本文選題：內(nèi)皮祖細胞 + wnt3a　； 參考：《安徽醫(yī)科大學》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胞,在體外誘導分化培養(yǎng)得到EPCs;購買沉默型pEGFP-shRNA-wnt3a重組質粒,將其轉染入EPCs,檢測其在EPCs中的表達,并利用MTT法檢測EPCs的增殖能力變化,為進一步的研究提供了基礎。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胞,于含10%FBS的EGM-2培養(yǎng)基中培養(yǎng),并經(jīng)免疫細胞化學CD133及VEGFR-2雙抗體熒光檢測和Dil-ac-LDL及FITC-UEA-1熒光檢測。②脂質體介導沉默型pEGFP-shRNA-wnt3a重組質粒轉染EPCs并檢測wnt3a蛋白表達變化:利用脂質體試劑LipofectamineTM 2000介導沉默型pEGFP-shRNA-wnt3a重組質粒轉染EPCs,并設置空白對照組。轉染48h后提取各組細胞總蛋白,Western blot法鑒定wnt3a蛋白在EPCs中的表達變化情況。③MTT法檢測轉染后EPCs的增殖活性:轉染48h后消化收集EPCs,重懸并計數(shù)接種到96孔板中培養(yǎng)72h,MTT法分析轉染后EPCs的增殖能力變化。結果①分離培養(yǎng)的細胞培養(yǎng)至第7天可觀察到條索狀特征,并經(jīng)免疫細胞化學CD133及VEGFR-2雙抗體熒光檢測和Dil-ac-LDL及FITC-UEA-1熒光檢測均為陽性,證實培養(yǎng)的細胞為EPCs。②沉默型pEGFP-shRNA-wnt3a重組質粒轉染EPCs 48h后,Western blot法鑒定wnt3a蛋白表達較空白對照組的EPCs明顯降低。③MTT結果表明,與空白對照組相比,沉默型pEGFP-shRNA-wnt3a轉染組中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�？捎行С聊珽PCs的wnt3a基因;沉默型pEGFP-shRNA-wnt3a重組質粒轉染后明顯抑制了EPCs的體外增殖能力。為進一步的研究提供了基礎。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R651.2

【參考文獻】

相關期刊論文 前1條

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

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本文編號：2026555