黄芪提取液对大鼠睾丸间质细胞体外培养的影响
发布时间：2019-05-24 14:33
【摘要】：为了研究黄芪提取液对大鼠睾丸间质细胞增殖及睾酮分泌的影响,并研究黄芪提取液对大鼠睾丸间质细胞总SOD及GSH-Px活性的影响,本试验用7-9周龄的雄性大鼠做试验对象,通过体外分离、纯化,并用3p-HSD染色法鉴定大鼠睾丸间质细胞。体外培养大鼠睾丸间质细胞,向培养液中添加不用浓度的黄芪提取液,用CCK-8及Elisa检测细胞增殖及睾酮分泌情况并检测不同浓度黄芪提取液处理后睾丸间质细胞总SOD和GSH-Px的活性。通过荧光定量PCR技术检测睾丸间质细胞中抑癌基因P53及凋亡基因Bax和Bcl-2的相对表达量。其试验结果及分析如下：(1)经鉴定,分离纯化培养的大鼠睾丸间质细胞纯度在95%以上。(2)黄芪能促进大鼠睾丸间质细胞增殖及睾酮分泌,并有一定的抗氧化作用。经CCK-8, Elisa及酶活性试验检测,添加黄芪提取液的试验组与对照组相比,吸光度逐渐升高,即间质细胞增殖呈上升趋势,数目逐渐增多；间质细胞分泌睾酮的浓度及SOD和GSH-PX的活性先升高后降低,黄芪提取液浓度为100μg/mL,150 μg/mL的试验组与对照组相比,细胞数目显著增多,后者差异极显著(P0.01)。其中浓度为20μg/mL, 50 μg/mL,100 μg/mL的试验组与对照组相比,睾酮浓度显著升高,差异极显著(P0.01)。黄芪提取液添加浓度为20μg/mL,50μg/mL的试验组,间质细胞总SOD和GSH-Px活性显著高于对照组(P0.01)。(3)黄芪具有抑制大鼠睾丸间质细胞凋亡的作用。经荧光定量试验检测,添加20μg/mL黄芪提取液的试验组间质细胞Bax, Bcl-2基因的表达量降低,且Bax基因的表达量与对照组相比显著降低(P0.01)。试验组与对照组相比,P53基因的表达量差异不显著(P0.05)。与对照组相比,试验组间质细胞,基因Bcl-2和Bax表达量的比值显著升高(P0.01)。本研究在动物繁殖学领域,为延缓体外培养的睾丸间质细胞衰老,延长其生物学功能等课题研究提供了理论依据和实践基础。
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
本文编号：2484942
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
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