【摘要】：為了研究黃芪提取液對大鼠睪丸間質(zhì)細(xì)胞增殖及睪酮分泌的影響,并研究黃芪提取液對大鼠睪丸間質(zhì)細(xì)胞總SOD及GSH-Px活性的影響,本試驗(yàn)用7-9周齡的雄性大鼠做試驗(yàn)對象,通過體外分離、純化,并用3p-HSD染色法鑒定大鼠睪丸間質(zhì)細(xì)胞。體外培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞,向培養(yǎng)液中添加不用濃度的黃芪提取液,用CCK-8及Elisa檢測細(xì)胞增殖及睪酮分泌情況并檢測不同濃度黃芪提取液處理后睪丸間質(zhì)細(xì)胞總SOD和GSH-Px的活性。通過熒光定量PCR技術(shù)檢測睪丸間質(zhì)細(xì)胞中抑癌基因P53及凋亡基因Bax和Bcl-2的相對表達(dá)量。其試驗(yàn)結(jié)果及分析如下：(1)經(jīng)鑒定,分離純化培養(yǎng)的大鼠睪丸間質(zhì)細(xì)胞純度在95%以上。(2)黃芪能促進(jìn)大鼠睪丸間質(zhì)細(xì)胞增殖及睪酮分泌,并有一定的抗氧化作用。經(jīng)CCK-8, Elisa及酶活性試驗(yàn)檢測,添加黃芪提取液的試驗(yàn)組與對照組相比,吸光度逐漸升高,即間質(zhì)細(xì)胞增殖呈上升趨勢,數(shù)目逐漸增多；間質(zhì)細(xì)胞分泌睪酮的濃度及SOD和GSH-PX的活性先升高后降低,黃芪提取液濃度為100μg/mL,150 μg/mL的試驗(yàn)組與對照組相比,細(xì)胞數(shù)目顯著增多,后者差異極顯著(P0.01)。其中濃度為20μg/mL, 50 μg/mL,100 μg/mL的試驗(yàn)組與對照組相比,睪酮濃度顯著升高,差異極顯著(P0.01)。黃芪提取液添加濃度為20μg/mL,50μg/mL的試驗(yàn)組,間質(zhì)細(xì)胞總SOD和GSH-Px活性顯著高于對照組(P0.01)。(3)黃芪具有抑制大鼠睪丸間質(zhì)細(xì)胞凋亡的作用。經(jīng)熒光定量試驗(yàn)檢測,添加20μg/mL黃芪提取液的試驗(yàn)組間質(zhì)細(xì)胞Bax, Bcl-2基因的表達(dá)量降低,且Bax基因的表達(dá)量與對照組相比顯著降低(P0.01)。試驗(yàn)組與對照組相比,P53基因的表達(dá)量差異不顯著(P0.05)。與對照組相比,試驗(yàn)組間質(zhì)細(xì)胞,基因Bcl-2和Bax表達(dá)量的比值顯著升高(P0.01)。本研究在動物繁殖學(xué)領(lǐng)域,為延緩體外培養(yǎng)的睪丸間質(zhì)細(xì)胞衰老,延長其生物學(xué)功能等課題研究提供了理論依據(jù)和實(shí)踐基礎(chǔ)。
[Abstract]:In order to study the effect of astragalus membranaceus extract on the proliferation and testosterone secretion of rat Leydig cells, and the effect of astragalus membranaceus extract on the activities of total SOD and GSH-Px in rat Leydig cells, In this experiment, male rats aged 7 to 9 weeks were used as subjects. The Leydig cells of rat testes were isolated and purified in vitro, and the Leydig cells of rat testes were identified by 3p-HSD staining. Rat Leydig cells were cultured in vitro, and astragalus membranaceus extract was added to the culture medium. The proliferation and testosterone secretion of testicular Leydig cells were detected by CCK-8 and Elisa, and the activities of total SOD and GSH-Px in Leydig cells treated with different concentrations of Astragalus membranaceus extract were detected. The relative expression of tumor inhibitor p53 and apoptosis genes Bax and Bcl-2 in testicular Leydig cells was detected by fluorescence quantitative PCR. The results and analysis are as follows: (1) the purity of rat Leydig cells isolated and purified is over 95%. (2) Astragalus membranaceus can promote the proliferation and testosterone secretion of rat Leydig cells, and has certain antioxidant effect. Compared with the control group, the absorbance of the experimental group with astragalus membranaceus extract increased gradually by CCK-8, Elisa and enzyme activity test, that is to say, the proliferation of Leydig cells increased and the number of Leydig cells increased gradually. The concentration of testosterone secreted by Leydig cells and the activities of SOD and GSH-PX increased at first and then decreased. Compared with the control group, the number of cells in the experimental group with 100 渭 g / mL,150 渭 g/mL of Astragalus membranaceus extract was significantly higher than that in the control group (P 0.01). The concentration of testosterone in the experimental group was 20 渭 g / mL, 50 渭 g / mL, and the testosterone concentration in the experimental group was significantly higher than that in the control group (P 0.01). The total SOD and GSH-Px activities of Leydig cells in the experimental group with 20 渭 g / mL, 50 渭 g / mL were significantly higher than those in the control group (P01). (3). Astragalus membranaceus could inhibit the apoptosis of Leydig cells in rats. Fluorescence quantitative test showed that the expression of Bax, Bcl-2 gene in stroma cells of the experimental group supplemented with 20 渭 g / mL astragalus extract was decreased, and the expression of Bax gene was significantly lower than that of the control group (P 0.01). Compared with the control group, there was no significant difference in the expression of p53 gene between the experimental group and the control group (P 0.05). Compared with the control group, the ratio of gene Bcl-2 and Bax expression in the experimental group was significantly higher than that in the control group (P 0.01). In the field of animal reproduction, this study provides a theoretical and practical basis for delaying the senescence of testicular Leydig cells and prolonging their biological function in vitro.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7

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