硫酸镍致原代培养大鼠睾丸间质细胞损伤的实验研究

发布时间：2018-06-19 06:55

本文选题：硫酸镍 + 睾丸间质细胞　；参考：《兰州大学》2012年硕士论文

【摘要】：目的建立体外分离、纯化和培养大鼠睾丸间质细胞的方法,探讨不同浓度、不同时间硫酸镍对大鼠睾丸间质细胞的毒作用及其机制。方法(1)采用胶原酶消化、Percoll分离液密度梯度离心和差速贴壁法体外分离、纯化和培养大鼠睾丸间质细胞,并用3β-HSD染色法进行纯度鉴定。(2)取对数生长期睾丸间质细胞,硫酸镍(NiSO4)O、62.5、125、25O、500和1000μmol/L分别处理细胞3、6、12、24、36和48h；采用MTT比色法和乳酸脱氢酶(LDH)法观察硫酸镍对睾丸间质细胞的毒作用。(3)取对数生长期睾丸间质细胞,硫酸镍(NiSO4)0、250、500和1000μmol/L分别处理细胞6、12和24h; Annexin V-FITC/PI流式细胞术检测睾丸间质细胞的凋亡情况；实时荧光定量PCR和Western blot技术检测凋亡调控基因c-kit、caspase-3mRNA及蛋白的表达水平。结果(1)体外分离、纯化的睾丸间质细胞经3p-HSD特异性染色鉴定,纯度可达到95%以上。(2)相同时间NiSO4各浓度组与对照组比较：处理12h后,NiSO4各浓度组均出现细胞增殖抑制(P0.01)；处理6h后,NiSO4各浓度组培养液中LDH活力均开始升高(P0.05或P0.01)。相同浓度各处理时间与对照组(0h)比较：NiSO4250μmol/L及其以上浓度组,各处理时间均可抑制间质细胞增殖(P0.01)；除NiSO462.5μmol/L处理3h组外,其他各浓度不同处理时间LDH活力均明显升高(P0.05或P0.01)。(3) Annexin V-FITC/PI流式细胞术检测结果显示：间质细胞凋亡主要发生于NiSO41000μmol/L处理12h和NiSO4250、500、1000μmol/L处理24h组。(4)实时荧光定量PCR结果显示：与对照组比较,NiSO4250μmol/L处理6h、NiSO4250和500μmo1/L处理12h后c-kit mRNA相对表达量上调(P0.05); NiSO4500和1000μmol/L处理24h后c-kitmRNA相对表达量下降(P0.05)。 NiSO4各浓度和各时间组caspase-3mRNA相对表达量均上调(P0.05)。(5) Western blot结果显示：与对照组比较,NiSO41000μmol/L处理6h、NiSO4各浓度分别处理12h和24h, c-kil蛋白表达呈下调趋势,而NiSO4各浓度不同处理时间caspase-3蛋白表达均呈上调趋势。结论建立了较理想的体外分离、纯化和培养大鼠睾丸间质细胞的方法；硫酸镍对原代培养的睾丸间质细胞具有明显的毒作用,并呈现一定的剂量-效应和时间-效应关系；硫酸镍可诱导原代培养大鼠睾丸间质细胞凋亡,且与凋亡调控基因c-kit、caspase-3mRNA和蛋白异常表达有关。
[Abstract]:Objective to establish a method for isolation, purification and culture of rat testicular stromal cells in vitro, and to investigate the toxic effect of nickel sulfate on rat testis stromal cells at different concentrations and at different times and its mechanism. Methods 1) Leydig cells of rat testis were purified and cultured by density gradient centrifugation with collagenase digesting Percoll isolate and differential adherent method in vitro, and purified by 3 尾 -HSD staining. The cytotoxic effect of nickel sulfate on stromal cells of testis was observed by MTT colorimetric assay and lactate dehydrogenase (LDH) method, and the cytotoxicity of nickel sulfate to the stromal cells of testis was observed by MTT colorimetry and lactate dehydrogenase (LDH) method, respectively, and 1000 渭 mol / L was used to determine the cytotoxicity of nickel sulfate to the stromal cells of testis in the logarithmic phase of testicular mesenchymal cells. The apoptosis of Leydig cells was detected by Annexin V-FITC / Pi flow cytometry, and the expression of c-kitc caspase-3 mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blot. Results 1) the purified Leydig cells were identified by 3p-HSD specific staining. The purity of the purified stromal cells was over 95%. Compared with the control group at the same time: after 12 hours of treatment, the proliferation inhibition (P0.01A) was found in all the NiSO4 concentration groups. After 6 hours of treatment, LDH activity in culture medium of each concentration of NiSO4 began to increase (P0.05 or P0.01). Compared with the control group for 0 h at the same concentration, the proliferation of stromal cells was inhibited in the concentration of 1: NiSO4 250 渭 mol / L and above, except for 3 h after treatment with NiSO462.5 渭 mol / L. The results of Annexin V-FITC / Pi flow cytometry showed that the apoptosis of mesenchymal cells mainly occurred in NiSO41000 渭 mol / L for 12h and NiSO4250,500,500,500 渭 mol / L for 24h. The results of real-time fluorescence quantitative PCR showed that: Compared with the control group, the relative expression of c-kit mRNA was up-regulated by NiSO4 250 渭 mol / L and 500 渭 mol / L for 12 h, and the relative expression of c-kit mRNA was decreased after 24 h treatment with NiSO4 500 and 1000 渭 mol / L respectively. The expression of caspase-3 mRNA was up-regulated in each concentration of NiSO4 and the relative expression of caspase-3 mRNA in each time group. The results of Western blot showed that compared with the control group, the expression of c-kil protein was down-regulated at 12h and 24h after treatment with NiSO41000 渭 mol / L for 6 h and 24 h, respectively. However, the expression of caspase-3 protein was up-regulated at different concentrations of NiSO4. Conclusion the method of isolation, purification and culture of rat testicular stromal cells in vitro was established, and nickel sulfate had obvious toxic effect on primary cultured testicular stromal cells, and showed a dose-effect and time-effect relationship. Nickel sulfate could induce apoptosis of primary cultured rat testicular interstitial cells, which was related to the abnormal expression of c-kita-caspase-3 mRNA and protein.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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