caveolin-1对成骨细胞MC3T3-E1增殖分化过程的调节作用
发布时间：2018-08-14 12:49
【摘要】：目的:观察成骨细胞不同生长阶段caveolin-1表达的时程变化,并研究caveolin-1对成骨细胞MC3T3-E1增殖和分化功能的影响。 方法:MC3T3-E1细胞在含β-甘油磷酸钠,抗坏血酸的培养基中培养18d,在细胞培养的不同时间(3,6,9,12,15,18 d) ,采用western blotting方法观察caveolin-1蛋白表达变化。采用经western blotting法筛选并确证有效的RNA干扰质粒pGenesil-1.1- cav-1质粒下调caveolin-1表达后,观察其对成骨细胞MC3T3-E1增殖和分化作用的影响:转染后第3天测定细胞存活率(MTT法),分别在转染后第3,6,9,12,15d测定细胞裂解液ALP活性(PNPP法)、培养上清骨钙素浓度(ELISA)。 结果:caveolin-1蛋白随MC3T3-E1细胞的分化,表达量逐渐降低(P 0. 01)各处理组对增殖期MC3T3-E1细胞存活率无明显影响(P 0. 05)。转染RNA干扰质粒pGenesil-1.1- cav-1组,ALP活性和骨钙素浓度与对照组相比峰值明显增大,且峰值前移。 结论:caveolin-1对成骨细胞MC3T3-E1分化具有负性调节作用,不影响增殖。
[Abstract]:Aim: to observe the temporal changes of caveolin-1 expression in osteoblasts at different growth stages and to study the effects of caveolin-1 on the proliferation and differentiation of MC3T3-E1 in osteoblasts. Methods MC3T3-E1 cells were cultured in a medium containing 尾 -glycerophosphate and ascorbic acid for 18 days, and the expression of caveolin-1 protein was observed by western blotting method at different time of cell culture. The RNA interference plasmid pGenesil-1.1-cav-1 was screened by western blotting method and confirmed to down-regulate the expression of caveolin-1. To observe its effect on the proliferation and differentiation of osteoblasts MC3T3-E1: the cell survival rate (MTT method) was measured on the 3rd day after transfection, the ALP activity of the cell lysate (PNPP method) was measured on the 3rd day after transfection and the osteocalcin concentration (ELISA).) in the culture supernatant was measured respectively. Results with the differentiation of MC3T3-E1 cells, the expression of 1: caveolin-1 protein decreased gradually (P 0. 0. 01) the survival rate of MC3T3-E1 cells in proliferative phase was not significantly affected by each treatment group (P 0. 0. ) The peak value of RNA activity and osteocalcin concentration in RNA interference plasmid pGenesil-1.1 cav-1 group was significantly higher than that in control group, and the peak value moved forward. Conclusion: 1: caveolin-1 has a negative regulatory effect on MC3T3-E1 differentiation of osteoblasts, and does not affect proliferation of osteoblasts.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
[Abstract]:Aim: to observe the temporal changes of caveolin-1 expression in osteoblasts at different growth stages and to study the effects of caveolin-1 on the proliferation and differentiation of MC3T3-E1 in osteoblasts. Methods MC3T3-E1 cells were cultured in a medium containing 尾 -glycerophosphate and ascorbic acid for 18 days, and the expression of caveolin-1 protein was observed by western blotting method at different time of cell culture. The RNA interference plasmid pGenesil-1.1-cav-1 was screened by western blotting method and confirmed to down-regulate the expression of caveolin-1. To observe its effect on the proliferation and differentiation of osteoblasts MC3T3-E1: the cell survival rate (MTT method) was measured on the 3rd day after transfection, the ALP activity of the cell lysate (PNPP method) was measured on the 3rd day after transfection and the osteocalcin concentration (ELISA).) in the culture supernatant was measured respectively. Results with the differentiation of MC3T3-E1 cells, the expression of 1: caveolin-1 protein decreased gradually (P 0. 0. 01) the survival rate of MC3T3-E1 cells in proliferative phase was not significantly affected by each treatment group (P 0. 0. ) The peak value of RNA activity and osteocalcin concentration in RNA interference plasmid pGenesil-1.1 cav-1 group was significantly higher than that in control group, and the peak value moved forward. Conclusion: 1: caveolin-1 has a negative regulatory effect on MC3T3-E1 differentiation of osteoblasts, and does not affect proliferation of osteoblasts.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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