硫化氢在SB203580影响肝星状细胞凋亡中的作用

发布时间：2018-03-19 13:04

  本文选题：H2S　切入点：SB203580　出处：《石河子大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的： 探讨硫化氢（H2S）、SB203580对大鼠肝星状细胞(HSC)增殖、凋亡的影响，研究P38MAPK信号通路在H2S影响大鼠肝星状细胞(HSC)增殖、凋亡中的作用，探讨H2S是否通过P38MAPK信号通路起到抗肝纤维化作用。 方法： (1) HSC-T6细胞的培养：以10%胎牛血清DMEM(高糖)培养基培养，接种后6-8h细胞贴壁，24h-48h换液，72h增殖达生长面积90%消化传代，细胞传代周期稳定用于实验。(2) MTT法检测HSC-T6细胞的增殖：分对照组与实验组，实验组按照加药浓度梯度分组：NaHS组分别按照25umol/L、50umol/L、75umol/L、100μmol/L、200μmol/L浓度梯度给药，SB203580组分别按照10umol/L、25umol/L、50umol/L、75umol/L、100umol/L的浓度梯度给药，均干预48h，加入MTT20μl暗处反应4h，加入DMSO150μl终止反应，使用酶标仪于570nm波长处检测各孔吸光度（A）值。（3）流式细胞术检测H2S与SB203580对HSC-T6细胞凋亡的影响：分5组，正常对照组、DMSO组、NaHS50μmol/L组、SB20358075μmol/L组(SB组)、SB20358075umol/L+NaHS50umol/L组(SB+NaHS组)，采用流式细胞仪经Annexin V-FITC/PI双染检测各组细胞HSC-T6细胞的凋亡率；同等实验条件，Hoechst33342染色，倒置荧光显微镜下观察并计算HSC-T6细胞的凋亡率。（4）逆转录PCR法检测HSC-T6细胞中Ⅰ、Ⅲ型胶原mRNA的表达：实验分对照组、DMSO组、NaHS组、SB组、SB+NaHS组，提取HSC-T6细胞中总RNA，逆转录PCR法检测细胞中Ⅰ、Ⅲ型胶原mRNA的表达。（5）Western blot法检测HSC-T6细胞中P-P38、Caspase-3蛋白的表达：实验分对照组、DMSO组、NaHS组、SB组、SB+NaHS组，Westernblot法检测细胞中P-P38、Caspase-3蛋白的表达。 结果： （1）HSC生长良好，总体死亡率5%，倒置显微镜下观察细胞形态，可见NaHS组细胞密度增大，SB组细胞增殖不明显，可见凋亡细胞。（2）按实验组干预HSC-T6细胞培养48h后，，与对照组相比，低浓度NaHS （25umol/L、50umol/L、75umol/L）促进HSC-T6细胞增殖，NaHS50umol/L促细胞增殖显著，差异有统计学意义(P0.05)；SB203580抑制HSC-T6细胞增殖，并促进细胞凋亡，差异有统计学意义(P0.05)。（3）低浓度NaHS对细胞凋亡无影响，SB203580可显著诱导HSC细胞凋亡，与NaHS联合促细胞凋亡增强，差异有统计学意义(P0.05)。（4）NaHS使HSC-T6细胞中Ⅰ、Ⅲ型胶原mRNA表达增强，SB203580促使HSC-T6细胞中Ⅰ、Ⅲ型胶原mRNA表达含量减少，二者联合使细胞中Ⅰ、Ⅲ型胶原mRNA表达量明显减少，差异有统计学意义(P0.05)。（5）NaHS使HSC-T6细胞中P-P38、Caspase-3蛋白表达增强，二者联合P-P38蛋白表达减少、Caspase-3蛋白表达增强。 结论： （1） H2S通过激活P38MAPK信号通路促进肝星状细胞的增殖。（2） P38MAPK信号通路被阻断后通过调节肝星状细胞的凋亡，进而延缓肝纤维化的发展。（3） H2S在P38MAPK信号通路被抑制后使刺激的肝星状细胞增殖受到抑制，凋亡得到促进。通过参与调节肝星状细胞中Ⅰ型、Ⅲ型胶原mRNA及P-P38、Caspase-3蛋白的表达，从而起到抗肝纤维化的作用。
[Abstract]:Objective:. To investigate the effects of H2SS-SB203580 on the proliferation and apoptosis of rat hepatic stellate cells (HSCC), to study the role of P38MAPK signaling pathway in H2S affecting the proliferation and apoptosis of rat hepatic stellate cells (HSCS), and to explore whether H2S may play an anti-fibrosis role through P38MAPK signaling pathway. Methods:. 1) HSC-T6 cell culture: 10% fetal bovine serum DMEM (high sugar) culture medium, 6-8 h after inoculation, the cell proliferation reached 90% digestion and passage after 24 h 48 h change of solution. Cell cycle stability was used to determine the proliferation of HSC-T6 cells by MTT: the control group and the experimental group were divided into two groups: the control group and the experimental group. The experimental group was divided into two groups according to the concentration gradient of 25 渭 mol / L, 50 渭 mol / L, 75 渭 mol / L, 100 渭 mol / L, 100 渭 mol / L, 100 渭 mol / L, 100 渭 mol / L, 100 渭 mol / L, respectively, according to the concentration gradient of 10 umoll / L 25 umololl / L 50 umololl / L 50 umololl / L 100 umol / L, respectively. All of them intervened for 48 h, added MTT20 渭 l for 4 h, and added DMSO150 渭 l to terminate the reaction. The effect of H 2S and SB203580 on the apoptosis of HSC-T6 cells was detected by flow cytometry at 570 nm wavelength. The effect of H 2S and SB203580 on the apoptosis of HSC-T6 cells was detected by flow cytometry: 5 groups were divided into 5 groups. The apoptosis rate of HSC-T6 cells was detected by flow cytometry with Annexin V-FITC / Pi double staining in SB20358075 渭 mol/L group and SB group SB20358075umol-L NaHS50umol/L group by flow cytometry and Hoechst33342 staining under the same experimental conditions. Reverse fluorescent microscope was used to observe and calculate the apoptosis rate of HSC-T6 cells. The expression of type 鈪

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