吸烟对雄性大鼠生殖毒性的实验研究
发布时间：2018-08-29 18:03
【摘要】：目的：研究吸烟对大鼠下丘脑-垂体-睾丸轴相关激素的影响及对睾丸细胞的毒性作用，通过对相关凋亡基因研究分析其可能的机制；研究香烟烟雾提取物对睾丸细胞的细胞毒性，分析其可能机制。方法：体内实验方法，选用5周SD大鼠80只，分为对照组与吸烟组，吸烟组采用静式染毒，每天1次，每次2h，连续8周；对照组采用空白对照。实验第2周、第4周、第6周与第8周分别处死对照组与吸烟组大鼠10只，测定各种指标。放射免疫法测定血清中睾酮、黄体生成激素、卵泡雌激素、胰岛素样生长因子-1及下丘脑促性腺激素释放激素；HE染色观察睾丸的结构形态；TUNEL法定性、定量测定睾丸的细胞凋亡；Western Blot检测各组大鼠睾丸组织中Fas/FasL与Bcl-2/Bax蛋白表达；RT-PCR检测各组大鼠睾丸组织中Fas/FasL与Bcl-2/BaxmRNA表达。用细胞培养研究香烟烟雾提取物对大鼠睾丸的支持细胞、间质细胞与生精细胞的影响。取对数生长期细胞用于试验，三种睾丸细胞培养，按照CSE干预浓度不同，将试验分为三组：0%CSE组、5%CSE组、10%CSE组。各组细胞培养12h后，采用MTT法检测CSE对细胞增殖的影响，流式细胞术检测细胞凋亡的情况，生物化学的方法检检测胞外乳酸脱氢酶的改变。结果：1）随着时间延长，吸烟组大鼠体重明显低于对照（P＜0.05）；2）大鼠血清中睾酮含量自第四周起吸烟组明显低于对照组（P＜0.05）；吸烟组大鼠血清T含量随染毒时间延长明显下降（P＜0.05）；3）大鼠血清中黄体生成素含量在对照组有随着时间延长逐渐增高，在吸烟组则随时间下降；第四周起吸烟组大鼠血清中黄体生成素含量明显低于对照组（P＜0.05）；4）大鼠血清中卵泡刺激素在对照组与吸烟组含量稳定，自第二周起吸烟组含量明显低于对照组（P＜0.05）；5）大鼠血清中胰岛素样生长因子-1含量随时间下降，吸烟组大鼠血清中胰岛素样生长因子-1在第八周含量明显低于第二周、第四周与第六周（P＜0.05），吸烟组第二周、第六周和第八周均明显低于对照组（P＜0.05）；6）大鼠下丘脑促性腺激素释放激素无论是对照组还是吸烟组随时间延长而升高，吸烟组与对照组比较差异无统计学意义（P＞0.05）；7）睾丸HE染色发现吸烟可以导致睾丸结构组织的破坏，随着时间的延长，组织改变明显加深。第八周组吸烟组睾丸组织出现明显的萎缩，大量的细胞裂解，细精管与间质组织出现明显的变性改变；8）TUNEL法测定睾丸细胞的凋亡率，吸烟组第四周、第六周、第八周细胞凋亡率明显增高（P＜0.05）；9）吸烟组大鼠睾丸组织中Fas/FasL与Bax蛋白上调，Bcl-2蛋白下调；Fas/FasL、Bax基因的mRNA表达水平有上调的趋势，结果与蛋白表达相同。吸烟第八周大鼠睾丸的Fas/FasL、Bax基因表达水平明显上升（P＜0.05），Bcl-2mRNA下调明显（P＜0.05）10）香烟烟雾提取物对支持细胞增值的影响没有显著性差异（P＞0.05）；对间质细胞、生精细胞增值随浓度的升高出现明显的下降（P＜0.05），，且呈现一定的剂量反应关系；香烟烟雾提取物组的睾丸支持细胞、间质细胞和生精细胞组胞外LDH活力明显高于对照组（P＜0.05）；11）流式细胞仪测定结果表明CSE导致支持细胞、间质细胞和生精细胞组胞凋亡率上升。结论：吸烟是青春期大鼠生殖系统发育的有害因素，不仅下丘脑-垂体-睾丸轴中重要的激素有影响，而且对睾丸组织与细胞有不利影响，其可能的机制是吸烟激活了细胞凋亡调控基因，导致相应蛋白表达增加，同时抑制了保护性蛋白的表达；香烟烟雾提取物中含有导致睾丸支持细胞、间质细胞和生精细胞凋亡的化学物质，这提示吸烟可能导致青春期大鼠生殖功能发育迟缓，精子数量和功能的减少。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114
本文编号：2211985
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114
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相关期刊论文 前2条
1 徐志强;唐宝松;周德众;;吸烟与不吸烟不育男子精液分析与性激素检测的比较[J];中国性科学;2006年01期
2 尚万兵;张杨;李文奇;樊爱英;;小鼠肢体创伤后血清乳酸脱氢酶活性的变化[J];新乡医学院学报;2011年01期
本文编号：2211985
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