论文部分内容阅读
目的:运用反复亚毒性剂量紫外线B(ultraviolet B,UVB)辐射人皮肤成纤维细胞,构建应激诱导早衰(stress-inducedpremature senescence,SIPS)模型,观察衰老相关生物学标志、衰老相关基因信号分子p53、p21、p16、沉默信息调节因子2相关酶1(sirtuin1,SIRT1)以及miR-34c的表达情况。方法:体外培养人皮肤成纤维细胞,予连续5次、每次剂量10 mJ/cm2的UVB辐射,细胞衰老β-半乳糖苷酶(SA-β-Gal)染色检测衰老细胞,流式细胞术检测细胞周期阻滞情况,实时荧光定量PCR检测miR-34c及衰老相关基因p53、p21、p16、SIRT1 mRNA表达水平变化,Western blot检测p53、p21、p16及SIRT1蛋白水平表达。结果:与对照组相比,SIPS组细胞SA-β-Gal染色呈强阳性[对照组(8.13±1.92)%,SIPS组(94.72±2.08)%,P<0.05];多数细胞阻滞于G1期[对照组(52.96±1.76)%,SIPS组(77.31±2.05)%,P<0.05];另外,实时荧光定量PCR显示miR-34c mRNA的表达升高(miR-34c-3p、miR-34c-5p分别为对照组0.93±0.09、1.03±0.03,SIPS组2.08±0.19、12.28±1.64,P<0.05);衰老相关基因p53、p21、p16、SIRT1 mRNA的表达亦升高(对照组分别为0.74±0.23、1.06±0.23、0.86±0.13、0.74±0.25;SIPS组分别为1.84±0.55、20.25±2.34、16.7±3.94、2.15±0.22,P<0.05);Western bolt表明p53、p21、p16及SIRT1蛋白水平表达明显升高,差异有统计学意义(P<0.05)。结论:miR-34c在反复多次亚毒性剂量UVB辐射人皮肤成纤维细胞后表达升高,对p53起正反馈调节作用,而SIRT1并未受miR-34c的抑制。
OBJECTIVE: To establish a model of stress-induced premature senescence (SIPS) by repeatedly sub-toxic doses of ultraviolet B (UVB) to human skin fibroblasts, and to observe the relationship between senescence-related biological markers, , P21, p16, sirtuin1 (SIRT1) and miR-34c expression. Methods: Human dermal fibroblasts were cultured in vitro. The cells were exposed to UVB radiation at a dose of 10 mJ / cm2 for 5 times in a row. The senescent cells were detected by the senescence assay of β-galactosidase (SA-β-Gal) The expression of p53, p21, p16 and SIRT1 mRNA was detected by real-time fluorescence quantitative PCR and the expressions of p53, p21, p16 and SIRT1 protein were detected by Western blot. Results: Compared with the control group, the expression of SA-β-Gal in SIPS cells was strongly positive (8.13 ± 1.92% in the control group and 94.72 ± 2.08% in the SIPS group, P <0.05] (52.96 ± 1.76)%, SIPS group (77.31 ± 2.05)%, P <0.05]. In addition, the expression of miR-34c mRNA and miR-34c -5p were 0.93 ± 0.09 and 1.03 ± 0.03 respectively in the control group and 2.08 ± 0.19 and 12.28 ± 1.64 in the SIPS group, P <0.05). The expressions of p53, p21, p16 and SIRT1 mRNA were also increased in the control group 0.74 ± 0.23,1.06 ± 0.23,0.86 ± 0.13,0.74 ± 0.25; SIPS group was 1.84 ± 0.55,20.25 ± 2.34,16.7 ± 3.94,2.15 ± 0.22, P <0.05); Western bolt showed that p53, p21, p16 and SIRT1 protein expression was significantly increased, the difference was statistically significant (P <0.05). CONCLUSION: miR-34c is up-regulated after repeated sub-toxic doses of UVB radiation on human dermal fibroblasts, and plays a positive feedback regulation role on p53, while SIRT1 is not inhibited by miR-34c.