SENP3对大鼠成骨细胞端粒酶活性及端粒长度的影响

发布时间：2018-12-20 22:15

【摘要】：目的:研究sentrin特异性蛋白酶3(SENP3)对大鼠成骨细胞端粒酶活性及端粒长度的影响。方法:首先0.2 mmol/L H_2O_2处理体外培养的大鼠成骨细胞后,Western blotting法检测SENP3及特异性蛋白1(Sp1)的表达。pc DNA3.0-SENP3转染成骨细胞,分别于24 h、48 h、72 h后采用四甲基偶氮唑蓝(MTT)法检测细胞活力的变化。转染48 h后,Western blotting法检测Sp1和端粒酶逆转录酶(TERT)的表达,PCR-TRAP法及PCR法检测端粒酶活性及端粒长度;ELISA检测上清中碱性磷酸酶(ALP)和骨桥蛋白(OPN)的含量;放射免疫法(RIA)检测骨钙蛋白(OCN)的含量。最后将pc DNA3.0-SENP3与siRNA-Sp1共转染成骨细胞,并检测以上指标。结果:H_2O_2处理成骨细胞后,SENP3和Sp1的表达显著上升。pc DNA3.0-SENP3转染成骨细胞后,Sp1和TERT的表达显著上升,细胞活力、ALP、OPN及OCN含量也都显著上升;端粒酶活性显著增加及端粒长度缩短显著延缓。而当pc DNA3.0-SENP3与siRNA-Sp1共转染成骨细胞后,细胞活力,ALP、OPN及OCN含量,端粒酶活性及端粒长度均未发生显著变化。结论:SENP1通过上调Sp1的表达促进TERT的表达,增加端粒酶活性上升及延缓端粒长度缩短,从而增强成骨细胞增殖能力。
[Abstract]:Aim: to study the effects of sentrin specific protease 3 (SENP3) on telomerase activity and telomere length in rat osteoblasts. Methods: the expression of SENP3 and specific protein 1 (Sp1) in cultured rat osteoblasts were detected by, Western blotting after 0. 2 mmol/L H_2O_2 treatment. The expression of SENP3 and specific protein 1 (Sp1) were detected by pc DNA3.0-SENP3 transfection into osteoblasts at 24 h and 48 h, respectively. After 72 hours, the changes of cell viability were detected by tetramethyl azolium blue (MTT) assay. 48 h after transfection, the expression of Sp1 and telomerase reverse transcriptase (TERT) was detected by, Western blotting, telomerase activity and telomere length were detected by PCR-TRAP and PCR, and the contents of alkaline phosphatase (ALP) and osteopontin (OPN) in supernatant were detected by ELISA. The content of osteocalcin (OCN) was detected by radioimmunoassay (RIA). Finally, pc DNA3.0-SENP3 and siRNA-Sp1 were co-transfected into osteoblasts and the above indexes were detected. Results: the expression of SENP3 and Sp1 increased significantly in osteoblasts treated with H_2O_2. After transfection of pc DNA3.0-SENP3 into osteoblasts, the expression of Sp1 and TERT increased significantly, and the cell viability, ALP,OPN and OCN contents also increased significantly. Telomerase activity increased significantly and telomere length decreased significantly. However, when pc DNA3.0-SENP3 and siRNA-Sp1 co-transfected osteoblasts, the cell viability, ALP,OPN and OCN contents, telomerase activity and telomere length did not change significantly. Conclusion: SENP1 can promote the expression of TERT by up-regulating the expression of Sp1, increase the telomerase activity and delay the shortening of telomere length, thus enhancing the proliferation ability of osteoblasts.
【作者单位】：河南中医药大学第一附属医院;河南中医药大学中医学博士后流动站第三附属医院博士后研发基地;
【分类号】：R580

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