TCDD对大鼠睾丸间质细胞凋亡的影响及其机制

发布时间：2019-05-30 00:58

【摘要】：目的探讨不同浓度2,3,7,8-四氯二苯并二VA英(2,3,7,8-Tetrachlorodibenzo-p-dioxin,TCDD)对雄性SD大鼠睾丸间质细胞(Leydig cell)凋亡的影响及其作用机制。从而探讨TCDD生殖毒性的可能机制,为预防相关性疾病提供理论依据。方法取8-10周龄的性成熟期SPF级雄性SD大鼠,在屏障环境下饲养2周后,进行了睾丸间质细胞分离培养以及细胞鉴定。通过建立的睾丸间质细胞体外培养模型,利用生长状态较好的第三代leydig细胞进行体外实验。根据TCDD作用浓度,本实验共分为5组:0 n M组(即对照组)、1 n M组、5 n M组、10 n M组以及50 n M组。采用噻唑蓝(methyl thiazolyl tetrazolium,MTT)法观察不同浓度TCDD组别处理大鼠睾丸间质细胞24h对细胞增殖的影响;利用荧光探针DCFH-DA对细胞内活性氧(Reactive oxygen species,ROS)水平进行检测;Annexin V-FITC/PI染色法联合激光共聚焦显微镜观察细胞凋亡情况;采用Western blot法检测细胞中凋亡相关蛋白Bax,Bcl-2,Caspase-3表达水平的变化。运用RT-PCR法检测细胞中凋亡相关基因Bax,Bcl-2,Caspase-3 m RNA表达的变化。结果通过3β-羟基类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)结果显示,细胞均被染成蓝黑色,说明该方法分离leydig细胞纯度较高。MTT结果显示,随着TCDD的浓度增加,leydig细胞吸光度值呈逐渐下降趋势(P0.05),并呈一定的剂量效应关系。ROS结果显示,随着浓度的增加,细胞内ROS水平呈上升趋势(P0.05)。显微镜下观察,随着染毒剂量的增加,细胞凋亡阳性信号比率也逐渐上升。蛋白印记(Western blot)结果显示,与对照组相比,5、10、50 n M染毒组大鼠睾丸间质细胞Bax、Caspase-3蛋白相对表达量明显增加,而Bcl-2蛋白水平随TCDD浓度增加呈降低趋势,差异具有统计学意义(P0.05)。RT-PCR结果与Western blot结果一致,随着浓度的增加Bax、Caspase-3 m RNA水平呈逐渐上升趋势,而Bcl-2水平随TCDD浓度的增加呈下降趋势,差异均具有统计学意义(P0.05)。结论TCDD能抑制大鼠睾丸间质细胞的增殖而诱导其凋亡,并增加细胞内ROS含量。其诱导凋亡调节机制可能是通过上调促凋亡蛋白Bax表达,下调抑凋亡蛋白Bcl-2表达,进而激活Caspase-3蛋白的表达,导致大鼠睾丸间质细胞凋亡。长时间染毒对凋亡的影响以及是否存在其他凋亡因子的调节需进一步的研究。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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