沉默PARP-1对氢醌所致大鼠骨髓间充质干细胞凋亡影响

发布时间：2018-05-27 08:46

本文选题：聚腺苷酸二磷酸核糖转移酶- + 氢醌　；参考：《中国职业医学》2017年01期

【摘要】：目的探讨沉默聚腺苷二磷酸核糖聚合酶-1(PARP-1)对氢醌所致大鼠骨髓间充质干细胞(BMMSCs)凋亡的影响。方法①PARP-1基因RNA干扰表达质粒转染BMMSCs后,经新霉素筛选获得PARP-1沉默BMMSCs,以免疫印迹法鉴定所构建的细胞。②以空载体BMMSCs为对照组,以PARP-1沉默BMMSCs为实验组。分别以质量浓度为0.0、2.5、5.0、10.0、20.0、40.0、80.0、160.0和320.0μmol/L的氢醌(溶于磷酸盐缓冲液)作用于2组细胞24 h,以噻唑蓝法检测细胞活力,根据细胞活力检测结果选择后续实验氢醌染毒剂量。③以浓度为0.0~20.0μmol/L的氢醌作用于2组细胞24 h后,以流式细胞技术检测细胞凋亡情况,以实时荧光定量-聚合酶链反应检测PARP-1 mRNA表达情况。结果①用质量浓度为400 mg/L的新霉素成功筛选出PARP-1沉默BMMSCs和空载体BMMSCs,PARP-1沉默BMMSCs中PARP-1蛋白表达量较BMMSCs下降85.00%,成功建立PARP-1沉默细胞。②根据细胞活力检测结果,后续实验选择氢醌染毒剂量为0.0~20.0μmol/L。3与氢醌染毒剂量为0.0μmol/L时比较,对照组及实验组细胞早期凋亡率分别在氢醌染毒剂量为10.0和5.0μmol/L时即出现有统计学意义的升高(P0.05);且在浓度为0.0~10.0μmol/L时,随着氢醌染毒剂量的增加,2组细胞早期凋亡率均增加(P0.01),均呈剂量-效应关系。③与氢醌染毒剂量为0.0μmol/L比较,对照组和实验组细胞的PARP-1 mRNA相对表达水平均在氢醌染毒剂量为5.0μmol/L时即出现有统计学意义的升高(P0.05),且在每个氢醌染毒剂量下实验组细胞PARP-1 mRNA相对表达水平均低于对照组(P0.05);在浓度为0.0~20.0μmol/L时,随着氢醌染毒剂量的增加,2组细胞PARP-1 mRNA相对表达水平均增加(P0.01),均呈剂量-效应关系。结论 PARP-1沉默BMMSCs在氢醌作用下更易发生凋亡,PARP-1可能参与了氢醌诱导的细胞凋亡。
[Abstract]:Objective to investigate the effect of silencing polyadenosine diphosphate (PARP-1) on apoptosis of rat bone marrow mesenchymal stem cells (BMMSCs) induced by hydroquinone. Methods after transfection of RNA interference expression plasmid of 1PARP-1 gene into BMMSCs, PARP-1 silencing BMMSCs was obtained by neomycin screening. The constructed cells were identified by Western blot as control group with empty vector BMMSCs as control group and PARP-1 silencing BMMSCs as experimental group. The cells in the two groups were treated with hydroquinone (dissolved in phosphate buffer solution) at a concentration of 0.02.55.00.0.0.0.0.0.0.0160.0 and 320.0 渭 mol/L for 24 h, respectively. The cell viability was detected by thiazolyl blue assay, and the cell viability was determined by thiazolyl blue assay. According to the results of cell viability test, the cell apoptosis was detected by flow cytometry after hydroquinone was treated with 0.020 渭 mol/L hydroquinone for 24 h. The expression of PARP-1 mRNA was detected by real-time fluorescence quantitative-polymerase chain reaction. Results 1 the expression of PARP-1 protein in PARP-1 silencing BMMSCs and empty vector BMMSCsPPARP-1 silencing BMMSCs was 85.00% lower than that of BMMSCs successfully. PARP-1 silencing cells were successfully established according to the results of cell viability test. In the following experiment, when the dose of hydroquinone was 20. 0 渭 mol/L.3 and the dose of hydroquinone was 0. 0 渭 mol/L, the early apoptosis rate of the control group and the experimental group were increased significantly at the dose of 10. 0 and 5. 0 渭 mol/L, respectively, and at the concentration of 0. 010 渭 mol/L. With the increase of the dose of hydroquinone, the early apoptotic rates of the two groups were increased with the dose-effect relationship of 0. 0 渭 mol/L and 0. 0 渭 mol/L, respectively. The relative expression level of PARP-1 mRNA in the control group and the experimental group was significantly increased when the dose of hydroquinone was 5.0 渭 mol/L, and the relative expression level of PARP-1 mRNA in the experimental group was lower than that in the control group under each dose of hydroquinone exposure. When the concentration was 0.00 渭 mol/L, the concentration of P0. 05 渭 mol/L was 0. 05 渭 mol/L. With the increase of the dose of hydroquinone, the relative expression of PARP-1 mRNA was increased in both groups, both in a dose-effect relationship. Conclusion PARP-1 silencing BMMSCs is more likely to induce apoptosis under the action of hydroquinone. PARP-1 may be involved in the apoptosis induced by hydroquinone.
【作者单位】：广东医科大学公共卫生学院东莞市环境医学重点实验室;广东医科大学公共卫生学院东莞市环境医学重点实验室;
【基金】：国家自然科学基金(81273116) 广东省自然科学基金(2015A030310532) 广东省医学科研基金(A2016246,A2014468) 广东医科大学科研基金(2XB13012)
【分类号】：R135.1

【相似文献】