硫酸镍致体外培养的大鼠睾丸间质细胞凋亡的机制研究
发布时间：2019-05-27 16:25
【摘要】：目的观察硫酸镍染毒体外培养的大鼠睾丸间质细胞其超微结构、凋亡调控基因Bax和Bcl-2蛋白表达以及氧化应激水平的变化,初步探讨硫酸镍致睾丸间质细胞凋亡的可能机制。 方法(1)选择健康雄性Wistar大鼠,采用胶原酶消化以及Percoll分离液密度梯度离心法,经体外贴壁培养获得高纯度大鼠睾丸间质细胞。(2)体外对数生长期的间质细胞经硫酸镍0、62.5、125、250、500和1000μmol/L处理6h,收集细胞并制备超薄切片,透射电镜观察睾丸间质细胞超微结构的变化。(3)取对数生长期大鼠睾丸间质细胞,用硫酸镍0、250、500和1000μmol/L分别处理6、12和24h,在各处理终点收集细胞,用Western blot技术检测凋亡调控基因Bax和Bcl-2的蛋白表达水平。(4)耿对数生长期大鼠睾丸间质细胞,硫酸镍0、250、500和1000μmol/L分别处理6、12和24h后收集细胞,超声处理后离心取上清液,采用分光光度法检测过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活力,羟自由基(·OH)抑制能力以及丙二醛(MDA)含量。 结果(1)透射电镜结果可见,62.5μmol/L硫酸镍组睾丸间质细胞仅有个别线粒体轻度肿胀,内质网无明显变化；125μmol/L硫酸镍组可见线粒体轻度肿胀,内质网扩张不明显；250μmol/L硫酸镍组可见线粒体肿胀,内质网部分扩张；500μmol/L硫酸镍组睾丸间质细胞线粒体肿胀明显并空泡化,粗面内质网出现脱颗粒现象；1000μmol/L硫酸镍组睾丸间质细胞线粒体严重肿胀,内质网明显扩张。(2)Western blot检测结果显示：与对照组比较,硫酸镍染毒各时间组,随着染毒浓度的不断增加,Bax蛋白表达水平呈上调趋势,而Bcl-2蛋白表达水平呈下调趋势。(3)硫酸镍呵引起睾丸间质细胞CAT和SOD活力下降,OH抑制能力降低以及MDA含量增加。相同染毒时间,硫酸镍各浓度组与对照组比较：染毒6h,硫酸镍1000μmol/L组CAT活力显著降低(P0.01),硫酸镍500μmol/L组·OH抑制能力也开始降低(P0.01)；染毒12h,硫酸镍500μmol/L组CAT活力开始下降(P0.01),各染毒组SOD活力和·OH抑制能力开始降低(P0.01)；染毒24h,各染毒组CAT和SOD活力以及·OH抑制能力降低,MDA含量升高(P0.05或P0.01)。相同浓度,硫酸镍各染毒时间组与0h组比较：硫酸镍250μmol/L染毒24h组,CAT活力下降而MDA含量升高(P0.01)；染毒12h及其以上组,SOD活力和·OH抑制能力均降低(P0.01)。硫酸镍500μmol/L染毒6h及其以上组,·OH抑制能力降低(P0.01)；染毒12h及其以上组,硫酸镍抑制CAT和SOD活力(P0.01)；染毒24h组,MDA含量升高(P0.01)。硫酸镍1000μmol/L,染毒6h及其以上组CAT活力和·OH抑制能力均降低(P0.01)；染毒12h及其以上组,SOD活力明显降低(P0.01)；染毒24h组,MDA含量升高(P0.01)。 结论硫酸镍所致的大鼠睾丸间质细胞线粒体超微结构、凋亡调控基Bax和Bcl-2蛋白表达以及氧化应激水平的异常变化可能与其诱导睾丸间质细胞凋亡有关。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【参考文献】
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1 李维仁;韩勃萱;刘涛;李广永;周峰;巩艳青;高U喼，
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