA對(duì)照組實(shí)驗(yàn)組誘導(dǎo)組。第10天,顯示縫隙連接通訊功能對(duì)照組實(shí)驗(yàn)組誘導(dǎo)組(均以P0.05為有顯著統(tǒng)計(jì)學(xué)意義)。第21天,鈣結(jié)節(jié)形成能力方面,肉眼觀察顯示實(shí)驗(yàn)組誘導(dǎo)組,對(duì)照組無鈣結(jié)節(jié)形成。 結(jié)論:18α-甘草次酸能通過阻滯由縫隙連接蛋白43介導(dǎo)的縫隙連接細(xì)胞間通訊能力,從而減弱成骨誘導(dǎo)劑對(duì)大鼠基質(zhì)細(xì)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 偽-glycyrrhizinic acid was used as gap junctional communication blocker. The cells of the three groups were cultured with 18 偽-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 偽-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).
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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