本文選題：丙烯酰胺 + 小鼠睪丸��； 參考：《山西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：實(shí)驗(yàn)?zāi)康慕A亞慢性染毒小鼠模型,研究AA對小鼠睪丸組織的生殖毒性作用,以及AA染毒后細(xì)胞骨架CK18與細(xì)胞凋亡相關(guān)因子Fas、FasL和Caspase-3表達(dá)的變化及其相互關(guān)系,進(jìn)而為研究生殖細(xì)胞凋亡通路及分子調(diào)控機(jī)制,以及探討AA的生殖毒性機(jī)制提供實(shí)驗(yàn)依據(jù)。 實(shí)驗(yàn)內(nèi)容與方法 將健康5-6周齡清潔級雄性昆明種小鼠40只,隨機(jī)分為4組,每組10只。用不同劑量的AA(0、10、20、40mg/kg)對小鼠連續(xù)腹腔染毒5周,每周6天,其中對照組給予等體積蒸餾水。每日測量體重,于首次染毒AA后第36天處死小鼠,計(jì)算睪丸系數(shù)。采用組織病理學(xué)技術(shù)(HE染色),RT-PCR技術(shù),免疫組織化學(xué)法,分別從組織學(xué)水平,mRNA水平,蛋白水平,分析AA對小鼠睪丸組織的生殖毒性作用、CK18與細(xì)胞凋亡相關(guān)因子Fas、FasL和Caspase-3表達(dá)的變化及其相互關(guān)系。 實(shí)驗(yàn)結(jié)果 1、AA染毒期間,小鼠體重、睪丸重量及其睪丸系數(shù)均呈下降趨勢,與對照組相比有統(tǒng)計(jì)學(xué)差異(P0.01)。 2、HE染色結(jié)果顯示,10mg/kg組與正常對照組比較,小鼠睪丸曲細(xì)精管排列基本規(guī)則,各級生精細(xì)胞未見明顯改變。20mg/kg和40mg/kg劑量組小鼠,曲細(xì)精管排列不規(guī)則,生精上皮層次減少,各級生精細(xì)胞減少,管腔內(nèi)成熟精子減少。 3、RT-PCR及免疫組化結(jié)果顯示,CK18mRNA及蛋白在正常小鼠睪丸組織有表達(dá)；20mg/kg和40mg/kg劑量組與正常對照組比較,CK18mRNA及蛋白表達(dá)均明顯降低(P0.01)。 4、小鼠睪丸組織Fas、FasL、Caspase-3的表達(dá),對照組生精小管中有散在分布的個(gè)別陽性細(xì)胞,10mg/kg組陽性細(xì)胞不明顯；20mg/kg組和40mg/kg組陽性細(xì)胞增多,IOD值較正常對照組均明顯增多,且有統(tǒng)計(jì)學(xué)差異(P0.01)。 結(jié)論 1、AA亞慢性染毒可致小鼠體重增長緩慢,睪丸系數(shù)降低。 2、AA亞慢性染毒可使小鼠睪丸結(jié)構(gòu)發(fā)生病理性改變,成熟精子減少,生精功能降低。 3、AA致小鼠睪丸組織CK18mRNA及蛋白的表達(dá)顯著減少。推測AA生殖毒性作用 的可能機(jī)制為AA染毒使CK18mRNA表達(dá)減少,從而導(dǎo)致CK18生成減少。 4、AA可誘導(dǎo)小鼠睪丸組織細(xì)胞凋亡相關(guān)因子Fas、FasL與Caspase-3表達(dá)增加,從而激活Fas/FasL凋亡通路。表明在AA生殖毒性中存在著Caspase-3的依賴性凋亡機(jī)制。 5、CK18mRNA和蛋白的表達(dá)與Fas、FasL、Caspase-3的表達(dá)均呈負(fù)相關(guān),表明AA誘導(dǎo)睪丸Fas、FasL、Caspase-3表達(dá)增加與CK18表達(dá)減少有一定的相關(guān)性。
[Abstract]:Objective to study the reproductive toxicity of AA to testis and the relationship between the expression of cytoskeleton CK18 and apoptosis-related factors FasL and Caspase-3. It provides experimental basis for studying germ cell apoptosis pathway, molecular regulation mechanism and the mechanism of reproductive toxicity of AA. Experimental contents and methods Forty male Kunming mice of 5-6 weeks old were randomly divided into 4 groups with 10 mice in each group. The mice were exposed to different doses of AAAZO 1010 ~ 20g / kg for 5 weeks, 6 days a week, and the control group was given distilled water of the same volume. The mice were killed on the 36th day after the first AA exposure and the testicular coefficient was calculated. By using histopathological technique, HE staining and RT-PCR, immunohistochemical method, mRNA and protein levels were determined, respectively. To analyze the reproductive toxicity of AA on testis of mice and the relationship between CK18 and the expression of FasL and Caspase-3 in apoptosis-related cytokines. Experimental results (1) the weight, testis weight and testicular coefficient of the mice decreased during the exposure to AA, and there was a significant difference compared with the control group (P 0.01). 2the results of HE staining showed that the basic rules of seminiferous tubules arrangement were observed in the 10 mg / kg group compared with the normal control group. The seminiferous cells of all levels of spermatogenic cells did not change significantly in the groups of .20mg / kg and 40mg/kg dosage. The seminiferous tubules arranged irregularly and the layers of seminiferous epithelium decreased. The number of spermatogenic cells and mature spermatozoa in the lumen decreased. 3The results of RT-PCR and immunohistochemistry showed that the expression of CK18 mRNA and protein in testis of normal mice was 20 mg / kg and the expression of CK18 mRNA and protein in 40mg/kg group was significantly lower than that in normal control group (P 0.01). (4) the expression of Fas-FasL- Caspase-3 in testicular tissues of mice, the positive cells in the seminiferous tubules of the control group were not significantly increased in 10 mg / kg group and 20 mg / kg group and 40mg/kg group were significantly higher than those in the normal control group, and there was a statistical difference between them (P0.01). Conclusion 1the weight gain and testis coefficient decreased after subchronic exposure to AA. 2Subchronic exposure to AA could induce pathological changes of testis structure, decrease of mature spermatozoa and decrease of spermatogenic function in mice. 3The expression of CK18mRNA and protein in testis of mice induced by AA was significantly decreased. Hypothetical reproductive toxicity of AA The possible mechanism is that AA exposure reduces the expression of CK18mRNA, resulting in reduced CK18 production. 4the expression of Fas-FasL and Caspase-3 in mouse testicular tissue was increased by AA-induced apoptosis, thus activating the apoptosis pathway of Fas/FasL. It is suggested that there is a mechanism of Caspase-3 dependent apoptosis in AA reproductive toxicity. 5The expression of CK18 mRNA and protein was negatively correlated with the expression of Fas-FasL- Caspase-3, which indicated that the increased expression of Caspase-3 in Fas-FasLL was correlated with the decrease of CK18 expression in the testis induced by AA.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

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