本文關鍵詞：PGC-1α在鉛致睪丸支持細胞氧化應激與能量代謝中的作用　出處：《武漢大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 醋酸鉛 PGC-1α 氧化應激 能量代謝 睪丸支持細胞

【摘要】：目的生殖系統(tǒng)對鉛毒性特別敏感,近年來,重金屬鉛的生殖毒性損傷備受關注,但是鉛對生殖系統(tǒng)產生的毒性作用機制尚不明確,本文通過醋酸鉛處理TM4細胞株和過表達PGC-1α的TM4細胞株(簡稱PGC-1α(+)TM4細胞株)及低表達PGC-1α的TM4細胞株(簡稱PGC-1α(-)TM4細胞株),來研究PGC-1α在鉛導致睪丸支持細胞的氧化應激與能量代謝中的作用。方法醋酸鉛處理PGC-1α(-)TM4、TM4和PGC-1α(+)TM4細胞株24小時后,用RT-PCR測細胞內PGC-1α、SIRT3 mRNA表達量;采用DHE熒光探針染色,分別用流式細胞儀、熒光酶標儀、熒光顯微鏡檢測細胞內ROS含量;分別用ATP含量測定試劑盒及LD含量測定試劑盒檢測細胞內ATP及LD含量;分別用LDH活性測定試劑盒、SDH活性測定試劑盒、Na+-K+-ATP酶活性測定試劑盒及Ca2+-Mg2+-ATP酶活性測定試劑盒檢測細胞內 LDH、SDH、Na+-K+-ATP 酶及 Ca2+-Mg2+-ATP 酶活性。結果①RT-PCR結果顯示,隨著染毒濃度的增加,TM4細胞和PGC-1α(+)TM4細胞中PGC-1α及SIRT3 mRNA水平呈先升高后降低的趨勢,而PGC-1α(-)TM4細胞中PGC-1α及SIRT3mRNA水平呈逐漸降低的趨勢,且與對照組有統(tǒng)計學差異,P0.05。相同濃度醋酸鉛處理后,TM4細胞內PGC-1α及SIRT3 mRNA表達明顯低于PGC-1α(+)TM4細胞,而高于PGC-1α(-)TM4細胞。②流式細胞儀結果顯示,隨著醋酸鉛濃度的升高,TM4、PGC-1α(-)TM4和PGC-1α(+)TM4細胞內ROS水平均呈逐漸升高的趨勢,顯示了醋酸鉛暴露與ROS水平升高的劑量效應關系,并且PGC-1α(+)TM4TM4PGC-1α(-)TM4(P0.05)。熒光酶標儀檢測ROS水平可以看到上述相同的趨勢,細胞的ROS均隨著醋酸鉛濃度的升高而升高(P0.05),但相同濃度的醋酸鉛處理后,PGC-1α(+)TM4細胞的ROS水平比TM4細胞降低了 33.7%～45.2%,而TM4細胞內ROS水平比PGC-1α(-)TM4細胞降低了65%～85%。熒光顯微鏡結果顯示,隨著醋酸鉛濃度的升高,與各自對照組相比,三組細胞內的熒光強度逐漸增強,說明細胞內的ROS水平升高,并且,相同濃度組中,TM4細胞內的熒光強度低于PGC-1α(-)TM4細胞,而高于PGC-1α(+)TM4細胞。③ATP含量測定結果顯示,與對照組相比,各濃度PGC-1α(-)TM4、TM4及PGC-1α(+)TM4細胞、內ATP的含量均下降,差異有統(tǒng)計學意義(P0.05);且隨著乙酸鉛染毒濃度升高,TM4及其過表達、低表達細胞內ATP的含量均呈下降趨勢。當乙酸鉛染毒濃度相同時,3種細胞內ATP的含量依次為PGC-1α(+)TM4TM4PGC-1α(-)TM4,差異均有統(tǒng)計學意義(P0.05)。④LD含量測定結果顯示,隨著醋酸鉛濃度升高,PGC-1α(-)TM4、TM4和PGC-1α(+)TM4細胞的胞內LD水平逐漸降低,與對照組相比,20μM～80μM醋酸鉛暴露使PGC-1α(-)TM4細胞內的LD水平降低約40%～70%(P0.05),TM4細胞內的LD水平降低約45%～75%(P0.05),PGC-1α(+)TM4細胞的胞內LD水平降低約30%～45%(P0.05)。相同濃度的醋酸鉛處理后,三種細胞的LD水平差異明顯,醋酸鉛0 μM～160μM 組,TM4 細胞的 LD 水平比 PGC-1α(-)TM4 細胞高15%～34%,PGC-1α(+)TM4 細胞的LD水平比TM4細胞高約20%～50%。當醋酸鉛濃度由0μM增加到160μM時,PGC-1α(+)TM4、TM4 及 PGC-1α(-)TM4 細胞內的 LD 含量分別由 127.60、100、71.22 nmol/mg pro 降低到 63.00、30.88、16.41 nmol/mg pro。⑤酶活性檢測結果顯示,醋酸鉛暴露會使細胞內的LDH、SDH、Na+-K+-ATP酶、及Ca2+-Mg2+-ATP酶活性降低。并且,當醋酸鉛濃度升高到160μM時,PGC-1α(-)TM4、TM4和PGC-1α(+)TM4細胞的胞內LDH活性分別由89.9%、100%、117.7%降低到37.7%、41.9%、62.7%。PGC-1α(-)TM4、TM4 和 PGC-1α(+)TM4 細胞的胞內 SDH 活性分別降低了6倍、5倍、5倍。相同濃度醋酸鉛處理細胞時,PGC-1α(+)TM4細胞的SDH活性比TM4細胞約增加54.7%～161.8%,而TM4細胞SDH活性比PGC-1α(-)TM4增加84.4%～50.3%。醋酸鉛暴露后,三種細胞內的Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性均降低,并且,用相同濃度醋酸鉛處理時,TM4細胞的Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性高于PGC-1α(-)TM4細胞,而低于PGC-1α(+)TM4細胞(P0.05)。結論醋酸鉛暴露可導致小鼠睪丸支持細胞內氧化損傷和能量代謝障礙。PGC-1α對鉛誘導的小鼠睪丸支持氧化應激和細胞能量代謝障礙具有一定的保護作用。
[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.
【學位授予單位】：武漢大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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