【摘要】：目的探討不同濃度2,3,7,8-四氯二苯并二VA英(2,3,7,8-Tetrachlorodibenzo-p-dioxin,TCDD)對雄性SD大鼠睪丸間質(zhì)細(xì)胞(Leydig cell)凋亡的影響及其作用機(jī)制。從而探討TCDD生殖毒性的可能機(jī)制,為預(yù)防相關(guān)性疾病提供理論依據(jù)。方法取8-10周齡的性成熟期SPF級雄性SD大鼠,在屏障環(huán)境下飼養(yǎng)2周后,進(jìn)行了睪丸間質(zhì)細(xì)胞分離培養(yǎng)以及細(xì)胞鑒定。通過建立的睪丸間質(zhì)細(xì)胞體外培養(yǎng)模型,利用生長狀態(tài)較好的第三代leydig細(xì)胞進(jìn)行體外實(shí)驗。根據(jù)TCDD作用濃度,本實(shí)驗共分為5組:0 n M組(即對照組)、1 n M組、5 n M組、10 n M組以及50 n M組。采用噻唑藍(lán)(methyl thiazolyl tetrazolium,MTT)法觀察不同濃度TCDD組別處理大鼠睪丸間質(zhì)細(xì)胞24h對細(xì)胞增殖的影響;利用熒光探針DCFH-DA對細(xì)胞內(nèi)活性氧(Reactive oxygen species,ROS)水平進(jìn)行檢測;Annexin V-FITC/PI染色法聯(lián)合激光共聚焦顯微鏡觀察細(xì)胞凋亡情況;采用Western blot法檢測細(xì)胞中凋亡相關(guān)蛋白Bax,Bcl-2,Caspase-3表達(dá)水平的變化。運(yùn)用RT-PCR法檢測細(xì)胞中凋亡相關(guān)基因Bax,Bcl-2,Caspase-3 m RNA表達(dá)的變化。結(jié)果通過3β-羥基類固醇脫氫酶(3β-hydroxysteroid dehydrogenase,3β-HSD)結(jié)果顯示,細(xì)胞均被染成藍(lán)黑色,說明該方法分離leydig細(xì)胞純度較高。MTT結(jié)果顯示,隨著TCDD的濃度增加,leydig細(xì)胞吸光度值呈逐漸下降趨勢(P0.05),并呈一定的劑量效應(yīng)關(guān)系。ROS結(jié)果顯示,隨著濃度的增加,細(xì)胞內(nèi)ROS水平呈上升趨勢(P0.05)。顯微鏡下觀察,隨著染毒劑量的增加,細(xì)胞凋亡陽性信號比率也逐漸上升。蛋白印記(Western blot)結(jié)果顯示,與對照組相比,5、10、50 n M染毒組大鼠睪丸間質(zhì)細(xì)胞Bax、Caspase-3蛋白相對表達(dá)量明顯增加,而Bcl-2蛋白水平隨TCDD濃度增加呈降低趨勢,差異具有統(tǒng)計學(xué)意義(P0.05)。RT-PCR結(jié)果與Western blot結(jié)果一致,隨著濃度的增加Bax、Caspase-3 m RNA水平呈逐漸上升趨勢,而Bcl-2水平隨TCDD濃度的增加呈下降趨勢,差異均具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論TCDD能抑制大鼠睪丸間質(zhì)細(xì)胞的增殖而誘導(dǎo)其凋亡,并增加細(xì)胞內(nèi)ROS含量。其誘導(dǎo)凋亡調(diào)節(jié)機(jī)制可能是通過上調(diào)促凋亡蛋白Bax表達(dá),下調(diào)抑凋亡蛋白Bcl-2表達(dá),進(jìn)而激活Caspase-3蛋白的表達(dá),導(dǎo)致大鼠睪丸間質(zhì)細(xì)胞凋亡。長時間染毒對凋亡的影響以及是否存在其他凋亡因子的調(diào)節(jié)需進(jìn)一步的研究。
[Abstract]:Objective to investigate the effect of different concentrations of 2,3,7,8-tetrachlorodibenzopdioxin (TCDD) on apoptosis of (Leydig cell) in testicular Leydig cells of male SD rats and its mechanism. In order to explore the possible mechanism of reproductive toxicity of TCDD and provide theoretical basis for the prevention of related diseases. Methods SPF male SD rats aged 8 脳 10 weeks were cultured in barrier environment for 2 weeks, and the Leydig cells were isolated and identified. Based on the culture model of testicular Leydig cells in vitro, the third generation leydig cells with good growth state were used to carry out the experiment in vitro. According to the concentration of TCDD, the experiment was divided into 5 groups: 0 NM group (control group), 1 NM group, 5 NM group, 10 NM group and 50 NM group. The effects of different concentrations of TCDD on the proliferation of rat Leydig cells were observed by thiazolyl blue (methyl thiazolyl tetrazolium,MTT assay, and the intracellular reactive oxygen species (Reactive oxygen species,ROS) levels were detected by fluorescence probe DCFH-DA. Apoptosis was observed by Annexin V-FITC/PI staining combined with laser confocal microscope, and the expression of apoptosis-related protein Bax,Bcl-2,Caspase-3 was detected by Western blot assay. The expression of apoptosis-related gene Bax,Bcl-2,Caspase-3 m RNA in cells was detected by RT-PCR assay. Results the results of 3 尾-hydroxysteroid dehydrogenase (3 尾-hydroxysteroid dehydrogenase,3 尾-HSD) showed that the cells were stained blue and black, indicating that the purity of leydig cells isolated by this method was high. The absorbance of leydig cells decreased gradually (P 0.05), and showed a dose-effect relationship. Ros results showed that with the increase of concentration, the intracellular ROS level showed an upward trend (P 0.05). Under microscope, with the increase of exposure dose, the positive signal ratio of apoptosis increased gradually. The results of protein imprinting (Western blot) showed that compared with the control group, the relative expression of Bax,Caspase-3 protein in testicular Leydig cells of rats exposed to 5, 10 and 50 NM was significantly increased, while the level of Bcl-2 protein decreased with the increase of TCDD concentration. The difference was statistically significant (P 0.05). The results of RT-PCR were consistent with those of Western blot. With the increase of concentration, the level of Bax,Caspase-3 m RNA increased gradually, while the level of Bcl-2 decreased with the increase of TCDD concentration. The difference was statistically significant (P 0.05). Conclusion TCDD can inhibit the proliferation of rat Leydig cells and induce apoptosis, and increase the intracellular ROS content. The regulatory mechanism of apoptosis induced by apoptosis may be to up-regulate the expression of apoptosis-promoting protein Bax, down-regulate the expression of apoptosis-inhibiting protein Bcl-2, and then activate the expression of Caspase-3 protein, which may lead to apoptosis of rat Leydig cells. The effect of long-term exposure on apoptosis and the regulation of other apoptosis factors need to be further studied.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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