[1]潘家钰,康刚劲,徐曼华,等.雌二醇通过激活SIRT1/P53通路对人晶状体上皮细胞抗凋亡作用研究[J].眼科新进展,2020,40(5):420-424.[doi:10.13389/j.cnki.rao.2020.0097]
　PAN Jiayu,KANG Gangjin,XU Manhua,et al.Anti-apoptotic effect of estradiol on human lens epithelial cells via SIRT1/P53 pathway[J].Recent Advances in Ophthalmology,2020,40(5):420-424.[doi:10.13389/j.cnki.rao.2020.0097]
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雌二醇通过激活SIRT1/P53通路对人晶状体上皮细胞抗凋亡作用研究/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年5期
页码:
420-424
栏目:
实验研究
出版日期:
2020-05-05

文章信息/Info

Title:
Anti-apoptotic effect of estradiol on human lens epithelial cells via SIRT1/P53 pathway
作者:
潘家钰; 康刚劲; 徐曼华; 彭正虹; 申苑莎
646000　四川省泸州市，西南医科大学附属医院眼科
Author(s):
PAN Jiayu; KANG Gangjin; XU Manhua; PENG Zhenghong; SHEN Yuansha
Department of Ophthalmology,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China
关键词:
雌二醇; 人晶状体上皮细胞; 沉默信息调节因子1; P53; 细胞凋亡
Keywords:
estradiol;  human lens epithelial cells;  silent information regulator 1;  P53;  cell apoptosis
分类号:
R776.1
DOI:
10.13389/j.cnki.rao.2020.0097
文献标志码:
A
摘要:
目的　探究雌二醇（estradiol,E2）对H2O2诱导的人晶状体上皮细胞（HLE-B3细胞）氧化损伤的保护作用及抗凋亡机制。方法　不同浓度H2O2作用于HLE-B3细胞，探索最佳浓度。将HLE-B3细胞随机分成5组：空白对照组，模型组（100 μmol·L-1 H2O2），低、中、高浓度E2组（100 μmol·L-1 H2O2+0.01 μmol·L-1、0.10 μmol·L-1、1.00 μmol·L-1 E2），观察各组细胞形态变化。采用CCK-8和流式细胞学检测细胞的增殖与凋亡，观察不同浓度的E2对HLE-B3细胞的影响。RT-qPCR检测沉默信息调节因子1（silent information regulator 1，SIRT1）mRNA、P53 mRNA表达。Western blot检测SIRT1、肿瘤抑制蛋白P53 、乙酰化P53（acetylate P53，Ac-P53）蛋白表达，免疫荧光化学检测SIRT1定位及荧光强度。结果　100 μmol·L-1H2O2为诱导HLE-B3细胞氧化应激的最佳浓度。CCK-8检测结果显示：低、中、高浓度E2组的细胞增殖率均高于模型组(均为P<0.05)。流式细胞学检测结果显示：模型组细胞凋亡率均高于其他各组，两两比较：空白对照组<高浓度E2组<中浓度E2组<低浓度E2组<模型组(均为P<0.05)。RT-qPCR及Western blot检测结果表明：SIRT1表达随着E2浓度的升高而增加，高浓度E2组>中浓度E2组>低浓度E2组>模型组>空白对照组(均为P<0.05)。Ac-P53在空白对照组表达与高浓度E2组差异无统计学意义(P>0.05)，其余组间Ac-P53表达比较：模型组>低浓度E2组>中浓度E2组>高浓度E2组(均为P<0.05)。空白对照组中P53表达均低于其他各组(均为P<0.05)，其余各组之间两两比较，差异均无统计学意义(均为P>0.05)。共聚焦免疫荧光化学检测结果示：SIRT1荧光强度随着E2浓度升高而增强。结论　E2对HLE-B3细胞的保护作用与SIRT1/ P53通路相关，在生理浓度范围化学检测内，随着E2浓度的增加，SIRT1表达增强，抑制Ac-P53，减少HLE-B3细胞凋亡。
Abstract:
Objective　To investigate the protective and anti-apoptotic mechanisms of estradiol (E2) on H2O2-induced oxidative damage in human lens epithelial cells (HLE-B3 cells). Methods　 HLE-B3 cells were treated with different concentrations of H2O2, and the optimal concentration was selected. HLE-B3 cells were randomly divided into 5 groups: normal control group, model group (100 μmol·L-1 H2O2), low, medium and high concentration of E2 group (100 μmol·L-1 H2O2+0.01, 0.10 and 1.00 μmol·L-1 E2), to observe the morphological changes of cells in each group. The cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry, to observe the effect of different concentrations of E2 on HLE-B3 cells. The expression of silent information regulator 1 (SIRT1) mRNA and P53 mRNA were detected by RT-qPCR. Protein expression of SIRT1, tumor suppressor protein P53 and acetylate P53 (Ac-P53) were detected by Western blot. The distribution and fluorescence intensity of SIRT1 were detected by immunofluorescence chemistry. Results　 100 μmol·L-1 H2O2was the optimal 〖JP2〗concentration to induce oxidative stress in HLE-B3 cells. CCK-8〖JP〗 detection results showed: The proliferation rates of low, medium and high concentrations of estradiol groups were higher than that in model group (all P<0.05). Flow cytometry test showed that the apoptosis rate in the model group was higher than that in other group: normal control group < high concentration of E2 group < medium concentration of E2 group < low concentration of E2 group < model group (all P<0.05). RT-qPCR and Western blot test showed that the expression of SIRT1 increased with the rising of E2 concentration: high concentration of E2 group > medium concentration of E2 group > low concentration of E2 group > model group > normal control group (all P<0.05). The expression of Ac-P53 in normal control group was higher than that in high concentration of E2 group, and no statistical difference was found (P> 0.05): model group > low concentration of E2 group > medium concentration of E2 group > high concentration of E2 group (all P<0.05). The expression of P53 in normal control group was lower than that in other group (all P<0.05), and no statistical difference was found by pairwise comparison among other groups (all P>0.05). Confocal immunofluorescence chemistry showed that the fluorescence intensity of SIRT1 increased with rising of estradiol concentration. Conclusion　E2 plays protective effect on HLE-B3 cells via SIRT1/P53 pathway, to inhibit Ac-P53 and decrease HLE-B3 cell apoptosis; the expression of SIRT1 increases with rising of E2 concentration in the physiological concentration range.

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备注/Memo

备注/Memo:
泸州市科技局基金资助［编号：2015LZCYD-S05(4/12)］
更新日期/Last Update: 2020-05-05