VEGF基因转染人脐带间充质干细胞向神经样细胞诱导分化的研究
发布时间：2018-08-14 13:00
【摘要】：目的 将血管内皮生长因子基因(vascular endothelial growth factor,VEGF)转染到人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cell,hUCMSCs),检测VEGF转染后的人脐带间充质干细胞向神经样细胞诱导分化能力的变化。 方法 将纯化的第4代hUCMSCs分为四组,常规培养的细胞组;不转染质粒但进行转染实验组;转染pEGFP-N3质粒组;转染GFP-VEGF质粒组,用Lipofectamine(多阴离子转染脂质体)法,将克隆入真核表达载体pEGFP-N3中的VEGF基因转染入hUCMSCs。然后用Real-time PCR检测VEGF基因表达,进行相对表达量分析。Western blot检测VEGF蛋白表达,以GAPDH作为内参,进行蛋白相对表达量分析,观察VEGF基因的表达对hUCMSCs的影响。 用β-巯基乙醇为诱导剂的定向诱导法将转染后的hUCMSCs定向诱导成神经样细胞,并且从细胞形态学观察和免疫组化神经标记物检测(神经元特异性标志NSE、神经干细胞标记物神经巢蛋白Nestin、星形胶质细胞特异性标志GFAP的检测)的方法来观察转染后的hUCMSCs向神经样细胞诱导能力的变化,评价VEGF基因转染对hUCMSCs诱导为神经样细胞能力的影响。 结果 1.RT-PCR结果显示,VEGF基因在hUCMSCs-VEGF细胞中的表达量与其他三组细胞有显著差异(P0.01),而未转染细胞,空转染细胞,转化空质粒细胞相比较,无明显差异。 2. Western Blot检测结果显示,未转染细胞,空转染细胞,转化空质粒细胞,转染VEGF细胞中的VEGF蛋白表达量有明显变化,未转染细胞,空转染细胞,转化空质粒细胞,转染VEGF细胞中VEGF的表达量明显上升(P0.01)。 3.诱导后细胞形态学观察利用β-巯基乙醇为诱导剂的定向诱导法将转染后的hUCMSCs定向诱导成神经样细胞,未诱导细胞呈长梭形或成纤维细胞样,诱导后细胞形态出现明显变化,细胞收缩,逐渐向神经细胞转化,诱导6h后,多数细胞诱导为典型的神经样细胞,诱导24h后细胞收缩为锥形,三角形或者不规则形,折光性好,有些长突起末端形成长锥样及丝状。 4.通过免疫组化实验,鉴定诱导分化出来的神经样细胞,分别进行了神经元特异性标志NSE、神经干细胞标记物神经巢蛋白Nestin和星形胶质细胞特异性标志GFAP的检测,发现NSE和Nestin染色有阳性结果,有荧光, GFAP染色和对照组无阳性结果,定量计数分析,NSE阳性的细胞约为(43.25±4.1)%,Nestin阳性的细胞约为(38.63±3.6)%。 结论 VEGF基因导入hUCMSCs,可以稳定表达,诱导后hUCMSCs-VEGF出现明显神经样细胞变化,并且转染了VEGF的hUCMSCs向神经样细胞诱导分化的能力未受到明显影响。
[Abstract]:Objective to transfect the vascular endothelial growth factor gene (vascular endothelial growth factor-VEGF) into human umbilical cord mesenchymal stem cells (human umbilical cord derived mesenchymal stem cells) and detect the differentiation ability of VEGF transfected human umbilical cord mesenchymal stem cells into neural like cells. Methods the purified fourth passage hUCMSCs was divided into four groups: the normal cultured cells group, the non-transfected plasmid group, the pEGFP-N3 plasmid group, the GFP-VEGF plasmid group, the Lipofectamine (polyanion transfection liposome) method, The VEGF gene cloned into eukaryotic expression vector pEGFP-N3 was transfected into hUCMSCs. Then the expression of VEGF gene was detected by Real-time PCR, the expression of VEGF protein was detected by relative expression analysis. The expression of VEGF protein was detected by using GAPDH as internal reference. The effect of VEGF gene expression on hUCMSCs was observed. HUCMSCs transfected with 尾 -mercaptoethanol as inducer was induced into neuron-like cells. The transfection of NSE, Nestin, GFAP, a neural stem cell marker, was observed by the methods of morphological observation and immunohistochemical detection of nerve markers (NSE, NSE, Nestin, and GFAP), which were the markers of neural stem cells. The ability of hUCMSCs to induce neuronal cells was changed after the treatment. To evaluate the effect of VEGF gene transfection on the ability of hUCMSCs to induce neuronal cells. Results the expression of 1.RT-PCR gene in hUCMSCs-VEGF cells was significantly different from that in the other three groups (P0.01), but there was no significant difference between untransfected cells, empty transfected cells and transformed empty plasmid cells. The results of Western Blot analysis showed that the expression of VEGF protein in untransfected cells, empty transfected cells, transformed empty plasmid cells and transfected VEGF cells had obvious changes, and the untransfected cells, empty transfected cells, and empty plasmid cells were transformed into empty plasmid cells. The expression of VEGF in transfected VEGF cells increased significantly (P0.01). After induction, the transfected hUCMSCs cells were induced into neuron-like cells by using 尾 -mercaptoethanol as the inducer. The cells were not induced to be fusiform or fibroblast-like, and the morphology of the cells changed obviously after induction. After 6 hours of induction, most of the cells were induced to be typical neuron-like cells, and after 24 hours, the cells contracted into conical, triangular or irregular shape, with good refraction. Some long protrusions end to form long cones and filaments. 4. NSE, neural stem cell marker (NSE), neural stem cell marker (NSE) and astrocyte-specific marker (GFAP) were detected by immunohistochemistry. It was found that NSE and Nestin staining were positive and fluorescent, GFAP staining and control group had no positive results. Quantitative analysis showed that the positive cells were (43.25 卤4.1) nestin positive cells and (38.63 卤3.6) positive cells. Conclusion VEGF gene can be expressed stably when it is introduced into hUCMSCs.After induction of hUCMSCs-VEGF, there are obvious changes in neuron-like cells, and the ability of hUCMSCs transfected with VEGF to differentiate into neuron-like cells has not been significantly affected.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2182932
[Abstract]:Objective to transfect the vascular endothelial growth factor gene (vascular endothelial growth factor-VEGF) into human umbilical cord mesenchymal stem cells (human umbilical cord derived mesenchymal stem cells) and detect the differentiation ability of VEGF transfected human umbilical cord mesenchymal stem cells into neural like cells. Methods the purified fourth passage hUCMSCs was divided into four groups: the normal cultured cells group, the non-transfected plasmid group, the pEGFP-N3 plasmid group, the GFP-VEGF plasmid group, the Lipofectamine (polyanion transfection liposome) method, The VEGF gene cloned into eukaryotic expression vector pEGFP-N3 was transfected into hUCMSCs. Then the expression of VEGF gene was detected by Real-time PCR, the expression of VEGF protein was detected by relative expression analysis. The expression of VEGF protein was detected by using GAPDH as internal reference. The effect of VEGF gene expression on hUCMSCs was observed. HUCMSCs transfected with 尾 -mercaptoethanol as inducer was induced into neuron-like cells. The transfection of NSE, Nestin, GFAP, a neural stem cell marker, was observed by the methods of morphological observation and immunohistochemical detection of nerve markers (NSE, NSE, Nestin, and GFAP), which were the markers of neural stem cells. The ability of hUCMSCs to induce neuronal cells was changed after the treatment. To evaluate the effect of VEGF gene transfection on the ability of hUCMSCs to induce neuronal cells. Results the expression of 1.RT-PCR gene in hUCMSCs-VEGF cells was significantly different from that in the other three groups (P0.01), but there was no significant difference between untransfected cells, empty transfected cells and transformed empty plasmid cells. The results of Western Blot analysis showed that the expression of VEGF protein in untransfected cells, empty transfected cells, transformed empty plasmid cells and transfected VEGF cells had obvious changes, and the untransfected cells, empty transfected cells, and empty plasmid cells were transformed into empty plasmid cells. The expression of VEGF in transfected VEGF cells increased significantly (P0.01). After induction, the transfected hUCMSCs cells were induced into neuron-like cells by using 尾 -mercaptoethanol as the inducer. The cells were not induced to be fusiform or fibroblast-like, and the morphology of the cells changed obviously after induction. After 6 hours of induction, most of the cells were induced to be typical neuron-like cells, and after 24 hours, the cells contracted into conical, triangular or irregular shape, with good refraction. Some long protrusions end to form long cones and filaments. 4. NSE, neural stem cell marker (NSE), neural stem cell marker (NSE) and astrocyte-specific marker (GFAP) were detected by immunohistochemistry. It was found that NSE and Nestin staining were positive and fluorescent, GFAP staining and control group had no positive results. Quantitative analysis showed that the positive cells were (43.25 卤4.1) nestin positive cells and (38.63 卤3.6) positive cells. Conclusion VEGF gene can be expressed stably when it is introduced into hUCMSCs.After induction of hUCMSCs-VEGF, there are obvious changes in neuron-like cells, and the ability of hUCMSCs transfected with VEGF to differentiate into neuron-like cells has not been significantly affected.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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