【摘要】：目的觀察硫酸鎳染毒體外培養(yǎng)的大鼠睪丸間質(zhì)細(xì)胞其超微結(jié)構(gòu)、凋亡調(diào)控基因Bax和Bcl-2蛋白表達(dá)以及氧化應(yīng)激水平的變化,初步探討硫酸鎳致睪丸間質(zhì)細(xì)胞凋亡的可能機(jī)制。 方法(1)選擇健康雄性Wistar大鼠,采用膠原酶消化以及Percoll分離液密度梯度離心法,經(jīng)體外貼壁培養(yǎng)獲得高純度大鼠睪丸間質(zhì)細(xì)胞。(2)體外對(duì)數(shù)生長(zhǎng)期的間質(zhì)細(xì)胞經(jīng)硫酸鎳0、62.5、125、250、500和1000μmol/L處理6h,收集細(xì)胞并制備超薄切片,透射電鏡觀察睪丸間質(zhì)細(xì)胞超微結(jié)構(gòu)的變化。(3)取對(duì)數(shù)生長(zhǎng)期大鼠睪丸間質(zhì)細(xì)胞,用硫酸鎳0、250、500和1000μmol/L分別處理6、12和24h,在各處理終點(diǎn)收集細(xì)胞,用Western blot技術(shù)檢測(cè)凋亡調(diào)控基因Bax和Bcl-2的蛋白表達(dá)水平。(4)耿對(duì)數(shù)生長(zhǎng)期大鼠睪丸間質(zhì)細(xì)胞,硫酸鎳0、250、500和1000μmol/L分別處理6、12和24h后收集細(xì)胞,超聲處理后離心取上清液,采用分光光度法檢測(cè)過氧化氫酶(CAT)和超氧化物歧化酶(SOD)活力,羥自由基(·OH)抑制能力以及丙二醛(MDA)含量。 結(jié)果(1)透射電鏡結(jié)果可見,62.5μmol/L硫酸鎳組睪丸間質(zhì)細(xì)胞僅有個(gè)別線粒體輕度腫脹,內(nèi)質(zhì)網(wǎng)無(wú)明顯變化；125μmol/L硫酸鎳組可見線粒體輕度腫脹,內(nèi)質(zhì)網(wǎng)擴(kuò)張不明顯；250μmol/L硫酸鎳組可見線粒體腫脹,內(nèi)質(zhì)網(wǎng)部分?jǐn)U張；500μmol/L硫酸鎳組睪丸間質(zhì)細(xì)胞線粒體腫脹明顯并空泡化,粗面內(nèi)質(zhì)網(wǎng)出現(xiàn)脫顆粒現(xiàn)象；1000μmol/L硫酸鎳組睪丸間質(zhì)細(xì)胞線粒體嚴(yán)重腫脹,內(nèi)質(zhì)網(wǎng)明顯擴(kuò)張。(2)Western blot檢測(cè)結(jié)果顯示：與對(duì)照組比較,硫酸鎳染毒各時(shí)間組,隨著染毒濃度的不斷增加,Bax蛋白表達(dá)水平呈上調(diào)趨勢(shì),而Bcl-2蛋白表達(dá)水平呈下調(diào)趨勢(shì)。(3)硫酸鎳呵引起睪丸間質(zhì)細(xì)胞CAT和SOD活力下降,OH抑制能力降低以及MDA含量增加。相同染毒時(shí)間,硫酸鎳各濃度組與對(duì)照組比較：染毒6h,硫酸鎳1000μmol/L組CAT活力顯著降低(P0.01),硫酸鎳500μmol/L組·OH抑制能力也開始降低(P0.01)；染毒12h,硫酸鎳500μmol/L組CAT活力開始下降(P0.01),各染毒組SOD活力和·OH抑制能力開始降低(P0.01)；染毒24h,各染毒組CAT和SOD活力以及·OH抑制能力降低,MDA含量升高(P0.05或P0.01)。相同濃度,硫酸鎳各染毒時(shí)間組與0h組比較：硫酸鎳250μmol/L染毒24h組,CAT活力下降而MDA含量升高(P0.01)；染毒12h及其以上組,SOD活力和·OH抑制能力均降低(P0.01)。硫酸鎳500μmol/L染毒6h及其以上組,·OH抑制能力降低(P0.01)；染毒12h及其以上組,硫酸鎳抑制CAT和SOD活力(P0.01)；染毒24h組,MDA含量升高(P0.01)。硫酸鎳1000μmol/L,染毒6h及其以上組CAT活力和·OH抑制能力均降低(P0.01)；染毒12h及其以上組,SOD活力明顯降低(P0.01)；染毒24h組,MDA含量升高(P0.01)。 結(jié)論硫酸鎳所致的大鼠睪丸間質(zhì)細(xì)胞線粒體超微結(jié)構(gòu)、凋亡調(diào)控基Bax和Bcl-2蛋白表達(dá)以及氧化應(yīng)激水平的異常變化可能與其誘導(dǎo)睪丸間質(zhì)細(xì)胞凋亡有關(guān)。
[Abstract]:Objective To observe the changes of the expression of Bax and Bcl-2 protein and the level of oxidative stress in the rat's testis interstitial cells cultured in vitro with nickel sulfate. Methods (1) Healthy male Wistar rats were selected, collagenase digestion and Percoll separation liquid density gradient centrifugation were used to obtain high-purity rat testis. (2) Interstitial cells in the logarithmic growth phase in vitro were treated with nickel sulfate 0, 62.5,125,250,500 and 1000. m u.mol/ L for 6 h, the cells were collected and the ultrathin sections were prepared, and the ultrastructural changes of the testicular interstitial cells were observed by transmission electron microscopy. (3) The expression of Bax and Bcl-2 was detected by Western blot, and the expression of Bax and Bcl-2 was detected by Western blot. (4) After treatment for 6,12 and 24 h, the cells were collected after 6,12 and 24 hours respectively. After the ultrasonic treatment, the supernatant was collected by centrifugation, and the activity of catalase (CAT) and superoxide dismutase (SOD) was detected by spectrophotometry. Force, hydroxyl radical (路 OH) inhibitory ability and malondialdehyde (MDA) content Results (1) The results of transmission electron microscopy (TEM) showed that there were only slight swelling of the mitochondria in the interstitial cells of the 62.5. m u.mol/ L nickel sulfate group, and there was no obvious change in the endoplasmic reticulum. The swelling of the body and the partial expansion of the endoplasmic reticulum; the swelling of the mitochondria in the interstitial cells of the testis of the 500. m u.mol/ L nickel sulfate group was obvious and vacuolated, and the endoplasmic reticulum of the rough surface appeared to be defragmented; the mitochondria of the testis interstitial cells of the 1000. m u.mol/ L nickel sulfate group were severely swollen, and the endoplasmic reticulum was bright. (2) The results of Western blot showed that the expression of Bax protein was up-regulated with the increase of the concentration of Bax and the level of the expression of Bcl-2 protein was lower than that of the control group. (3) The activity of CAT and SOD in the interstitial cells of the testis caused by nickel sulfate decreased, the inhibition ability of OH was decreased, and the content of MDA was decreased. In the same time, the concentration of the nickel sulfate in the nickel sulfate group was significantly lower than that of the control group (P 0.01), the activity of the nickel sulfate 500. mu.mol/ L group 路 OH was decreased (P0.01), and the CAT activity in the 500. mu.mol/ L group of the nickel sulfate was decreased (P0.01). The activity of SOD and 路 OH in each group was decreased (P0.01). The activity of CAT and SOD and 路 OH inhibition ability of each group were decreased and the content of MDA was increased (P0.05 or P0). The results showed that the activity of CAT was decreased and the content of MDA increased (P0.01). The activity of SOD and 路 OH was decreased in 12 h and above group (P 0.01). The results showed that the content of MDA in the group was higher than that of the group (P 0.01). The content of MDA in the group was higher than that of the group (P.01). The content of MDA in the group was higher than that of the group (P.01). The activity of CAT and the inhibition of the activity of 路 OH were decreased (P0.01). The activity of SOD and the activity of SOD decreased significantly (P0.01). The activity of SOD in the group was significantly lower than that of the group (P 0.01). The content of MDA in the group was higher than that of the group (P <0.01). Conclusion The ultrastructure of the mitochondria, the expression of Bax and Bcl-2 and the abnormal changes of the level of oxidative stress in the rat's testis can be induced by the nickel sulfate.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

【參考文獻(xiàn)】

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1 李維仁;韓勃萱;劉濤;李廣永;周峰;鞏艷青;高U喼，

本文編號(hào)：2486303