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  本文選題：內(nèi)皮祖細胞　切入點：腺病毒載體　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：背景 內(nèi)皮祖細胞(Endothelial progenitor cells, EPCs)是血管內(nèi)皮細胞的 體細胞,參與血管再生和內(nèi)皮損傷后的修復(fù)過程。EPCs衰老及其功能紊亂可致血管內(nèi)皮功能失調(diào),后者與動脈粥樣硬化的發(fā)生、發(fā)展密切相關(guān)。餐后高甘油三酯血癥患者血漿中增多的殘粒脂蛋白(Remnant-like particles, RLPs)與血管內(nèi)皮功能失調(diào)密切相關(guān),可加速人外周血單個核細胞來源EPCs的衰老進程,但其機制尚不明確。微小RNA (microRNA, miRNA)是一類在進化過程中高度保守的內(nèi)源性非編碼單鏈RNA,通過在轉(zhuǎn)錄后水平抑制真核生物的基因表達而參與調(diào)控細胞分化、生長發(fā)育、凋亡、腫瘤形成等復(fù)雜的病理生理過程。近年來,許多研究發(fā)現(xiàn)miRNA參與調(diào)控細胞衰老。我們通過前期研究發(fā)現(xiàn)：RLPs誘導(dǎo)的衰老小鼠EPCs中miRNA-27b (miR-27b)表達顯著下調(diào)。推測miR-27b可能參與調(diào)控RLPs誘導(dǎo)的EPCs衰老。 目的 利用細菌內(nèi)同源重組法構(gòu)建以綠色熒光蛋白(EGFP)為報告基因、含miR-27b基因的重組腺病毒,導(dǎo)入小鼠內(nèi)皮祖細胞中表達,為進一步研究miR-27b在RLPs誘導(dǎo)的EPCs衰老中的作用奠定基礎(chǔ)。 方法 利用密度梯度離心法分離小鼠骨髓中單個核細胞,差速貼壁結(jié)合EBM-2培養(yǎng)基擴增細胞,誘導(dǎo)單個核細胞分化為EPCs。流式細胞技術(shù)鑒定EPCs。將含有目的基因miR-27b的質(zhì)粒與目的載體pDC316-siRNA分別進行酶切。酶切產(chǎn)物瓊脂糖電泳回收后進行定向連接,連接產(chǎn)物轉(zhuǎn)入細菌感受態(tài)細胞BJ5183。先對生長的克隆進行菌落PCR鑒定,再對PCR鑒定陽性的克隆進行測序和比對分析,比對分析正確的克隆即為構(gòu)建成功的目的質(zhì)粒。將重組質(zhì)粒在人胚腎293細胞(HEK293)中包裝、擴增出miR-27b重組腺病毒載體(Ad-miR-27b),檢測其滴度；以腺病毒載體綠色熒光蛋白(Ad-EGFP)為陰性對照,分別感染EPCs,RT-PCR檢測感染后miR-27b的表達情況。 結(jié)果 經(jīng)密度梯度離心和差速貼壁法分離所得的細胞經(jīng)EBM-2專用培養(yǎng)基培養(yǎng)后,培養(yǎng)到第12d行流式細胞儀檢測其CD34、CD133、Flk-1、 CD31的陽性率分別為(65+4)%、(48±3)%、(37±3)%和(51±4)%。目的基因miR-27b的質(zhì)粒與目的載體pDC316-siRNA雙酶切后共轉(zhuǎn)化BJ5183感受態(tài)菌,可獲得陽性重組體細菌克隆。重組質(zhì)粒在人胚腎293細胞(HEK293)中包裝、擴增出miR-27b重組腺病毒載體(Ad-miR-27b),檢測其滴度重組質(zhì)粒病毒滴度1.5×109pfU/mL。 Ad-miR-27b感染EPCs后24h miR-27b表達較陰性對照組顯著增加。 結(jié)論 成功制備的重組腺病毒Ad-miR-27b感染體外培養(yǎng)的EPCs后,在體外能有效表達目的基因產(chǎn)物。
[Abstract]:Background. Endothelial progenitor cells (EPCs) are vascular endothelial cells. Somatic cells, involved in the repair process after vascular regeneration and endothelial injury. Aging and dysfunction of EPCs may lead to vascular endothelial dysfunction, which is associated with atherosclerosis. The increase of residual lipoprotein in plasma of patients with postprandial hypertriglyceridemia is closely related to vascular endothelial dysfunction, which can accelerate the aging process of EPCs derived from human peripheral blood mononuclear cells. However, the mechanism of microRNAs is still unclear. MicroRNAs are a class of highly conserved endogenous non-coding single-stranded RNAs that regulate cell differentiation, growth, development and apoptosis by inhibiting gene expression in eukaryotes at the post-transcriptional level. Tumor formation and other complex pathophysiological processes. In recent years, Many studies have found that miRNA is involved in the regulation of cellular senescence. We have found that the expression of miRNA-27b miR-27b in EPCs induced by miRNA-27b RLPs is significantly down-regulated. It is suggested that miR-27b may be involved in the regulation of EPCs senescence induced by RLPs. Purpose. The recombinant adenovirus containing miR-27b gene was constructed by homologous recombination in bacteria. The recombinant adenovirus containing miR-27b gene was introduced into mouse endothelial progenitor cells and expressed in mouse endothelial progenitor cells, which laid a foundation for further study on the role of miR-27b in EPCs senescence induced by RLPs. Method. Mononuclear cells were isolated from mouse bone marrow by density gradient centrifugation. The cells were amplified by differential adhesion and EBM-2 medium. The plasmid containing the target gene miR-27b and the target vector pDC316-siRNA were digested respectively. The ligation product was transferred into the bacterial competent cell BJ5183.The clones were identified by colony PCR, then the positive clones identified by PCR were sequenced and compared with each other. The recombinant plasmid was packaged in human embryonic kidney 293 cell line (HEK293) to amplify the recombinant adenovirus vector Ad-miR-27bN and detect its titer, and the adenovirus vector Ad-EGFP was used as negative control. The expression of miR-27b was detected by RT-PCR. Results. The cells isolated by density gradient centrifugation and differential adhesion method were cultured on EBM-2 medium. On the 12th day of culture, flow cytometry was used to detect the positive rates of CD34, CD133, Flk-1 and CD31, respectively. The positive rates of CD31 were 48 鹵3% and 51 鹵4%, respectively. The plasmid of miR-27b was digested with the target vector pDC316-siRNA and then transformed into BJ5183 receptive bacteria. The recombinant plasmid was packaged in human embryonic kidney 293 cell line (HEK293) and the recombinant adenovirus vector Ad-miR-27b was amplified. The titer of the recombinant plasmid was 1.5 脳 10 9 pfU 路mL. Ad-miR-27b. 24 h after EPCs infection, the expression of miR-27b was significantly increased compared with the negative control group. Conclusion. The recombinant adenovirus Ad-miR-27b was successfully infected with EPCs in vitro, and the target gene product could be expressed effectively in vitro.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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