本文選題：H2S　切入點(diǎn)：SB203580　出處：《石河子大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 探討硫化氫（H2S）、SB203580對(duì)大鼠肝星狀細(xì)胞(HSC)增殖、凋亡的影響，研究P38MAPK信號(hào)通路在H2S影響大鼠肝星狀細(xì)胞(HSC)增殖、凋亡中的作用，探討H2S是否通過P38MAPK信號(hào)通路起到抗肝纖維化作用。 方法： (1) HSC-T6細(xì)胞的培養(yǎng)：以10%胎牛血清DMEM(高糖)培養(yǎng)基培養(yǎng)，接種后6-8h細(xì)胞貼壁，24h-48h換液，72h增殖達(dá)生長(zhǎng)面積90%消化傳代，細(xì)胞傳代周期穩(wěn)定用于實(shí)驗(yàn)。(2) MTT法檢測(cè)HSC-T6細(xì)胞的增殖：分對(duì)照組與實(shí)驗(yàn)組，實(shí)驗(yàn)組按照加藥濃度梯度分組：NaHS組分別按照25umol/L、50umol/L、75umol/L、100μmol/L、200μmol/L濃度梯度給藥，SB203580組分別按照10umol/L、25umol/L、50umol/L、75umol/L、100umol/L的濃度梯度給藥，均干預(yù)48h，加入MTT20μl暗處反應(yīng)4h，加入DMSO150μl終止反應(yīng)，使用酶標(biāo)儀于570nm波長(zhǎng)處檢測(cè)各孔吸光度（A）值。（3）流式細(xì)胞術(shù)檢測(cè)H2S與SB203580對(duì)HSC-T6細(xì)胞凋亡的影響：分5組，正常對(duì)照組、DMSO組、NaHS50μmol/L組、SB20358075μmol/L組(SB組)、SB20358075umol/L+NaHS50umol/L組(SB+NaHS組)，采用流式細(xì)胞儀經(jīng)Annexin V-FITC/PI雙染檢測(cè)各組細(xì)胞HSC-T6細(xì)胞的凋亡率；同等實(shí)驗(yàn)條件，Hoechst33342染色，倒置熒光顯微鏡下觀察并計(jì)算HSC-T6細(xì)胞的凋亡率。（4）逆轉(zhuǎn)錄PCR法檢測(cè)HSC-T6細(xì)胞中Ⅰ、Ⅲ型膠原mRNA的表達(dá)：實(shí)驗(yàn)分對(duì)照組、DMSO組、NaHS組、SB組、SB+NaHS組，提取HSC-T6細(xì)胞中總RNA，逆轉(zhuǎn)錄PCR法檢測(cè)細(xì)胞中Ⅰ、Ⅲ型膠原mRNA的表達(dá)。（5）Western blot法檢測(cè)HSC-T6細(xì)胞中P-P38、Caspase-3蛋白的表達(dá)：實(shí)驗(yàn)分對(duì)照組、DMSO組、NaHS組、SB組、SB+NaHS組，Westernblot法檢測(cè)細(xì)胞中P-P38、Caspase-3蛋白的表達(dá)。 結(jié)果： （1）HSC生長(zhǎng)良好，總體死亡率5%，倒置顯微鏡下觀察細(xì)胞形態(tài)，可見NaHS組細(xì)胞密度增大，SB組細(xì)胞增殖不明顯，可見凋亡細(xì)胞。（2）按實(shí)驗(yàn)組干預(yù)HSC-T6細(xì)胞培養(yǎng)48h后，，與對(duì)照組相比，低濃度NaHS （25umol/L、50umol/L、75umol/L）促進(jìn)HSC-T6細(xì)胞增殖，NaHS50umol/L促細(xì)胞增殖顯著，差異有統(tǒng)計(jì)學(xué)意義(P0.05)；SB203580抑制HSC-T6細(xì)胞增殖，并促進(jìn)細(xì)胞凋亡，差異有統(tǒng)計(jì)學(xué)意義(P0.05)。（3）低濃度NaHS對(duì)細(xì)胞凋亡無影響，SB203580可顯著誘導(dǎo)HSC細(xì)胞凋亡，與NaHS聯(lián)合促細(xì)胞凋亡增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(P0.05)。（4）NaHS使HSC-T6細(xì)胞中Ⅰ、Ⅲ型膠原mRNA表達(dá)增強(qiáng)，SB203580促使HSC-T6細(xì)胞中Ⅰ、Ⅲ型膠原mRNA表達(dá)含量減少，二者聯(lián)合使細(xì)胞中Ⅰ、Ⅲ型膠原mRNA表達(dá)量明顯減少，差異有統(tǒng)計(jì)學(xué)意義(P0.05)。（5）NaHS使HSC-T6細(xì)胞中P-P38、Caspase-3蛋白表達(dá)增強(qiáng)，二者聯(lián)合P-P38蛋白表達(dá)減少、Caspase-3蛋白表達(dá)增強(qiáng)。 結(jié)論： （1） H2S通過激活P38MAPK信號(hào)通路促進(jìn)肝星狀細(xì)胞的增殖。（2） P38MAPK信號(hào)通路被阻斷后通過調(diào)節(jié)肝星狀細(xì)胞的凋亡，進(jìn)而延緩肝纖維化的發(fā)展。（3） H2S在P38MAPK信號(hào)通路被抑制后使刺激的肝星狀細(xì)胞增殖受到抑制，凋亡得到促進(jìn)。通過參與調(diào)節(jié)肝星狀細(xì)胞中Ⅰ型、Ⅲ型膠原mRNA及P-P38、Caspase-3蛋白的表達(dá)，從而起到抗肝纖維化的作用。
[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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