本文選題：硫酸鎳 + 睪丸間質細胞��； 參考：《蘭州大學》2012年碩士論文

【摘要】：目的建立體外分離、純化和培養(yǎng)大鼠睪丸間質細胞的方法,探討不同濃度、不同時間硫酸鎳對大鼠睪丸間質細胞的毒作用及其機制。 方法(1)采用膠原酶消化、Percoll分離液密度梯度離心和差速貼壁法體外分離、純化和培養(yǎng)大鼠睪丸間質細胞,并用3β-HSD染色法進行純度鑒定。(2)取對數(shù)生長期睪丸間質細胞,硫酸鎳(NiSO4)O、62.5、125、25O、500和1000μmol/L分別處理細胞3、6、12、24、36和48h；采用MTT比色法和乳酸脫氫酶(LDH)法觀察硫酸鎳對睪丸間質細胞的毒作用。(3)取對數(shù)生長期睪丸間質細胞,硫酸鎳(NiSO4)0、250、500和1000μmol/L分別處理細胞6、12和24h; Annexin V-FITC/PI流式細胞術檢測睪丸間質細胞的凋亡情況；實時熒光定量PCR和Western blot技術檢測凋亡調(diào)控基因c-kit、caspase-3mRNA及蛋白的表達水平。 結果(1)體外分離、純化的睪丸間質細胞經(jīng)3p-HSD特異性染色鑒定,純度可達到95%以上。(2)相同時間NiSO4各濃度組與對照組比較：處理12h后,NiSO4各濃度組均出現(xiàn)細胞增殖抑制(P0.01)；處理6h后,NiSO4各濃度組培養(yǎng)液中LDH活力均開始升高(P0.05或P0.01)。相同濃度各處理時間與對照組(0h)比較：NiSO4250μmol/L及其以上濃度組,各處理時間均可抑制間質細胞增殖(P0.01)；除NiSO462.5μmol/L處理3h組外,其他各濃度不同處理時間LDH活力均明顯升高(P0.05或P0.01)。(3) Annexin V-FITC/PI流式細胞術檢測結果顯示：間質細胞凋亡主要發(fā)生于NiSO41000μmol/L處理12h和NiSO4250、500、1000μmol/L處理24h組。(4)實時熒光定量PCR結果顯示：與對照組比較,NiSO4250μmol/L處理6h、NiSO4250和500μmo1/L處理12h后c-kit mRNA相對表達量上調(diào)(P0.05); NiSO4500和1000μmol/L處理24h后c-kitmRNA相對表達量下降(P0.05)。 NiSO4各濃度和各時間組caspase-3mRNA相對表達量均上調(diào)(P0.05)。(5) Western blot結果顯示：與對照組比較,NiSO41000μmol/L處理6h、NiSO4各濃度分別處理12h和24h, c-kil蛋白表達呈下調(diào)趨勢,而NiSO4各濃度不同處理時間caspase-3蛋白表達均呈上調(diào)趨勢。 結論建立了較理想的體外分離、純化和培養(yǎng)大鼠睪丸間質細胞的方法；硫酸鎳對原代培養(yǎng)的睪丸間質細胞具有明顯的毒作用,并呈現(xiàn)一定的劑量-效應和時間-效應關系；硫酸鎳可誘導原代培養(yǎng)大鼠睪丸間質細胞凋亡,且與凋亡調(diào)控基因c-kit、caspase-3mRNA和蛋白異常表達有關。
[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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114

【引證文獻】

相關碩士學位論文 前1條

1 鄭菁;硫酸鎳致體外培養(yǎng)的大鼠睪丸間質細胞凋亡的機制研究[D];蘭州大學;2013年

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本文編號：2039012