何首乌饮对衰老大鼠生精细胞胰岛素通路IRS1、IGF-1、IGFBP3基因表达的影响

发布时间：2018-05-16 16:27

本文选题：何首乌饮 + 衰老　；参考：《河北大学》2017年硕士论文

【摘要】：目的:探讨何首乌饮对衰老大鼠生精细胞胰岛素信号通路IRS1、IGF-1、IGFBP3基因表达的影响。方法:一、体内实验1.实验分组及处理选用30只清洁级12月龄Wistar雄性大鼠随机分为:青年对照组(YCG),自然衰老组(NAG),何首乌饮组(SWYG),每组各10只。青年对照组适应性饲养一周后取材;自然衰老组在饲养到16月龄后灌胃等量蒸馏水60 d;何首乌饮组在饲养到16月龄后,灌胃何首乌饮(4.8 g/100 g)60 d;自然衰老组和何首乌饮组均于18月龄时取材,备用。2.观察睾丸组织IRS1、IGF-1、IGFBP3基因表达采用免疫荧光染色观察IRS1、IGF-1、IGFBP3在睾丸组织的表达位置,Western blot和实时荧光定量PCR检测睾丸组织IRS1、IGF-1、IGFBP3蛋白和mRNA表达的变化。二、体外实验1.细胞的分离、纯化、培养和鉴定组合酶法分离细胞,Percoll梯度密度离心和差异贴壁法纯化细胞,利用苏丹Ⅳ染液鉴定支持细胞并计算支持细胞的纯度;分别利用碱性磷酸酶染色、孚耳根染色、HE染色鉴定生精细胞。2.衰老模型的建立将培养至7 d的生精细胞,加入终浓度为50μmol/L的H2O2和100μmol/L FeSO4,连续干预8 h,换液,继续培养72 h;采用β-半乳糖甘酶特异性染色试剂盒检测实验各组中生精细胞β-半乳糖甘酶的表达情况,以判断衰老模型是否建立成功。3.实验分组及处理根据实验目的分为:正常对照组(NCG),将培养至7 d的生精细胞,继续培养80 h;衰老模型组(AMG),将培养至7 d的生精细胞,加入终浓度为50μmol/L的H2O2和100μmol/L的FeSO4,连续干预8 h,换液,继续培养72 h;何首乌饮组(SWYG),将培养至7 d的生精细胞,加入终浓度分别为50μmol/L的H2O2和100μmol/L FeSO4,连续干预8 h,换液,加入终浓度为10%的何首乌饮含药血清,继续培养72h。4.采用westernblot和实时荧光定量pcr检测基因irs1、igf-1、igfbp3在生精细胞的蛋白表达变化以及其mrna的相对表达水平。结果:一、体内实验结果1.免疫荧光检测结果irs1阳性产物发红色荧光,定位在细胞膜,细胞核采用dapi染色呈蓝色,irs1阳性产物主要表达在精母细胞、支持细胞和肌样细胞。与青年对照组相比,自然衰老组irs1阳性率明显降低(p0.01),何首乌饮干预后,与自然衰老组相比,何首乌饮用药组irs1阳性率明显升高(p0.01)。igf-1阳性产物发绿色荧光,定位在细胞质,细胞核采用dapi染色呈蓝色,igf-1阳性产物主要表达在精子细胞。与青年对照组相比,自然衰老组igf-1阳性率明显降低(p0.01),何首乌饮干预后,与自然衰老组相比,何首乌饮用药组igf-1阳性率明显升高(p0.01)。igfbp3阳性产物发绿色荧光,定位在细胞质,细胞核采用dapi染色呈蓝色,igfbp3阳性产物在精原细胞和少量间质细胞均有表达。经统计分析,与青年对照组相比,自然衰老组igfbp3阳性率明显升高(p0.01),何首乌饮干预后,与自然衰老组相比,何首乌饮用药组阳性率明显降低(p0.01)。2.westernblot检测结果结果显示,与青年对照组相比,自然衰老组igfbp3表达明显增加(p0.01),irs1、igf-1表达明显减少(p0.01);与自然衰老组相比,何首乌饮用药组igfbp3表达量明显减少(p0.01),irs1、igf-1表达量明显增加(p0.01)。3.qrt-pcr检测结果结果显示,与青年对照组相比,自然衰老组igfbp3mrna表达明显上调(p0.01),irs1、igf-1的表达明显下调(p0.01);与自然衰老组相比,何首乌饮用药组igfbp3表达明显下调(p0.01),irs1、igf-1的表达明显上调(p0.01)。以上结果表明,何首乌饮可以通过调节胰岛素信号通路关键基因irs1、igf-1、igfbp3的表达延缓大鼠睾丸组织的衰老,为了进一步分析何首乌饮对衰老大鼠生精功能的调控机制,我们采用支持细胞和生精细胞共培养的方法,检测基因irs1、igf-1、igfbp3在生精细胞的表达变化。二、体外实验结果:1.苏丹Ⅳ染色结果显示支持细胞纯度可达90%以上。2.β-半乳糖甘酶特异性染色结果β-半乳糖甘酶特异性染色结果显示,β-半乳糖苷酶阳性产物定位在生精细胞质中。衰老模型组β-半乳糖苷酶阳性率明显高于正常对照组(P0.01);何首乌饮含药血清干预后,与衰老模型组相比,何首乌饮组阳性率明显降低(P0.01)。3.Western blot检测结果Western blot检测生精细胞IRS1、IGF-1、IGFBP3蛋白表达结果显示,与正常对照组相比,衰老模型组IRS1、IGF-1表达下调,IGFBP3表达上调(P0.01),何首乌饮含药血清干预后,与衰老模型组相比,何首乌饮组IRS1、IGF-1表达上调,IGFBP3表达下调(P0.01)。4.qRT-PCR检测结果qRT-PCR检测生精细胞IRS1、IGF-1、IGFBP3在mRNA水平的改变,与正常对照组相比,衰老模型组IRS1、IGF-1的表达明显下调(P0.01),IGFBP3的表达上调(P0.01);何首乌饮含药血清干预以后,与衰老模型组相比较,IRS1、IGF-1的表达明显上调(P0.01),IGFBP3的表达有所下调(P0.01)。结论:1.何首乌饮可以通过提高衰老大鼠睾丸组织胰岛素通路关键基因IRS1、IGF-1的表达,抑制IGFBP3的表达,延缓大鼠睾丸组织的衰老。2.何首乌饮能够降低衰老标志物β-半乳糖苷酶在生精细胞的表达。3.何首乌饮可以通过促进生精细胞胰岛素通路关键基因IRS1、IGF-1的表达,抑制IGFBP3的表达,延缓大鼠生精细胞的衰老。综上所述,何首乌饮通过调节胰岛素信号通路关键基因IRS1、IGF-1、IGFBP3的表达延缓大鼠睾丸组织生精细胞的衰老。
[Abstract]:Objective: To investigate the effect of Ho Chi Wu Decoction on the expression of insulin signaling pathway IRS1, IGF-1 and IGFBP3 in the spermatogenic cells of aging rats. Methods: 1. In vivo experiment 1. experimental groups and treatment of 30 clean grade 12 month old Wistar male rats were randomly divided into young control group (YCG), natural aging group (NAG), Polygonum multiflorum group (SWYG), 10 rats in each group. The control group was adapted for one week after feeding, and the natural aging group was fed to the same amount of distilled water of 60 d after feeding to 16 month old, and after feeding to 16 month old, the gavage of Polygonum multiflorum (4.8 g/100 g) 60 d, the natural senescence group and the ho Shu Wu drink group were obtained at 18 month old, and the reserve.2. was used to observe the IRS1, IGF-1, IGFBP3 gene expression of the testis tissue. The expression of IRS1, IGF-1, IGFBP3 in the testis tissue was observed by immunofluorescence staining. The changes in the expression of IRS1, IGF-1, IGFBP3 protein and mRNA in testis tissues were detected by Western blot and real-time quantitative PCR. Two, the separation, purification, culture and identification of the 1. cells in vitro were isolated, purified, cultured and identified by combination enzyme method, and Percoll gradient density centrifugation and differential adherence method The purified cells were used to identify the support cells and calculate the purity of the support cells by using Sultan IV dye solution. Using alkaline phosphatase staining, Fu ear staining and HE staining, the.2. senescence model of spermatogenic cells was established to be cultured to 7 d spermatogenic cells, adding H2O2 and 100 mu mol/L FeSO4 with a final concentration of 50 mu mol/L, continuously intervening 8 h, changing liquid and continuing. 72 h was cultured, and beta galactog specific staining kit was used to detect the expression of beta galactog in the spermatogenic cells of the experimental group, so as to determine whether the aging model was established successfully in the.3. experiment group and the control group was divided into normal control group (NCG), the cultured spermatogenic cells cultured to 7 d, and continued to cultivate 80 h; aging model group ( AMG), the spermatogenic cells, which were cultured to 7 d, were added to the H2O2 and FeSO4 of 100 mu mol/L with the final concentration of H2O2 and 100 mu mol/L. The 8 h was continuously intervened, the liquid was changed, and the 72 h continued to be cultivated, and the Polygonum multiflorum group (SWYG) would be cultured to 7 d spermatogenic cells, adding 50 mu mol/L H2O2 and 100 mu respectively. The serum of Uyin, 72h.4., Westernblot and real-time quantitative PCR were used to detect the protein expression of IRS1, IGF-1, IGFBP3 in spermatogenic cells and the relative expression level of mRNA. Results: 1. In vivo 1. immunofluorescence test results showed that IRS1 positive products were red fluorescence, located in cell membrane and nucleus adopted. DAPI staining was blue, IRS1 positive products were mainly expressed in spermatocytes, supporting cells and myolike cells. Compared with the young control group, the positive rate of IRS1 in the natural aging group was significantly lower (P0.01). The IRS1 positive rate of Polygonum multiflorum drinking medicine group was significantly higher than that in the natural aging group (P0.01), the positive rate of.Igf-1 positive products in the Polygonum multiflorum group was significantly higher than that in the natural aging group (P0.01), the positive product of.Igf-1 was green fluorescence. The cytoplasm was located in the cytoplasm, the nucleus was stained blue with DAPI staining, and the positive products of IGF-1 were mainly expressed in the spermatozoa. Compared with the young control group, the positive rate of IGF-1 in the natural aging group was significantly lower (P0.01). The IGF-1 positive rate of the Radix Polygoni Multiflori drinking group was significantly higher than that in the natural aging group (P0.01), the positive rate of the positive product of.Igfbp3 was greenish (P0.01). The color fluorescence was located in the cytoplasm, the nucleus was blue with DAPI staining, and the positive products of IGFBP3 were expressed in the spermatogonia and a few stromal cells. The positive rate of IGFBP3 in the natural aging group was significantly higher than that in the young control group (P0.01). The positive rate of the Polygonum multiflorum drinking group was compared with the natural aging group. The results of significantly reduced (P0.01).2.westernblot detection showed that the expression of IGFBP3 in the natural aging group was significantly increased (P0.01), and the expression of IRS1 and IGF-1 decreased significantly (P0.01), compared with the young control group, and the IGFBP3 expression of Polygonum multiflorum drinking medicine group decreased significantly (P0.01), IRS1, IGF-1 expression was significantly increased (P0.01) inspection compared with the natural senescence group. The results showed that, compared with the young control group, the expression of igfbp3mrna in the natural aging group was significantly up (P0.01), and the expression of IRS1 and IGF-1 was significantly down (P0.01). Compared with the natural aging group, the expression of IGFBP3 in the drinking drug group of Polygonum multiflorum was obviously down regulated (P0.01), IRS1, and IGF-1 increased obviously (P0.01). The above results showed that the Polygonum multiflorum was able to pass through The expression of the key genes of insulin signaling pathway IRS1, IGF-1, and IGFBP3 delayed the aging of rat testicular tissue. In order to further analyze the regulation mechanism of Polygonum multiflorum on the function of spermatogenesis in aging rats, we used the method of co culture of supporting cells and spermatogenic cells to detect the expression changes of gene IRS1, IGF-1, IGFBP3 in spermatogenic cells. Two, In vitro experimental results: 1. Sultan IV staining results showed that the purity of the support cells was more than 90% of.2. beta galactosanase specific staining results of beta galactosanase specific staining results showed that beta galactosidase positive products were located in the cytoplasm of spermatogenic cytoplasm. The positive rate of beta galactosidase in the aging model group was significantly higher than that of the normal control group (P0 .01); compared with the aging model group, the positive rate of the Polygonum multiflorum group was significantly lower than that of the aging model group (P0.01).3.Western blot detection results. The results of IRS1, IGF-1, and IGFBP3 protein expression of Western blot detected by Western blot showed that, compared with the normal control group, IRS1, IGF-1 expression was down and IGFBP3 expression was up-regulated. Compared with the aging model group, the expression of IRS1, IGF-1 expression, IGFBP3 expression down regulation (P0.01).4.qRT-PCR detection results, qRT-PCR detection of IRS1, IGF-1, IGFBP3 at mRNA level, compared with the normal control group, IRS1 in the aging model group, the expression of IGF-1 was obviously down. After the intervention, the expression of IRS1 and IGF-1 was significantly up (P0.01) and the expression of IGFBP3 was down (P0.01). Conclusion: 1. the 1. Polygonum multiflorum decoction can improve the expression of the key gene of insulin in the testicular tissue of aging rats, the expression of IGF-1, inhibit the expression of IGFBP3, and postpone the large amount of expression. The senescence of the rat testicular tissue.2. Ho Chi Wu Yin can reduce the expression of senescence marker beta galactosidase in spermatogenic cells.3. ho Hong Wu drink can promote the expression of IRS1, IGF-1 expression, inhibit the expression of IGFBP3 and delay the senescence of spermatogenic cells in spermatogenic cells. The expression of IRS1, IGF-1 and IGFBP3, a key signaling pathway, delay the aging of rat spermatogenic cells.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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