wnt3a基因沉默对内皮祖细胞增殖的影响

发布时间：2018-06-16 11:48

  本文选题：内皮祖细胞 + wnt3a　； 参考：《安徽医科大学》2015年硕士论文

【摘要】：目的通过密度梯度离心法获取大鼠骨髓血单核细胞,在体外诱导分化培养得到EPCs;购买沉默型pEGFP-shRNA-wnt3a重组质粒,将其转染入EPCs,检测其在EPCs中的表达,并利用MTT法检测EPCs的增殖能力变化,为进一步的研究提供了基础。方法①EPCs的分离、培养及鉴定:密度梯度离心法获取大鼠骨髓血单核细胞,于含10%FBS的EGM-2培养基中培养,并经免疫细胞化学CD133及VEGFR-2双抗体荧光检测和Dil-ac-LDL及FITC-UEA-1荧光检测。②脂质体介导沉默型pEGFP-shRNA-wnt3a重组质粒转染EPCs并检测wnt3a蛋白表达变化:利用脂质体试剂LipofectamineTM 2000介导沉默型pEGFP-shRNA-wnt3a重组质粒转染EPCs,并设置空白对照组。转染48h后提取各组细胞总蛋白,Western blot法鉴定wnt3a蛋白在EPCs中的表达变化情况。③MTT法检测转染后EPCs的增殖活性:转染48h后消化收集EPCs,重悬并计数接种到96孔板中培养72h,MTT法分析转染后EPCs的增殖能力变化。结果①分离培养的细胞培养至第7天可观察到条索状特征,并经免疫细胞化学CD133及VEGFR-2双抗体荧光检测和Dil-ac-LDL及FITC-UEA-1荧光检测均为阳性,证实培养的细胞为EPCs。②沉默型pEGFP-shRNA-wnt3a重组质粒转染EPCs 48h后,Western blot法鉴定wnt3a蛋白表达较空白对照组的EPCs明显降低。③MTT结果表明,与空白对照组相比,沉默型pEGFP-shRNA-wnt3a转染组中EPCs的体外增殖能力受到明显的抑制(0.364±0.014 vs 0.402±0.013,t=3.952,P0.05)。结论通过密度梯度离心法于体外分离大鼠骨髓血成功培养出EPCs;沉默型pEGFP-shRNA-wnt3a重组质粒可有效沉默EPCs的wnt3a基因;沉默型pEGFP-shRNA-wnt3a重组质粒转染后明显抑制了EPCs的体外增殖能力。为进一步的研究提供了基础。
[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 卤0.014 vs 0.402 卤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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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相关期刊论文 前1条

1 杜公文;杜怡斌;张辉;尹宗生;;共培养下内皮祖细胞对神经干细胞增殖及分化的影响[J];安徽医科大学学报;2013年12期

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