PGC-1α在铅致睾丸支持细胞氧化应激与能量代谢中的作用

发布时间：2017-12-28 04:06

本文关键词：PGC-1α在铅致睾丸支持细胞氧化应激与能量代谢中的作用　出处：《武汉大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的生殖系统对铅毒性特别敏感,近年来,重金属铅的生殖毒性损伤备受关注,但是铅对生殖系统产生的毒性作用机制尚不明确,本文通过醋酸铅处理TM4细胞株和过表达PGC-1α的TM4细胞株(简称PGC-1α(+)TM4细胞株)及低表达PGC-1α的TM4细胞株(简称PGC-1α(-)TM4细胞株),来研究PGC-1α在铅导致睾丸支持细胞的氧化应激与能量代谢中的作用。方法醋酸铅处理PGC-1α(-)TM4、TM4和PGC-1α(+)TM4细胞株24小时后,用RT-PCR测细胞内PGC-1α、SIRT3 mRNA表达量;采用DHE荧光探针染色,分别用流式细胞仪、荧光酶标仪、荧光显微镜检测细胞内ROS含量;分别用ATP含量测定试剂盒及LD含量测定试剂盒检测细胞内ATP及LD含量;分别用LDH活性测定试剂盒、SDH活性测定试剂盒、Na+-K+-ATP酶活性测定试剂盒及Ca2+-Mg2+-ATP酶活性测定试剂盒检测细胞内 LDH、SDH、Na+-K+-ATP 酶及 Ca2+-Mg2+-ATP 酶活性。结果①RT-PCR结果显示,随着染毒浓度的增加,TM4细胞和PGC-1α(+)TM4细胞中PGC-1α及SIRT3 mRNA水平呈先升高后降低的趋势,而PGC-1α(-)TM4细胞中PGC-1α及SIRT3mRNA水平呈逐渐降低的趋势,且与对照组有统计学差异,P0.05。相同浓度醋酸铅处理后,TM4细胞内PGC-1α及SIRT3 mRNA表达明显低于PGC-1α(+)TM4细胞,而高于PGC-1α(-)TM4细胞。②流式细胞仪结果显示,随着醋酸铅浓度的升高,TM4、PGC-1α(-)TM4和PGC-1α(+)TM4细胞内ROS水平均呈逐渐升高的趋势,显示了醋酸铅暴露与ROS水平升高的剂量效应关系,并且PGC-1α(+)TM4TM4PGC-1α(-)TM4(P0.05)。荧光酶标仪检测ROS水平可以看到上述相同的趋势,细胞的ROS均随着醋酸铅浓度的升高而升高(P0.05),但相同浓度的醋酸铅处理后,PGC-1α(+)TM4细胞的ROS水平比TM4细胞降低了 33.7%～45.2%,而TM4细胞内ROS水平比PGC-1α(-)TM4细胞降低了65%～85%。荧光显微镜结果显示,随着醋酸铅浓度的升高,与各自对照组相比,三组细胞内的荧光强度逐渐增强,说明细胞内的ROS水平升高,并且,相同浓度组中,TM4细胞内的荧光强度低于PGC-1α(-)TM4细胞,而高于PGC-1α(+)TM4细胞。③ATP含量测定结果显示,与对照组相比,各浓度PGC-1α(-)TM4、TM4及PGC-1α(+)TM4细胞、内ATP的含量均下降,差异有统计学意义(P0.05);且随着乙酸铅染毒浓度升高,TM4及其过表达、低表达细胞内ATP的含量均呈下降趋势。当乙酸铅染毒浓度相同时,3种细胞内ATP的含量依次为PGC-1α(+)TM4TM4PGC-1α(-)TM4,差异均有统计学意义(P0.05)。④LD含量测定结果显示,随着醋酸铅浓度升高,PGC-1α(-)TM4、TM4和PGC-1α(+)TM4细胞的胞内LD水平逐渐降低,与对照组相比,20μM～80μM醋酸铅暴露使PGC-1α(-)TM4细胞内的LD水平降低约40%～70%(P0.05),TM4细胞内的LD水平降低约45%～75%(P0.05),PGC-1α(+)TM4细胞的胞内LD水平降低约30%～45%(P0.05)。相同浓度的醋酸铅处理后,三种细胞的LD水平差异明显,醋酸铅0 μM～160μM 组,TM4 细胞的 LD 水平比 PGC-1α(-)TM4 细胞高15%～34%,PGC-1α(+)TM4 细胞的LD水平比TM4细胞高约20%～50%。当醋酸铅浓度由0μM增加到160μM时,PGC-1α(+)TM4、TM4 及 PGC-1α(-)TM4 细胞内的 LD 含量分别由 127.60、100、71.22 nmol/mg pro 降低到 63.00、30.88、16.41 nmol/mg pro。⑤酶活性检测结果显示,醋酸铅暴露会使细胞内的LDH、SDH、Na+-K+-ATP酶、及Ca2+-Mg2+-ATP酶活性降低。并且,当醋酸铅浓度升高到160μM时,PGC-1α(-)TM4、TM4和PGC-1α(+)TM4细胞的胞内LDH活性分别由89.9%、100%、117.7%降低到37.7%、41.9%、62.7%。PGC-1α(-)TM4、TM4 和 PGC-1α(+)TM4 细胞的胞内 SDH 活性分别降低了6倍、5倍、5倍。相同浓度醋酸铅处理细胞时,PGC-1α(+)TM4细胞的SDH活性比TM4细胞约增加54.7%～161.8%,而TM4细胞SDH活性比PGC-1α(-)TM4增加84.4%～50.3%。醋酸铅暴露后,三种细胞内的Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性均降低,并且,用相同浓度醋酸铅处理时,TM4细胞的Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性高于PGC-1α(-)TM4细胞,而低于PGC-1α(+)TM4细胞(P0.05)。结论醋酸铅暴露可导致小鼠睾丸支持细胞内氧化损伤和能量代谢障碍。PGC-1α对铅诱导的小鼠睾丸支持氧化应激和细胞能量代谢障碍具有一定的保护作用。
[Abstract]:The purpose of the reproductive system is especially sensitive to the toxicity of lead in recent years, the reproductive toxicity of heavy metal injury concern, but the toxicity mechanism of lead on the reproductive system is not clear, the lead acetate treated TM4 cells and expression of PGC-1 in TM4 cell line (PGC-1 alpha (+) TM4 cell line) TM4 cell lines and low expression of PGC-1 alpha (PGC-1 alpha (-) TM4 cell line), to study the PGC-1 alpha lead to oxidative stress and energy metabolism of Sertoli cells in the role of lead. Method of lead acetate PGC-1 alpha (-) TM4, TM4 and PGC-1 alpha (+) TM4 cells after 24 hours, the expression of PGC-1 alpha and SIRT3 mRNA measured the amount of intracellular RT-PCR; stained by DHE fluorescent probe, respectively by flow cytometry and fluorescence microplate and fluorescence microscopy in detection of cell ROS content; each assay kit to detect ATP and LD content by ATP assay kit and LD content; respectively by LDH activity assay kit, SDH activity assay kit, Na+-K+-ATP enzyme activity assay kit and Ca2+-Mg2+-ATP enzyme activity assay kit for detection of intracellular LDH, SDH, Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity. Results RT-PCR results showed that with the increase in concentration of TM4 cells, and PGC-1 alpha (+) increased first and then decreased with PGC-1 alpha and SIRT3 mRNA level in TM4 cells, and PGC-1 alpha (-) decreased gradually with PGC-1 alpha and SIRT3mRNA levels in TM4 cells, and there is significant difference with the control group P0.05. After the same concentration of lead acetate, the expression of PGC-1 alpha and SIRT3 mRNA in TM4 cells was significantly lower than that of PGC-1 alpha (+) TM4 cells, but higher than that of PGC-1 alpha (-) TM4 cells. The flow cytometry results showed that, with increasing concentration of lead acetate, TM4, alpha PGC-1 (-) TM4 and PGC-1 (+) ROS alpha level in TM4 cells was increased, showed lead acetate dose effect relationship with elevated ROS levels of exposure, and the PGC-1 (+) TM4TM4PGC-1 (alpha alpha - TM4 (P0.05)). Fluorescence microplate reader to detect the level of ROS can see the same trend, ROS cells were increased with increasing concentration of lead acetate and (P0.05), but the same concentration of lead acetate after treatment with PGC-1 alpha (+) TM4 cell ROS level decreased by 33.7% ~ 45.2% than TM4 cells, while the level of ROS TM4 cells in alpha than PGC-1 (-) TM4 cells decreased from 65% to 85%. Fluorescence microscopy results showed that with increasing concentration of lead acetate, compared with the control group, the fluorescence intensity of the three groups of cells were gradually increased, indicating the intracellular ROS level increased, and the same concentration, the fluorescence intensity of TM4 cells was lower than that of the PGC-1 alpha (-) TM4 cells, but higher than that of PGC-1 (alpha +) TM4 cells. The results showed that the content of ATP compared with the control group, the concentration of PGC-1 (-) TM4, alpha TM4 alpha and PGC-1 (+) TM4 cells, ATP content was decreased, the difference was statistically significant (P0.05); and with lead acetate concentration, TM4 content and low ATP expression in cells were decreased expression. When the concentration of lead acetate was the same, the content of ATP in the 3 cells was PGC-1 alpha (+) TM4TM4PGC-1 alpha (-) TM4 in turn, and the difference was statistically significant (P0.05). The results showed that the content of LD increased with the concentration of lead acetate, alpha PGC-1 (-) TM4, TM4 and PGC-1 (+) LD alpha level of TM4 cells decreased, compared with the control group, 20 M ~ 80 M lead acetate exposure to alpha PGC-1 (-) TM4 intracellular LD level reduced about 40% ~ 70% TM4 (P0.05), intracellular LD level decreased about 45% ~ 75% (P0.05), alpha PGC-1 (+) TM4 cell intracellular LD level decreased about 30% ~ 45% (P0.05). After treatment with the same concentration of lead acetate, the LD level of three kinds of cells was significantly different. The level of LD in TM4 cells was 15% to 34% higher than that in PGC-1 (TM4) cells, and the LD level of PGC-1 (+) TM4 cells was about 20% to 50% higher than that of TM4 cells in the lead acetate group of 0 M to 160 M. When the concentration of lead acetate increased from 0 M to 160 M, the LD content in PGC-1 alpha (+) TM4, TM4 and PGC-1 alpha (-) TM4 cells decreased from 127.60, 100, 71.22 nmol/mg pro to 63, 30.88, 16.41 nmol/mg nmol/mg, respectively. The results of enzyme activity test showed that lead acetate exposure could reduce the activity of LDH, SDH, Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in the cells. Moreover, when the concentration of lead acetate increased to 160 mu M, the intracellular LDH activity of PGC-1 alpha (-) TM4, TM4 and PGC-1 alpha (+) TM4 cells decreased from 89.9%, 100% and 117.7% to 37.7%, 41.9% and 62.7%, respectively. The intracellular SDH activity of PGC-1 alpha (-) TM4, TM4 and PGC-1 alpha (+) TM4 cells decreased by 6 times, 5 times and 5 times respectively. The SDH activity of PGC-1 + (TM4) cells increased by 54.7% ~ 161.8% compared with TM4 cells at the same concentration of lead acetate, while SDH activity of TM4 cells increased by 84.4% ~ 50.3% compared with PGC-1 (-) TM4. After exposure to lead acetate, the activities of Na+-K+-ATP enzymes and Ca2+-Mg2+-ATP enzymes in three kinds of cells decreased, and the activities of Na+-K+-ATP and Ca2+-Mg2+-ATP enzymes in TM4 cells were higher than those in PGC-1 alpha (-) TM4 cells, but lower than those in PGC-1 (+) TM4 cells (P0.05). Conclusion lead acetate exposure can lead to internal oxidative damage and energy metabolism in mouse testis support cells. PGC-1 - alpha has protective effect on lead induced mouse testis to support oxidative stress and cell energy metabolism disorder.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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