本文關(guān)鍵詞：PGC-1α在鉛致睪丸支持細(xì)胞氧化應(yīng)激與能量代謝中的作用　出處：《武漢大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 醋酸鉛 PGC-1α 氧化應(yīng)激 能量代謝 睪丸支持細(xì)胞

【摘要】：目的生殖系統(tǒng)對(duì)鉛毒性特別敏感,近年來,重金屬鉛的生殖毒性損傷備受關(guān)注,但是鉛對(duì)生殖系統(tǒng)產(chǎn)生的毒性作用機(jī)制尚不明確,本文通過醋酸鉛處理TM4細(xì)胞株和過表達(dá)PGC-1α的TM4細(xì)胞株(簡(jiǎn)稱PGC-1α(+)TM4細(xì)胞株)及低表達(dá)PGC-1α的TM4細(xì)胞株(簡(jiǎn)稱PGC-1α(-)TM4細(xì)胞株),來研究PGC-1α在鉛導(dǎo)致睪丸支持細(xì)胞的氧化應(yīng)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(xì)胞株24小時(shí)后,用RT-PCR測(cè)細(xì)胞內(nèi)PGC-1α、SIRT3 mRNA表達(dá)量;采用DHE熒光探針染色,分別用流式細(xì)胞儀、熒光酶標(biāo)儀、熒光顯微鏡檢測(cè)細(xì)胞內(nèi)ROS含量;分別用ATP含量測(cè)定試劑盒及LD含量測(cè)定試劑盒檢測(cè)細(xì)胞內(nèi)ATP及LD含量;分別用LDH活性測(cè)定試劑盒、SDH活性測(cè)定試劑盒、Na+-K+-ATP酶活性測(cè)定試劑盒及Ca2+-Mg2+-ATP酶活性測(cè)定試劑盒檢測(cè)細(xì)胞內(nèi) LDH、SDH、Na+-K+-ATP 酶及 Ca2+-Mg2+-ATP 酶活性。結(jié)果①RT-PCR結(jié)果顯示,隨著染毒濃度的增加,TM4細(xì)胞和PGC-1α(+)TM4細(xì)胞中PGC-1α及SIRT3 mRNA水平呈先升高后降低的趨勢(shì),而PGC-1α(-)TM4細(xì)胞中PGC-1α及SIRT3mRNA水平呈逐漸降低的趨勢(shì),且與對(duì)照組有統(tǒng)計(jì)學(xué)差異,P0.05。相同濃度醋酸鉛處理后,TM4細(xì)胞內(nèi)PGC-1α及SIRT3 mRNA表達(dá)明顯低于PGC-1α(+)TM4細(xì)胞,而高于PGC-1α(-)TM4細(xì)胞。②流式細(xì)胞儀結(jié)果顯示,隨著醋酸鉛濃度的升高,TM4、PGC-1α(-)TM4和PGC-1α(+)TM4細(xì)胞內(nèi)ROS水平均呈逐漸升高的趨勢(shì),顯示了醋酸鉛暴露與ROS水平升高的劑量效應(yīng)關(guān)系,并且PGC-1α(+)TM4TM4PGC-1α(-)TM4(P0.05)。熒光酶標(biāo)儀檢測(cè)ROS水平可以看到上述相同的趨勢(shì),細(xì)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(xì)胞的ROS水平比TM4細(xì)胞降低了 33.7%～45.2%,而TM4細(xì)胞內(nèi)ROS水平比PGC-1α(-)TM4細(xì)胞降低了65%～85%。熒光顯微鏡結(jié)果顯示,隨著醋酸鉛濃度的升高,與各自對(duì)照組相比,三組細(xì)胞內(nèi)的熒光強(qiáng)度逐漸增強(qiáng),說明細(xì)胞內(nèi)的ROS水平升高,并且,相同濃度組中,TM4細(xì)胞內(nèi)的熒光強(qiáng)度低于PGC-1α(-)TM4細(xì)胞,而高于PGC-1α(+)TM4細(xì)胞。③ATP含量測(cè)定結(jié)果顯示,與對(duì)照組相比,各濃度PGC-1α(-)TM4、TM4及PGC-1α(+)TM4細(xì)胞、內(nèi)ATP的含量均下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05);且隨著乙酸鉛染毒濃度升高,TM4及其過表達(dá)、低表達(dá)細(xì)胞內(nèi)ATP的含量均呈下降趨勢(shì)。當(dāng)乙酸鉛染毒濃度相同時(shí),3種細(xì)胞內(nèi)ATP的含量依次為PGC-1α(+)TM4TM4PGC-1α(-)TM4,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。④LD含量測(cè)定結(jié)果顯示,隨著醋酸鉛濃度升高,PGC-1α(-)TM4、TM4和PGC-1α(+)TM4細(xì)胞的胞內(nèi)LD水平逐漸降低,與對(duì)照組相比,20μM～80μM醋酸鉛暴露使PGC-1α(-)TM4細(xì)胞內(nèi)的LD水平降低約40%～70%(P0.05),TM4細(xì)胞內(nèi)的LD水平降低約45%～75%(P0.05),PGC-1α(+)TM4細(xì)胞的胞內(nèi)LD水平降低約30%～45%(P0.05)。相同濃度的醋酸鉛處理后,三種細(xì)胞的LD水平差異明顯,醋酸鉛0 μM～160μM 組,TM4 細(xì)胞的 LD 水平比 PGC-1α(-)TM4 細(xì)胞高15%～34%,PGC-1α(+)TM4 細(xì)胞的LD水平比TM4細(xì)胞高約20%～50%。當(dāng)醋酸鉛濃度由0μM增加到160μM時(shí),PGC-1α(+)TM4、TM4 及 PGC-1α(-)TM4 細(xì)胞內(nèi)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(cè)結(jié)果顯示,醋酸鉛暴露會(huì)使細(xì)胞內(nèi)的LDH、SDH、Na+-K+-ATP酶、及Ca2+-Mg2+-ATP酶活性降低。并且,當(dāng)醋酸鉛濃度升高到160μM時(shí),PGC-1α(-)TM4、TM4和PGC-1α(+)TM4細(xì)胞的胞內(nèi)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(xì)胞的胞內(nèi) SDH 活性分別降低了6倍、5倍、5倍。相同濃度醋酸鉛處理細(xì)胞時(shí),PGC-1α(+)TM4細(xì)胞的SDH活性比TM4細(xì)胞約增加54.7%～161.8%,而TM4細(xì)胞SDH活性比PGC-1α(-)TM4增加84.4%～50.3%。醋酸鉛暴露后,三種細(xì)胞內(nèi)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(shí),TM4細(xì)胞的Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性高于PGC-1α(-)TM4細(xì)胞,而低于PGC-1α(+)TM4細(xì)胞(P0.05)。結(jié)論醋酸鉛暴露可導(dǎo)致小鼠睪丸支持細(xì)胞內(nèi)氧化損傷和能量代謝障礙。PGC-1α對(duì)鉛誘導(dǎo)的小鼠睪丸支持氧化應(yīng)激和細(xì)胞能量代謝障礙具有一定的保護(hù)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(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 路博玉;李倫;代洪儒;朱尚嫻;宋文祥;欒俊勝;許弘范;康范龍;王佐峰;;124例急性醋酸鉛中毒的臨床分析[J];吉林醫(yī)學(xué);1984年01期

2 薛蓮,周建華,時(shí)錫金;醋酸鉛體外誘發(fā)人外周血淋巴細(xì)胞HPRT基因位點(diǎn)突變的研究[J];工業(yè)衛(wèi)生與職業(yè)病;2004年02期

3 賈慶華;蔣軍軍;哈小琴;;醋酸鉛對(duì)體外培養(yǎng)人腎小管上皮細(xì)胞的毒性效應(yīng)[J];山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年06期

4 陳鵬;楊昆;;醋酸鉛對(duì)未成年雄性大鼠骨髓間充質(zhì)干細(xì)胞生長(zhǎng)的影響[J];毒理學(xué)雜志;2010年05期

5 賈慶華;哈小琴;楊志華;呂同德;;醋酸鉛對(duì)體外培養(yǎng)人腎小球系膜細(xì)胞超微結(jié)構(gòu)的影響[J];工業(yè)衛(wèi)生與職業(yè)病;2012年01期

6 _5定芳;;醋酸鉛濁度試驗(yàn)對(duì)肝臟功能測(cè)定的實(shí)驗(yàn)觀察[J];山西醫(yī)學(xué)雜志;1963年03期

7 ;用“優(yōu)選法”選取醋酸鉛的最佳用量[J];新醫(yī)藥通訊;1972年04期

8 陳惠君,李冰深;醋酸鉛外洗致鉛中毒1例報(bào)告[J];中國(guó)工業(yè)醫(yī)學(xué)雜志;1991年02期

9 李建秀,王曉梅,高麗珍,劉青;醋酸鉛染毒雄性小鼠生殖器官的形態(tài)改變[J];邯鄲醫(yī)學(xué)高等專科學(xué)校學(xué)報(bào);2002年02期

10 靳翠紅;蔡原;劉秋芳;王軍明;趙劍;王述森;;醋酸鉛對(duì)大鼠成骨細(xì)胞凋亡影響[J];中國(guó)公共衛(wèi)生;2006年10期

相關(guān)會(huì)議論文 前7條

1 任雁;楊冬華;李麗;邢立國(guó);;醋酸鉛的神經(jīng)發(fā)育毒性機(jī)制[A];中國(guó)毒理學(xué)會(huì)第六屆全國(guó)毒理學(xué)大會(huì)論文摘要[C];2013年

2 馬繼霞;畢瑞明;羅大珍;;富硒康口服液對(duì)醋酸鉛誘發(fā)小鼠遺傳損傷的防護(hù)作用[A];中國(guó)營(yíng)養(yǎng)學(xué)會(huì)第八屆微量元素營(yíng)養(yǎng)學(xué)術(shù)會(huì)議論文摘要匯編[C];2003年

3 張惠英;陳玉柱;汪嶺;張鵬舉;丁鵬;;排鉛產(chǎn)品的驅(qū)鉛實(shí)驗(yàn)研究[A];中國(guó)營(yíng)養(yǎng)學(xué)會(huì)第十屆微量元素營(yíng)養(yǎng)學(xué)術(shù)會(huì)議論文摘要匯編[C];2009年

4 黃邵鑫;余君;周郎環(huán);金珊珊;陳莉;王紅;汪春紅;;GSH和MRP1調(diào)節(jié)鉛在睪丸支持細(xì)胞中的排出[A];中國(guó)毒理學(xué)會(huì)第六屆全國(guó)毒理學(xué)大會(huì)論文摘要[C];2013年

5 梁建成;石慧娟;孟妍妍;汪春紅;;醋酸鉛對(duì)原代培養(yǎng)小鼠肝細(xì)胞超氧化物歧化酶及Cu-Zn SOD mRNA表達(dá)的影響[A];湖北省、武漢市營(yíng)養(yǎng)學(xué)會(huì)第十一屆學(xué)術(shù)會(huì)議論文匯編[C];2007年

6 梁建成;汪春紅;張妍;張春蓮;王程強(qiáng);;維生素C、B_1聯(lián)合作用對(duì)醋酸鉛致小鼠肝細(xì)胞DNA損傷的影響[A];湖北省、武漢市營(yíng)養(yǎng)學(xué)會(huì)第十一屆學(xué)術(shù)會(huì)議論文匯編[C];2007年

7 鄒積艷;李守柔;;葉酸對(duì)醋酸鉛胚胎發(fā)育毒性的拮抗研究[A];第五屆全國(guó)優(yōu)生科學(xué)大會(huì)論文匯編[C];2000年

相關(guān)博士學(xué)位論文 前1條

1 張瑩;鉛誘導(dǎo)的中樞神經(jīng)系統(tǒng)星形膠質(zhì)細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)[D];中國(guó)醫(yī)科大學(xué);2008年

相關(guān)碩士學(xué)位論文 前10條

1 蘇紅;鐵調(diào)素在納米硫化鉛和醋酸鉛暴露對(duì)仔鼠神經(jīng)損傷中的實(shí)驗(yàn)研究[D];華北理工大學(xué);2015年

2 張誼;HIF-1α介導(dǎo)的ROCK信號(hào)通路在人參皂苷Rb1保護(hù)醋酸鉛誘導(dǎo)神經(jīng)細(xì)胞損傷中的作用[D];江蘇大學(xué);2016年

3 馮佩佩;醋酸鉛與納米硫化鉛暴露對(duì)大鼠血腦脊液屏障損傷的比較研究[D];華北理工大學(xué);2016年

4 高曉燕;長(zhǎng)期低劑量鉛染毒對(duì)雛雞生長(zhǎng)發(fā)育和主要組織臟器結(jié)構(gòu)及功能的影響[D];河南農(nóng)業(yè)大學(xué);2016年

5 劉茜;PGC-1α在鉛致睪丸支持細(xì)胞氧化應(yīng)激與能量代謝中的作用[D];武漢大學(xué);2017年

6 張榮;醋酸鉛對(duì)大鼠腦組織中神經(jīng)營(yíng)養(yǎng)因子及其受體表達(dá)的影響以及甲狀腺激素調(diào)節(jié)作用的研究[D];河北醫(yī)科大學(xué);2004年

7 蔣軍軍;醋酸鉛致腦損傷及腎損傷的細(xì)胞機(jī)制探討[D];甘肅農(nóng)業(yè)大學(xué);2010年

8 葉小鶴;四醋酸鉛在間接有機(jī)電合成中的應(yīng)用[D];華東師范大學(xué);2005年

9 吝春紅;醋酸鉛對(duì)骨髓造血微環(huán)境造血調(diào)控機(jī)制影響的研究[D];天津醫(yī)科大學(xué);2009年

10 時(shí)金玲;醋酸鉛對(duì)體外培養(yǎng)小鼠睪丸間質(zhì)細(xì)胞的毒性作用及凋亡機(jī)理的研究[D];河南農(nóng)業(yè)大學(xué);2014年

，

本文編號(hào)：1344486