【摘要】：目的：研究吸煙對大鼠下丘腦-垂體-睪丸軸相關(guān)激素的影響及對睪丸細(xì)胞的毒性作用，通過對相關(guān)凋亡基因研究分析其可能的機(jī)制；研究香煙煙霧提取物對睪丸細(xì)胞的細(xì)胞毒性，分析其可能機(jī)制。方法：體內(nèi)實(shí)驗(yàn)方法，選用5周SD大鼠80只，分為對照組與吸煙組，吸煙組采用靜式染毒，每天1次，每次2h，連續(xù)8周；對照組采用空白對照。實(shí)驗(yàn)第2周、第4周、第6周與第8周分別處死對照組與吸煙組大鼠10只，測定各種指標(biāo)。放射免疫法測定血清中睪酮、黃體生成激素、卵泡雌激素、胰島素樣生長因子-1及下丘腦促性腺激素釋放激素；HE染色觀察睪丸的結(jié)構(gòu)形態(tài)；TUNEL法定性、定量測定睪丸的細(xì)胞凋亡；Western Blot檢測各組大鼠睪丸組織中Fas/FasL與Bcl-2/Bax蛋白表達(dá)；RT-PCR檢測各組大鼠睪丸組織中Fas/FasL與Bcl-2/BaxmRNA表達(dá)。用細(xì)胞培養(yǎng)研究香煙煙霧提取物對大鼠睪丸的支持細(xì)胞、間質(zhì)細(xì)胞與生精細(xì)胞的影響。取對數(shù)生長期細(xì)胞用于試驗(yàn)，三種睪丸細(xì)胞培養(yǎng)，按照CSE干預(yù)濃度不同，將試驗(yàn)分為三組：0%CSE組、5%CSE組、10%CSE組。各組細(xì)胞培養(yǎng)12h后，采用MTT法檢測CSE對細(xì)胞增殖的影響，流式細(xì)胞術(shù)檢測細(xì)胞凋亡的情況，生物化學(xué)的方法檢檢測胞外乳酸脫氫酶的改變。結(jié)果：1）隨著時(shí)間延長，吸煙組大鼠體重明顯低于對照（P＜0.05）；2）大鼠血清中睪酮含量自第四周起吸煙組明顯低于對照組（P＜0.05）；吸煙組大鼠血清T含量隨染毒時(shí)間延長明顯下降（P＜0.05）；3）大鼠血清中黃體生成素含量在對照組有隨著時(shí)間延長逐漸增高，在吸煙組則隨時(shí)間下降；第四周起吸煙組大鼠血清中黃體生成素含量明顯低于對照組（P＜0.05）；4）大鼠血清中卵泡刺激素在對照組與吸煙組含量穩(wěn)定，自第二周起吸煙組含量明顯低于對照組（P＜0.05）；5）大鼠血清中胰島素樣生長因子-1含量隨時(shí)間下降，吸煙組大鼠血清中胰島素樣生長因子-1在第八周含量明顯低于第二周、第四周與第六周（P＜0.05），吸煙組第二周、第六周和第八周均明顯低于對照組（P＜0.05）；6）大鼠下丘腦促性腺激素釋放激素?zé)o論是對照組還是吸煙組隨時(shí)間延長而升高，吸煙組與對照組比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）；7）睪丸HE染色發(fā)現(xiàn)吸煙可以導(dǎo)致睪丸結(jié)構(gòu)組織的破壞，隨著時(shí)間的延長，組織改變明顯加深。第八周組吸煙組睪丸組織出現(xiàn)明顯的萎縮，大量的細(xì)胞裂解，細(xì)精管與間質(zhì)組織出現(xiàn)明顯的變性改變；8）TUNEL法測定睪丸細(xì)胞的凋亡率，吸煙組第四周、第六周、第八周細(xì)胞凋亡率明顯增高（P＜0.05）；9）吸煙組大鼠睪丸組織中Fas/FasL與Bax蛋白上調(diào)，Bcl-2蛋白下調(diào)；Fas/FasL、Bax基因的mRNA表達(dá)水平有上調(diào)的趨勢，結(jié)果與蛋白表達(dá)相同。吸煙第八周大鼠睪丸的Fas/FasL、Bax基因表達(dá)水平明顯上升（P＜0.05），Bcl-2mRNA下調(diào)明顯（P＜0.05）10）香煙煙霧提取物對支持細(xì)胞增值的影響沒有顯著性差異（P＞0.05）；對間質(zhì)細(xì)胞、生精細(xì)胞增值隨濃度的升高出現(xiàn)明顯的下降（P＜0.05），，且呈現(xiàn)一定的劑量反應(yīng)關(guān)系；香煙煙霧提取物組的睪丸支持細(xì)胞、間質(zhì)細(xì)胞和生精細(xì)胞組胞外LDH活力明顯高于對照組（P＜0.05）；11）流式細(xì)胞儀測定結(jié)果表明CSE導(dǎo)致支持細(xì)胞、間質(zhì)細(xì)胞和生精細(xì)胞組胞凋亡率上升。結(jié)論：吸煙是青春期大鼠生殖系統(tǒng)發(fā)育的有害因素，不僅下丘腦-垂體-睪丸軸中重要的激素有影響，而且對睪丸組織與細(xì)胞有不利影響，其可能的機(jī)制是吸煙激活了細(xì)胞凋亡調(diào)控基因，導(dǎo)致相應(yīng)蛋白表達(dá)增加，同時(shí)抑制了保護(hù)性蛋白的表達(dá)；香煙煙霧提取物中含有導(dǎo)致睪丸支持細(xì)胞、間質(zhì)細(xì)胞和生精細(xì)胞凋亡的化學(xué)物質(zhì)，這提示吸煙可能導(dǎo)致青春期大鼠生殖功能發(fā)育遲緩，精子數(shù)量和功能的減少。
[Abstract]:AIM: To study the effects of smoking on hormones related to hypothalamus-pituitary-testicular axis in rats and the toxic effects on testicular cells, and to analyze the possible mechanism of apoptosis by studying the related genes; to study the cytotoxicity of cigarette smoke extract on testicular cells and analyze its possible mechanism. The rats in the control group and the smoking group were sacrificed at the 2nd, 4th, 6th and 8th week of the experiment, and 10 rats in the control group and the smoking group were sacrificed respectively. The serum levels of testosterone, luteinizing hormone, follicular estrogen and pancreas were determined by radioimmunoassay. Islet-like growth factor-1 and hypothalamic gonadotropin-releasing hormone; HE staining was used to observe the structure and morphology of testis; TUNEL assay was used to quantify the apoptosis of testis cells; Western Blot was used to detect the expression of Fas/FasL and Bcl-2/Bax protein in testicular tissue of rats in each group; RT-PCR was used to detect Fas/FasL and Bcl-2/Bax mRNA in testicular tissue of rats in each group. The effects of cigarette smoke extract on Sertoli cells, Leydig cells and spermatogenic cells of rat testis were studied by cell culture. The logarithmic growth phase cells were used in the experiment. Three kinds of testicular cells were cultured and divided into three groups according to the different concentration of CSE intervention: 0% CSE group, 5% CSE group and 10% CSE group. The effects of CSE on cell proliferation, apoptosis and extracellular lactate dehydrogenase were measured by flow cytometry and biochemical methods. Results: 1) The body weight of smoking rats was significantly lower than that of the control group (P < 0.05); 2) The serum testosterone content of smoking rats was significantly lower than that of the control group from the fourth week (P < 0.0). The content of serum T in smoking group decreased significantly with the prolongation of exposure time (P The levels of FSH-1 in serum of rats in smoking group were significantly lower than those in control group (P The levels of gonadotropin-releasing hormone in the hypothalamus of smoking group were significantly lower than those of control group at the second, sixth and eighth weeks (P In the eighth week group, the testicular tissue atrophy, a large number of cell lysis, seminiferous tubules and interstitial tissue degeneration were observed; 8) TUNEL method was used to determine the apoptosis rate of testicular cells, and the apoptosis rate was clear in the fourth, sixth and eighth weeks of smoking group. 9) Fas/FasL and Bax protein were up-regulated and Bcl-2 protein was down-regulated in the testicular tissues of smoking rats, and Fas/FasL and Bax gene mRNA expression levels were up-regulated with the same results as protein expression. 10) There was no significant difference in the effect of cigarette smoke extract on Sertoli cell proliferation (P > 0.05); the proliferation of spermatogenic cells in Leydig cells decreased significantly with the increase of concentration (P < 0.05), and there was a dose-response relationship; the LDH activity of testicular Sertoli cells, Leydig cells and spermatogenic cells in cigarette smoke extract group was significantly decreased (P < 0.05). The results of flow cytometry showed that CSE induced apoptosis of Sertoli cells, interstitial cells and spermatogenic cells. CONCLUSION: Smoking is a harmful factor for the development of reproductive system in adolescent rats. It not only affects the important hormones in hypothalamus-pituitary-testicular axis, but also affects the testicular tissue and fineness. Cigarette smoke extracts contain chemicals that induce apoptosis of Sertoli cells, interstitial cells and spermatogenic cells, suggesting that smoking may lead to puberty in rats. The development of reproductive function is slow, the number and function of spermatozoa are reduced.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R114

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相關(guān)期刊論文 前2條

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