本文選題：鉛　切入點：睪丸間質(zhì)細胞　出處：《暨南大學(xué)》2013年碩士論文

【摘要】：目的: 研究重金屬鉛對睪丸間質(zhì)細胞的損傷作用，定位到線粒體，闡明其毒理作用機制，為臨床男性性功能低下的治療提供實驗依據(jù)。 方法: 以100uM濃度的醋酸鉛作用于睪丸間質(zhì)細胞株R2C，加入25μM的羥基膽固醇作為孕酮合成底物，在1、3、6、12和24h分別收取正常和鉛暴露細胞樣品，應(yīng)用放射免疫學(xué)技術(shù)檢測培養(yǎng)上清中孕酮的含量；應(yīng)用Real time RT-PCR和Western blot的方法驗證孕酮合成相關(guān)酶的表達水平；應(yīng)用流式細胞儀技術(shù)檢測線粒體膜電位，并以透射電鏡的技術(shù)觀察線粒體形態(tài)學(xué)變化；應(yīng)用ELISA的方法檢測細胞內(nèi)的cAMP水平，，并通過Western blot的方法檢測cAMP-PKA/PKC-ERK1/2通路上相關(guān)蛋白的表達及其磷酸化水平，最后又通過免疫熒光技術(shù)檢測了MEK/ERK與線粒體共定位的變化。 結(jié)果: 對比同一時間點的正常對照組，重金屬鉛（100μM）使R2C細胞培養(yǎng)上清中的孕酮含量明顯減少，并呈現(xiàn)時效關(guān)系，在12h（P0.01）與24h（P0.001）具有統(tǒng)計學(xué)差異。檢測孕酮合成相關(guān)酶的基因和蛋白表達水平結(jié)果顯示，StAR基因表達從1h開始就出現(xiàn)明顯的下調(diào)趨勢，CYP11A1基因表達從6h開始出現(xiàn)明顯的下調(diào)趨勢，而3β-HSD基因表達從3h開始出現(xiàn)明顯的下調(diào)趨勢；StAR蛋白從1h開始就出現(xiàn)明顯的下調(diào)趨勢，CYP11A1蛋白從1h就開始下調(diào)，3β-HSD蛋白從12h開始出現(xiàn)明顯的下調(diào)趨勢。流式細胞儀技術(shù)檢測結(jié)果顯示，重金屬鉛作用1h就能夠觀察到線粒體膜電位降低9.37%，隨后3和6h略有回升，接著在12h和24h保持下降10%的水平，與正常組比較均具有統(tǒng)計學(xué)差異。電鏡結(jié)果發(fā)現(xiàn)醋酸鉛作用1h開始就發(fā)生了損傷，直到24h呈現(xiàn)時間效應(yīng)關(guān)系。cAMP-PKA/PKC-ERK1/2信號通路相關(guān)蛋白檢測顯示，鉛暴露使細胞內(nèi)cAMP水平自1h開始顯著下降，PKA和PKC表達下降，ERK和MEK蛋白磷酸化水平也相應(yīng)顯著下調(diào)。免疫熒光結(jié)果發(fā)現(xiàn)重金屬鉛作用后，MEK/ERK與線粒體共定位減少。 結(jié)論: 重金屬鉛能夠抑制睪丸間質(zhì)細胞分泌孕酮的功能，能夠在基因和蛋白水平上抑制孕酮合成關(guān)鍵酶StAR、CYP11A1和3β-HSD的表達。鉛導(dǎo)致線粒體發(fā)生損傷，使線粒體膜電位下降。重金屬鉛對睪丸間質(zhì)細胞的毒理作用與cAMP-PKA/PKC-ERK1/2信號通路的抑制密切相關(guān)。鉛促使睪丸間質(zhì)細胞內(nèi)cAMP水平下降，cAMP的下降同時抑制PKA和PKC的蛋白表達，顯示PKA和PKC都同時參與了該類固醇激素合成通路；PKA/PKC的下調(diào)抑制了MEK1/2和ERK1/2的磷酸化，進而影響StAR的活化，抑制孕酮的合成和分泌。這一機制的提出為部分因雄激素缺乏而導(dǎo)致的男性功能衰減的治療和男性性腺功能衰退的治療提供了實驗依據(jù)。
[Abstract]:Objective:. To study the damage effect of heavy metal lead on Leydig cells of testis, locate mitochondria, elucidate its toxicological mechanism, and provide experimental basis for the treatment of male sexual dysfunction. Methods:. The Leydig cell line R2C was treated with 100uM concentration of lead acetate, and 25 渭 M hydroxyl cholesterol was added as the synthesis substrate of progesterone. Normal and lead exposed cell samples were collected at 1 ~ (3) O ~ (6) O _ (12) and 24 h, respectively. Radioimmunoassay was used to detect progesterone content in culture supernatant; Real time RT-PCR and Western blot were used to verify the expression of progesterone synthase; flow cytometry was used to detect mitochondrial membrane potential. The morphological changes of mitochondria were observed by transmission electron microscopy (TEM), the level of cAMP in cells was detected by ELISA, and the expression and phosphorylation of related proteins in cAMP-PKA/PKC-ERK1/2 pathway were detected by Western blot. Finally, the co-localization of MEK/ERK and mitochondria was detected by immunofluorescence technique. Results:. Compared with the normal control group at the same time point, the concentration of progesterone in the supernatant of R2C cells was significantly decreased by heavy metal lead (100 渭 M). The gene and protein expression level of progesterone synthase were detected. The results showed that the expression of star gene was down-regulated from 1h to 6h, and CYP11A1 gene expression was down-regulated from 6h to 6h. However, the expression of 3 尾 -HSD gene showed a down-regulation trend from 3 h to 1 h. The CYP11A1 protein began to down-regulate from 1 h to 12 h. Flow cytometry showed that the expression of 3 尾 -HSD protein was down-regulated from 1 h to 12 h, and the results of flow cytometry showed that the expression of CYP11A1 protein was down-regulated from 1 h to 12 h. After exposure to heavy metal lead for 1 hour, the mitochondrial membrane potential decreased by 9.377.The mitochondrial membrane potential increased slightly at 3 and 6 h, and then decreased by 10% at 12 h and 24 h, respectively. The results of electron microscope showed that the damage occurred at 1 h after exposure to lead acetate, until 24 hours after exposure to lead acetate. The results showed that the signal pathway related proteins of cAMP-PKA / PKC-ERK1 / 2 showed a time-dependent relationship. The level of cAMP and PKC expression decreased significantly from 1 h after lead exposure, and the phosphorylation of ERK and MEK protein were also significantly down-regulated. The results of immunofluorescence showed that the co-localization of MEK / ERK and mitochondria was decreased after heavy metal lead exposure. Conclusion:. Heavy metal lead can inhibit the secretion of progesterone in the interstitial cells of testis, and inhibit the expression of the key enzymes of progesterone synthesis, StARP11A1 and 3 尾 -HSD, at the gene and protein levels. The toxic effect of heavy metal lead on Leydig cells was closely related to the inhibition of cAMP-PKA/PKC-ERK1/2 signaling pathway. Lead induced the decrease of cAMP level in Leydig cells and inhibited the expression of PKA and PKC proteins. The results showed that both PKA and PKC were involved in the downregulation of PKA / PKC in the steroid hormone synthesis pathway, which inhibited the phosphorylation of MEK1/2 and ERK1/2, thus affecting the activation of StAR. Inhibition of progesterone synthesis and secretion. This mechanism provides experimental evidence for the treatment of male functional decline caused partly by androgen deficiency and the treatment of male gonadal dysfunction.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114

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