【摘要】：目的:通過體外培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞(Leydig細(xì)胞),觀察硫酸鎳(NiSO_4)致大鼠Leydig細(xì)胞活性氧(ROS)水平及睪酮合成酶mRNA和蛋白表達(dá)水平的變化,探討ROS在鎳致大鼠Leydig細(xì)胞睪酮合成障礙中的作用。方法:(1)采用0.25%膠原酶消化、Percoll連續(xù)梯度離心法和體外貼壁培養(yǎng)提取和純化Leydig細(xì)胞,并通過3β-HSD(3β-羥類固醇脫氫酶)對純化后的細(xì)胞進(jìn)行純度鑒定。(2)細(xì)胞處理取處于對數(shù)生長期的大鼠Leydig細(xì)胞,給予不同濃度(0、250、500和1000μmol/L)NiSO_4及1 IU/ml人絨毛膜促性腺激素(hCG)分別處理0、6、12和24 h;干預(yù)實(shí)驗(yàn)細(xì)胞處理取對數(shù)生長期Leydig細(xì)胞,分別經(jīng)活性氧抑制劑NAC和TEMPO提前干預(yù)1 h后,再行1000μmol/L NiSO_4處理24 h。(3)用酶聯(lián)免疫吸附法(ELISA)檢測培養(yǎng)上清液中睪酮濃度。(4)采用實(shí)時(shí)熒光定量PCR(RT-qPCR)和Western blot技術(shù)分別檢測Leydig細(xì)胞類固醇合成快速調(diào)節(jié)蛋白(StAR)、細(xì)胞色素P450膽固醇側(cè)鏈裂解酶(CYP11A1)、類固醇17α-羥化酶(CYP17A1)、3β-HSD和17β-羥類固醇脫氫酶(17β-HSD)的mRNA和蛋白表達(dá)水平。(5)采用DCFH-DA探針法檢測大鼠Leydig細(xì)胞內(nèi)ROS水平。結(jié)果:(1)3β-HSD染色法鑒定結(jié)果顯示,Percoll純化及細(xì)胞貼壁生長24h后,可得到純度較高的Leydig細(xì)胞。(2)ELISA法檢測結(jié)果顯示:與0 h組比較,hCG+0μmol/L NiSO_4處理Leydig細(xì)胞12 h和24 h后,培養(yǎng)液中睪酮濃度均明顯升高(P0.05)。與0μmol/L NiSO_4組(對照組)比較,hCG+500μmol/L NiSO_4處理Leydig細(xì)胞24 h,以及hCG+1000μmol/L NiSO_4處理細(xì)胞12和24h后,細(xì)胞培養(yǎng)液中睪酮濃度均顯著降低(P0.05)。RT-qPCR結(jié)果顯示:與對照組比較,500μmol/L NiSO_4處理Leydig細(xì)胞24 h后,睪酮合成酶CYP11A1和17β-HSD mRNA表達(dá)水平均下降(P0.05)。與對照組比較,1000μmol/L NiSO_4處理Leydig細(xì)胞24 h后,睪酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD mRNA表達(dá)水平均下降(P0.05)。Western Blot結(jié)果顯示:與對照組比較,1000μmol/L NiSO_4處理Leydig細(xì)胞24 h后,睪酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD的蛋白表達(dá)水平均下降(P0.05)。(3)DCFH-DA探針法檢測結(jié)果顯示:與對照組比較,1000μmol/L NiSO_4處理Leydig細(xì)胞24 h(NiSO_4組),熒光顯微鏡下可見細(xì)胞中ROS水平明顯升高。與NiSO_4組比較,經(jīng)5 mmol/L NAC(NAC組)和1 mmol/L TEMPO(TEMPO組)干預(yù)后,Leydig細(xì)胞中ROS水平明顯下降(P0.05)。ELISA法檢測結(jié)果顯示:與對照組比較,hCG+1000μmol/L NiSO_4處理細(xì)胞24 h后,細(xì)胞培養(yǎng)液中的睪酮濃度顯著降低(P0.05)。與NiSO_4組比較,NAC組和TEMPO組Leydig細(xì)胞培養(yǎng)液中的睪酮濃度則顯著升高(P0.05)。RT-qPCR和Western Blot分析結(jié)果顯示:與對照組比較,NiSO_4組Leydig細(xì)胞睪酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD的mRNA和蛋白表達(dá)水平均明顯降低(P0.05)。與NiSO_4組比較,NAC組細(xì)胞上述基因mRNA和蛋白表達(dá)水平均升高(P0.05),而TEMPO組除CYP17A1和17β-HSD外,其他蛋白表達(dá)水平也顯著升高(P0.05)。結(jié)論:NiSO_4可致大鼠Leydig細(xì)胞睪酮合成障礙,這可能與其誘導(dǎo)Leydig細(xì)胞ROS大量生成有關(guān)。NiSO_4可降低大鼠Leydig細(xì)胞睪酮分泌和下調(diào)睪酮合成酶相關(guān)基因和蛋白表達(dá)水平,且前述變化均可被活性氧抑制劑NAC和TEMPO所抑制,表明ROS在NiSO_4誘導(dǎo)的大鼠Leydig細(xì)胞睪酮合成障礙中發(fā)揮重要作用。
[Abstract]:AIM: To observe the changes of reactive oxygen species (ROS) and testosterone synthase mRNA and protein expression in rat Leydig cells induced by nickel sulfate (NiSO_4), and to explore the role of ROS in nickel-induced testosterone dyssynthesis in rat Leydig cells. Leydig cells were extracted and purified by gradient centrifugation and adherent culture in vitro, and purified by 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). (2) Rat Leydig cells in logarithmic growth phase were treated with NiSO_4 and 1 IU/ml human chorionic gonadotropin (hCG) at different concentrations (0,250,500 and 1000 micromol/L). Leydig cells were treated for 0, 6, 12, and 24 hours respectively. After 1 hour of intervention with active oxygen inhibitor NAC and TEMPO, the cells were treated with 1 000 micromol/L NiSO_4 for 24 hours. (3) Testosterone concentration in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). (4) Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used. The levels of mRNA and protein expression of steroid synthesis rapid regulator protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1), steroid 17 alpha hydroxylase (CYP17A1), 3 beta-HSD and 17 beta-hydroxysteroid dehydrogenase (17beta-HSD) in Leydig cells of rats were detected by DCFH-DA probe. (2) The results of ELISA showed that the concentration of testosterone in Leydig cells treated with hCG+0 micromol/L NiSO_4 for 12 h and 24 h was significantly higher than that of the control group (P 0.05). The levels of testosterone synthase CYP11A1 and 17 beta-HSD mRNA expression in cultured Leydig cells treated with CG+500 micromol/L NiSO_4 for 24 hours and hCG+1000 micromol/L NiSO_4 for 12 and 24 hours were significantly decreased (P 0.05). Compared with the control group, the expression levels of testosterone synthase StAR, CYP11A1, CYP17A1, 3-beta-HSD and 17-beta-HSD mRNA in Leydig cells treated with 1 000 micromol/L NiSO_4 for 24 hours decreased (P 0.05). The Western Blot results showed that, compared with the control group, the eggs of testosterone synthase StAR, CYP11A1, CYP17A1, 3-beta-HSD and 17-HSD were treated with 1 000 micromol/L NiSO_4 for 24 hours. The results of DCFH-DA probe assay showed that compared with the control group, the ROS level of Leydig cells treated with 1 000 micromol/L NiSO_4 for 24 hours (NiSO_4 group) was significantly increased under fluorescence microscope. Compared with NiSO_4 group, the ROS level of Leydig cells was significantly increased after 5 mmol/L NAC (NAC group) and 1 mmol/L TEMPO (TEMPO group) intervention. The results of ELISA showed that the concentration of testosterone in the cell culture medium decreased significantly (P 0.05) after 24 hours of treatment with hCG + 1000 micromol / L NiSO_4 compared with the control group. Compared with NiSO_4 group, the concentration of testosterone in the Leydig cell culture medium of NAC group and TEMPO group increased significantly (P 0.05). RT-q PCR and Western Blot analysis showed that the concentration of testosterone in the cell culture medium was significantly higher (P 0.05). Compared with the control group, the levels of mRNA and protein expression of testosterone synthase StAR, CYP11A1, CYP17A1, 3beta-HSD and 17beta-HSD were significantly decreased in NiSO_4 group (P 0.05). Compared with NiSO_4 group, the levels of mRNA and protein expression of these genes in NAC group were increased (P 0.05), while those of TEMPO group were significantly increased except CYP17A1 and 17beta-HSD. CONCLUSION: NiSO_4 may induce the production of ROS in rat Leydig cells. NiSO_4 can decrease the secretion of testosterone and down-regulate the expression of testosterone synthase-related genes and proteins in rat Leydig cells, and these changes can be inhibited by the inhibitors of reactive oxygen species NAC and TEMPO. Ming ROS plays an important role in NiSO_4 induced Leydig cell testosterone synthesis disorder in rats.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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