本文選題：何首烏飲 + 衰老��； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：目的:探討何首烏飲對衰老大鼠生精細(xì)胞胰島素信號通路IRS1、IGF-1、IGFBP3基因表達(dá)的影響。方法:一、體內(nèi)實(shí)驗(yàn)1.實(shí)驗(yàn)分組及處理選用30只清潔級12月齡Wistar雄性大鼠隨機(jī)分為:青年對照組(YCG),自然衰老組(NAG),何首烏飲組(SWYG),每組各10只。青年對照組適應(yīng)性飼養(yǎng)一周后取材;自然衰老組在飼養(yǎng)到16月齡后灌胃等量蒸餾水60 d;何首烏飲組在飼養(yǎng)到16月齡后,灌胃何首烏飲(4.8 g/100 g)60 d;自然衰老組和何首烏飲組均于18月齡時(shí)取材,備用。2.觀察睪丸組織IRS1、IGF-1、IGFBP3基因表達(dá)采用免疫熒光染色觀察IRS1、IGF-1、IGFBP3在睪丸組織的表達(dá)位置,Western blot和實(shí)時(shí)熒光定量PCR檢測睪丸組織IRS1、IGF-1、IGFBP3蛋白和mRNA表達(dá)的變化。二、體外實(shí)驗(yàn)1.細(xì)胞的分離、純化、培養(yǎng)和鑒定組合酶法分離細(xì)胞,Percoll梯度密度離心和差異貼壁法純化細(xì)胞,利用蘇丹Ⅳ染液鑒定支持細(xì)胞并計(jì)算支持細(xì)胞的純度;分別利用堿性磷酸酶染色、孚耳根染色、HE染色鑒定生精細(xì)胞。2.衰老模型的建立將培養(yǎng)至7 d的生精細(xì)胞,加入終濃度為50μmol/L的H2O2和100μmol/L FeSO4,連續(xù)干預(yù)8 h,換液,繼續(xù)培養(yǎng)72 h;采用β-半乳糖甘酶特異性染色試劑盒檢測實(shí)驗(yàn)各組中生精細(xì)胞β-半乳糖甘酶的表達(dá)情況,以判斷衰老模型是否建立成功。3.實(shí)驗(yàn)分組及處理根據(jù)實(shí)驗(yàn)?zāi)康姆譃?正常對照組(NCG),將培養(yǎng)至7 d的生精細(xì)胞,繼續(xù)培養(yǎng)80 h;衰老模型組(AMG),將培養(yǎng)至7 d的生精細(xì)胞,加入終濃度為50μmol/L的H2O2和100μmol/L的FeSO4,連續(xù)干預(yù)8 h,換液,繼續(xù)培養(yǎng)72 h;何首烏飲組(SWYG),將培養(yǎng)至7 d的生精細(xì)胞,加入終濃度分別為50μmol/L的H2O2和100μmol/L FeSO4,連續(xù)干預(yù)8 h,換液,加入終濃度為10%的何首烏飲含藥血清,繼續(xù)培養(yǎng)72h。4.采用westernblot和實(shí)時(shí)熒光定量pcr檢測基因irs1、igf-1、igfbp3在生精細(xì)胞的蛋白表達(dá)變化以及其mrna的相對表達(dá)水平。結(jié)果:一、體內(nèi)實(shí)驗(yàn)結(jié)果1.免疫熒光檢測結(jié)果irs1陽性產(chǎn)物發(fā)紅色熒光,定位在細(xì)胞膜,細(xì)胞核采用dapi染色呈藍(lán)色,irs1陽性產(chǎn)物主要表達(dá)在精母細(xì)胞、支持細(xì)胞和肌樣細(xì)胞。與青年對照組相比,自然衰老組irs1陽性率明顯降低(p0.01),何首烏飲干預(yù)后,與自然衰老組相比,何首烏飲用藥組irs1陽性率明顯升高(p0.01)。igf-1陽性產(chǎn)物發(fā)綠色熒光,定位在細(xì)胞質(zhì),細(xì)胞核采用dapi染色呈藍(lán)色,igf-1陽性產(chǎn)物主要表達(dá)在精子細(xì)胞。與青年對照組相比,自然衰老組igf-1陽性率明顯降低(p0.01),何首烏飲干預(yù)后,與自然衰老組相比,何首烏飲用藥組igf-1陽性率明顯升高(p0.01)。igfbp3陽性產(chǎn)物發(fā)綠色熒光,定位在細(xì)胞質(zhì),細(xì)胞核采用dapi染色呈藍(lán)色,igfbp3陽性產(chǎn)物在精原細(xì)胞和少量間質(zhì)細(xì)胞均有表達(dá)。經(jīng)統(tǒng)計(jì)分析,與青年對照組相比,自然衰老組igfbp3陽性率明顯升高(p0.01),何首烏飲干預(yù)后,與自然衰老組相比,何首烏飲用藥組陽性率明顯降低(p0.01)。2.westernblot檢測結(jié)果結(jié)果顯示,與青年對照組相比,自然衰老組igfbp3表達(dá)明顯增加(p0.01),irs1、igf-1表達(dá)明顯減少(p0.01);與自然衰老組相比,何首烏飲用藥組igfbp3表達(dá)量明顯減少(p0.01),irs1、igf-1表達(dá)量明顯增加(p0.01)。3.qrt-pcr檢測結(jié)果結(jié)果顯示,與青年對照組相比,自然衰老組igfbp3mrna表達(dá)明顯上調(diào)(p0.01),irs1、igf-1的表達(dá)明顯下調(diào)(p0.01);與自然衰老組相比,何首烏飲用藥組igfbp3表達(dá)明顯下調(diào)(p0.01),irs1、igf-1的表達(dá)明顯上調(diào)(p0.01)。以上結(jié)果表明,何首烏飲可以通過調(diào)節(jié)胰島素信號通路關(guān)鍵基因irs1、igf-1、igfbp3的表達(dá)延緩大鼠睪丸組織的衰老,為了進(jìn)一步分析何首烏飲對衰老大鼠生精功能的調(diào)控機(jī)制,我們采用支持細(xì)胞和生精細(xì)胞共培養(yǎng)的方法,檢測基因irs1、igf-1、igfbp3在生精細(xì)胞的表達(dá)變化。二、體外實(shí)驗(yàn)結(jié)果:1.蘇丹Ⅳ染色結(jié)果顯示支持細(xì)胞純度可達(dá)90%以上。2.β-半乳糖甘酶特異性染色結(jié)果β-半乳糖甘酶特異性染色結(jié)果顯示,β-半乳糖苷酶陽性產(chǎn)物定位在生精細(xì)胞質(zhì)中。衰老模型組β-半乳糖苷酶陽性率明顯高于正常對照組(P0.01);何首烏飲含藥血清干預(yù)后,與衰老模型組相比,何首烏飲組陽性率明顯降低(P0.01)。3.Western blot檢測結(jié)果Western blot檢測生精細(xì)胞IRS1、IGF-1、IGFBP3蛋白表達(dá)結(jié)果顯示,與正常對照組相比,衰老模型組IRS1、IGF-1表達(dá)下調(diào),IGFBP3表達(dá)上調(diào)(P0.01),何首烏飲含藥血清干預(yù)后,與衰老模型組相比,何首烏飲組IRS1、IGF-1表達(dá)上調(diào),IGFBP3表達(dá)下調(diào)(P0.01)。4.qRT-PCR檢測結(jié)果qRT-PCR檢測生精細(xì)胞IRS1、IGF-1、IGFBP3在mRNA水平的改變,與正常對照組相比,衰老模型組IRS1、IGF-1的表達(dá)明顯下調(diào)(P0.01),IGFBP3的表達(dá)上調(diào)(P0.01);何首烏飲含藥血清干預(yù)以后,與衰老模型組相比較,IRS1、IGF-1的表達(dá)明顯上調(diào)(P0.01),IGFBP3的表達(dá)有所下調(diào)(P0.01)。結(jié)論:1.何首烏飲可以通過提高衰老大鼠睪丸組織胰島素通路關(guān)鍵基因IRS1、IGF-1的表達(dá),抑制IGFBP3的表達(dá),延緩大鼠睪丸組織的衰老。2.何首烏飲能夠降低衰老標(biāo)志物β-半乳糖苷酶在生精細(xì)胞的表達(dá)。3.何首烏飲可以通過促進(jìn)生精細(xì)胞胰島素通路關(guān)鍵基因IRS1、IGF-1的表達(dá),抑制IGFBP3的表達(dá),延緩大鼠生精細(xì)胞的衰老。綜上所述,何首烏飲通過調(diào)節(jié)胰島素信號通路關(guān)鍵基因IRS1、IGF-1、IGFBP3的表達(dá)延緩大鼠睪丸組織生精細(xì)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.

【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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