本文選題：內(nèi)皮祖細(xì)胞 + wnt3a��； 參考：《安徽醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的通過密度梯度離心法獲取大鼠骨髓血單核細(xì)胞,在體外誘導(dǎo)分化培養(yǎng)得到EPCs;購買沉默型pEGFP-shRNA-wnt3a重組質(zhì)粒,將其轉(zhuǎn)染入EPCs,檢測(cè)其在EPCs中的表達(dá),并利用MTT法檢測(cè)EPCs的增殖能力變化,為進(jìn)一步的研究提供了基礎(chǔ)。方法①EPCs的分離、培養(yǎng)及鑒定:密度梯度離心法獲取大鼠骨髓血單核細(xì)胞,于含10%FBS的EGM-2培養(yǎng)基中培養(yǎng),并經(jīng)免疫細(xì)胞化學(xué)CD133及VEGFR-2雙抗體熒光檢測(cè)和Dil-ac-LDL及FITC-UEA-1熒光檢測(cè)。②脂質(zhì)體介導(dǎo)沉默型pEGFP-shRNA-wnt3a重組質(zhì)粒轉(zhuǎn)染EPCs并檢測(cè)wnt3a蛋白表達(dá)變化:利用脂質(zhì)體試劑LipofectamineTM 2000介導(dǎo)沉默型pEGFP-shRNA-wnt3a重組質(zhì)粒轉(zhuǎn)染EPCs,并設(shè)置空白對(duì)照組。轉(zhuǎn)染48h后提取各組細(xì)胞總蛋白,Western blot法鑒定wnt3a蛋白在EPCs中的表達(dá)變化情況。③MTT法檢測(cè)轉(zhuǎn)染后EPCs的增殖活性:轉(zhuǎn)染48h后消化收集EPCs,重懸并計(jì)數(shù)接種到96孔板中培養(yǎng)72h,MTT法分析轉(zhuǎn)染后EPCs的增殖能力變化。結(jié)果①分離培養(yǎng)的細(xì)胞培養(yǎng)至第7天可觀察到條索狀特征,并經(jīng)免疫細(xì)胞化學(xué)CD133及VEGFR-2雙抗體熒光檢測(cè)和Dil-ac-LDL及FITC-UEA-1熒光檢測(cè)均為陽性,證實(shí)培養(yǎng)的細(xì)胞為EPCs。②沉默型pEGFP-shRNA-wnt3a重組質(zhì)粒轉(zhuǎn)染EPCs 48h后,Western blot法鑒定wnt3a蛋白表達(dá)較空白對(duì)照組的EPCs明顯降低。③MTT結(jié)果表明,與空白對(duì)照組相比,沉默型pEGFP-shRNA-wnt3a轉(zhuǎn)染組中EPCs的體外增殖能力受到明顯的抑制(0.364±0.014 vs 0.402±0.013,t=3.952,P0.05)。結(jié)論通過密度梯度離心法于體外分離大鼠骨髓血成功培養(yǎng)出EPCs;沉默型pEGFP-shRNA-wnt3a重組質(zhì)�？捎行С聊珽PCs的wnt3a基因;沉默型pEGFP-shRNA-wnt3a重組質(zhì)粒轉(zhuǎn)染后明顯抑制了EPCs的體外增殖能力。為進(jìn)一步的研究提供了基礎(chǔ)。
[Abstract]:Objective to obtain rat bone marrow mononuclear cells by density gradient centrifugation, and to obtain EPCs by inducing differentiation and culture in vitro, purchase the recombinant plasmid pEGFP-shRNA-wnt3a, transfect it into EPCsand detect its expression in EPCs. MTT assay was used to detect the proliferation ability of EPCs, which provided a basis for further research. Methods 1isolation, culture and identification of EPCs: rat bone marrow monocytes were obtained by density gradient centrifugation and cultured in EGM-2 medium containing 10s. The recombinant plasmid pEGFP-shRNA-wnt3a was transfected by immunocytochemistry CD133 and VEGFR-2 double antibody fluorescence detection, Dil-ac-LDL and FITC-UEA-1 fluorescence detection. 2 liposome-mediated silencing pEGFP-shRNA-wnt3a plasmid was transfected and the expression of wnt3a protein was detected. The silencing pEGFP-shRNA-wnt3a was mediated by liposome reagent LipofectamineTM 2000. The recombinant plasmid was transfected into EPCsand blank control group was set. After 48 hours of transfection, the total protein of each group was extracted to determine the expression of wnt3a protein in EPCs by Western blot. The proliferative activity of transfected EPCs was detected by MTT method. After 48 hours of transfection, the cells were digested and collected, then resuspended and counted and inoculated into 96 well plates. The proliferation ability of EPCs after transfection was analyzed by 72 h MTT assay. Results (1) the cells isolated and cultured on the 7th day were found to be striped and positive by immunocytochemistry, CD133 and VEGFR-2 double antibody fluorescence detection, Dil-ac-LDL and FITC-UEA-1 fluorescence detection. It was confirmed that the cultured cells were transfected with pEGFP-shRNA-wnt3a recombinant plasmid of EPCs.2 silenced pEGFP-shRNA-wnt3a for 48 h. The results showed that the expression of wnt3a protein was significantly lower than that of the blank control group by Western blot assay. The results showed that compared with the blank control group, the expression of wnt3a protein was significantly lower than that of the control group. In the silencing pEGFP-shRNA-wnt3a transfection group, the proliferation of EPCs was significantly inhibited by 0.364 鹵0.014 vs 0.402 鹵0.013 ~ 3.952% P0.05a. Conclusion EPCs were isolated from rat bone marrow blood by density gradient centrifugation, the silencing pEGFP-shRNA-wnt3a plasmid could effectively silence the wnt3a gene, and the silencing pEGFP-shRNA-wnt3a recombinant plasmid could inhibit the proliferation of EPCs in vitro. It provides a basis for further research.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 杜公文;杜怡斌;張輝;尹宗生;;共培養(yǎng)下內(nèi)皮祖細(xì)胞對(duì)神經(jīng)干細(xì)胞增殖及分化的影響[J];安徽醫(yī)科大學(xué)學(xué)報(bào);2013年12期

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本文編號(hào)：2026555