發(fā)布時(shí)間：2018-04-08 21:32

  本文選題：芝麻素　切入點(diǎn)：JAK1　出處：《延邊大學(xué)》2017年碩士論文

【摘要】：目的:本文通過研究及比較芝麻素與AG490在動(dòng)脈粥樣硬化形成過程中對(duì)兔主動(dòng)脈內(nèi)皮細(xì)胞JAK1表達(dá)的影響,探討芝麻素在抗動(dòng)脈粥樣硬化機(jī)制中的作用。方法:(1)兔主動(dòng)脈內(nèi)皮細(xì)胞的分離、培養(yǎng)及傳代。用傳代成功后的對(duì)數(shù)生長(zhǎng)期的主動(dòng)脈內(nèi)皮細(xì)胞進(jìn)行實(shí)驗(yàn)。(2)細(xì)胞分組及處理:①正常對(duì)照組:在無菌條件下,將實(shí)驗(yàn)細(xì)胞于無血清的DMEM培養(yǎng)基中培養(yǎng)24-48小時(shí);②ox-LDL組:將實(shí)驗(yàn)細(xì)胞在100mg/L濃度的ox-LDL培養(yǎng)基中培養(yǎng)24小時(shí),然后在無血清的DMEM培養(yǎng)基中培養(yǎng)24小時(shí);③AG490(JAK1抑制劑)組:將實(shí)驗(yàn)細(xì)胞培養(yǎng)于50 μ mol/L的AG490培養(yǎng)基中培養(yǎng)24小時(shí),然后在ox-LDL培養(yǎng)基中培養(yǎng)24小時(shí),最后在無血清的DMEM培養(yǎng)基中培養(yǎng)24小時(shí);④芝麻素組:將實(shí)驗(yàn)細(xì)胞在芝麻素培養(yǎng)基(60μmol/L)中培養(yǎng)24小時(shí),然后在100mg/L的ox-LDL培養(yǎng)基中培養(yǎng)24小時(shí),最后在無血清的DEME培養(yǎng)基中培養(yǎng)24小時(shí)。(3)采用Real-time PCR方法檢測(cè)兔主動(dòng)脈內(nèi)皮細(xì)胞增殖過程中芝麻素、AG490對(duì)JAK1表達(dá)的影響。結(jié)果:正常對(duì)照組:兔主動(dòng)脈內(nèi)皮細(xì)胞為梭形或多角形,胞漿豐富,邊界清楚,可見典型的呈"鋪路石狀"鑲嵌排列的細(xì)胞,單層分布。ox-LDL組:兔主動(dòng)脈內(nèi)皮細(xì)胞圓形細(xì)胞明顯增多,邊界不清,數(shù)目明顯減少,凋亡小體出現(xiàn),細(xì)胞碎片增多,細(xì)胞間隙變寬,偶有脫落。芝麻素組:兔主動(dòng)脈內(nèi)皮細(xì)胞呈多角形或梭形,邊界清楚,排列接近正常對(duì)照組,但細(xì)胞數(shù)少于正常對(duì)照組細(xì)胞且多于ox-LDL組。PCR產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳分析,在250bp處出現(xiàn)一條特異性條帶。成功擴(kuò)增出兔主動(dòng)脈內(nèi)皮細(xì)胞JAK1基因。以稀釋的重組質(zhì)粒作為PCR反應(yīng)的模板,分別以各參數(shù)的上、下游引物為引物,經(jīng)1%瓊脂糖凝膠電泳分析表明,在2945bp處出現(xiàn)一條特異性條帶。成功的擴(kuò)增出兔主動(dòng)脈內(nèi)皮細(xì)胞JAK1基因的克隆質(zhì)粒。ox-LDL組,芝麻素組,AG490組JAK1-mRNA表達(dá)明顯高于正常對(duì)照組,有顯著性差異P0.05;芝麻素組與AG490組JAK1-mRNA的表達(dá)明顯低于ox-LDL組P0.05;芝麻素與AG490比較有顯著性差異0.05。結(jié)論:芝麻素組與AG490組均可抑制JAK1-mRNA表達(dá)。芝麻素組抑制作用大于JAK1抑制劑AG490組,可作為防治心腦血管疾病新的靶點(diǎn)。
[Abstract]:Aim: to study and compare the effects of sesamin and AG490 on the expression of JAK1 in rabbit aortic endothelial cells during atherosclerosis, and to explore the role of sesamin in the mechanism of anti-atherosclerosis.Methods the rabbit aortic endothelial cells were isolated, cultured and subcultured.The aortic endothelial cells in logarithmic growth phase were divided into two groups and treated with 1: 1 normal control group: the experimental cells were cultured in serum-free DMEM medium for 24-48 hours under aseptic conditions.2ox-LDL group: the experimental cells were cultured in the ox-LDL medium of 100mg/L concentration for 24 hours and then in the serum-free DMEM medium for 24 hours. The experimental cells were cultured in 50 渭 mol/L AG490 medium for 24 hours.Then the cells were cultured in ox-LDL medium for 24 hours and in serum-free DMEM medium for 24 hours. The experimental cells were cultured in 60 渭 mol / L sesamin medium for 24 hours, and then in the ox-LDL medium of 100mg/L for 24 hours.Finally, cultured in serum-free DEME medium for 24 hours, Real-time PCR method was used to detect the effect of sesamin AG-490 on the expression of JAK1 during the proliferation of rabbit aortic endothelial cells.Results: in normal control group, the endothelial cells of rabbit aorta were fusiform or polygonal, with abundant cytoplasm and clear boundary.In the monolayer distribution of ox-LDL, the number of round cells in rabbit aortic endothelial cells was obviously increased, the boundary was not clear, the number of apoptotic bodies appeared, the number of apoptotic bodies appeared, the cell fragments increased, the cell gap became wider, and occasionally the cells fell off.In the sesamin group, the endothelial cells of rabbit aorta were polygonal or fusiform with clear boundary and close to the normal control group, but the number of cells was less than that of the normal control group and more than that of the ox-LDL group by 1% agarose gel electrophoresis.A specific band appeared at 250bp.The JAK1 gene of rabbit aortic endothelial cells was successfully amplified.The diluted recombinant plasmid was used as the template of PCR reaction. The primers of upstream and downstream of each parameter were used as primers respectively. The results of 1% agarose gel electrophoresis showed that there was a specific band in 2945bp.The cloned plasmid of JAK1 gene of rabbit aortic endothelial cells, ox-LDL group and sesamin group, were successfully amplified. The expression of JAK1-mRNA in AG490 group was significantly higher than that in normal control group.The expression of JAK1-mRNA in sesamin group and AG490 group was significantly lower than that in ox-LDL group (P 0.05), and there was a significant difference between sesamin group and AG490 group (P 0.05).Conclusion: both sesamin group and AG490 group can inhibit JAK1-mRNA expression.The inhibitory effect of sesamin group was greater than that of JAK1 inhibitor AG490 group, and could be used as a new target for prevention and treatment of cardiovascular and cerebrovascular diseases.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R543.5
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本文編號(hào)：1723420