【摘要】：目的:研究sentrin特異性蛋白酶3(SENP3)對大鼠成骨細(xì)胞端粒酶活性及端粒長度的影響。方法:首先0.2 mmol/L H_2O_2處理體外培養(yǎng)的大鼠成骨細(xì)胞后,Western blotting法檢測SENP3及特異性蛋白1(Sp1)的表達(dá)。pc DNA3.0-SENP3轉(zhuǎn)染成骨細(xì)胞,分別于24 h、48 h、72 h后采用四甲基偶氮唑藍(lán)(MTT)法檢測細(xì)胞活力的變化。轉(zhuǎn)染48 h后,Western blotting法檢測Sp1和端粒酶逆轉(zhuǎn)錄酶(TERT)的表達(dá),PCR-TRAP法及PCR法檢測端粒酶活性及端粒長度;ELISA檢測上清中堿性磷酸酶(ALP)和骨橋蛋白(OPN)的含量;放射免疫法(RIA)檢測骨鈣蛋白(OCN)的含量。最后將pc DNA3.0-SENP3與siRNA-Sp1共轉(zhuǎn)染成骨細(xì)胞,并檢測以上指標(biāo)。結(jié)果:H_2O_2處理成骨細(xì)胞后,SENP3和Sp1的表達(dá)顯著上升。pc DNA3.0-SENP3轉(zhuǎn)染成骨細(xì)胞后,Sp1和TERT的表達(dá)顯著上升,細(xì)胞活力、ALP、OPN及OCN含量也都顯著上升;端粒酶活性顯著增加及端粒長度縮短顯著延緩。而當(dāng)pc DNA3.0-SENP3與siRNA-Sp1共轉(zhuǎn)染成骨細(xì)胞后,細(xì)胞活力,ALP、OPN及OCN含量,端粒酶活性及端粒長度均未發(fā)生顯著變化。結(jié)論:SENP1通過上調(diào)Sp1的表達(dá)促進(jìn)TERT的表達(dá),增加端粒酶活性上升及延緩端粒長度縮短,從而增強(qiáng)成骨細(xì)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.
【作者單位】： 河南中醫(yī)藥大學(xué)第一附屬醫(yī)院;河南中醫(yī)藥大學(xué)中醫(yī)學(xué)博士后流動(dòng)站第三附屬醫(yī)院博士后研發(fā)基地;
【分類號】：R580

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