VEGF基因轉(zhuǎn)染人臍帶間充質(zhì)干細胞向神經(jīng)樣細胞誘導分化的研究
發(fā)布時間：2018-08-14 13:00
【摘要】：目的 將血管內(nèi)皮生長因子基因(vascular endothelial growth factor,VEGF)轉(zhuǎn)染到人臍帶間充質(zhì)干細胞(human umbilical cord derived mesenchymal stem cell,hUCMSCs),檢測VEGF轉(zhuǎn)染后的人臍帶間充質(zhì)干細胞向神經(jīng)樣細胞誘導分化能力的變化。 方法 將純化的第4代hUCMSCs分為四組,常規(guī)培養(yǎng)的細胞組;不轉(zhuǎn)染質(zhì)粒但進行轉(zhuǎn)染實驗組;轉(zhuǎn)染pEGFP-N3質(zhì)粒組;轉(zhuǎn)染GFP-VEGF質(zhì)粒組,用Lipofectamine(多陰離子轉(zhuǎn)染脂質(zhì)體)法,將克隆入真核表達載體pEGFP-N3中的VEGF基因轉(zhuǎn)染入hUCMSCs。然后用Real-time PCR檢測VEGF基因表達,進行相對表達量分析。Western blot檢測VEGF蛋白表達,以GAPDH作為內(nèi)參,進行蛋白相對表達量分析,觀察VEGF基因的表達對hUCMSCs的影響。 用β-巰基乙醇為誘導劑的定向誘導法將轉(zhuǎn)染后的hUCMSCs定向誘導成神經(jīng)樣細胞,并且從細胞形態(tài)學觀察和免疫組化神經(jīng)標記物檢測(神經(jīng)元特異性標志NSE、神經(jīng)干細胞標記物神經(jīng)巢蛋白Nestin、星形膠質(zhì)細胞特異性標志GFAP的檢測)的方法來觀察轉(zhuǎn)染后的hUCMSCs向神經(jīng)樣細胞誘導能力的變化,評價VEGF基因轉(zhuǎn)染對hUCMSCs誘導為神經(jīng)樣細胞能力的影響。 結(jié)果 1.RT-PCR結(jié)果顯示,VEGF基因在hUCMSCs-VEGF細胞中的表達量與其他三組細胞有顯著差異(P0.01),而未轉(zhuǎn)染細胞,空轉(zhuǎn)染細胞,轉(zhuǎn)化空質(zhì)粒細胞相比較,無明顯差異。 2. Western Blot檢測結(jié)果顯示,未轉(zhuǎn)染細胞,空轉(zhuǎn)染細胞,轉(zhuǎn)化空質(zhì)粒細胞,轉(zhuǎn)染VEGF細胞中的VEGF蛋白表達量有明顯變化,未轉(zhuǎn)染細胞,空轉(zhuǎn)染細胞,轉(zhuǎn)化空質(zhì)粒細胞,轉(zhuǎn)染VEGF細胞中VEGF的表達量明顯上升(P0.01)。 3.誘導后細胞形態(tài)學觀察利用β-巰基乙醇為誘導劑的定向誘導法將轉(zhuǎn)染后的hUCMSCs定向誘導成神經(jīng)樣細胞,未誘導細胞呈長梭形或成纖維細胞樣,誘導后細胞形態(tài)出現(xiàn)明顯變化,細胞收縮,逐漸向神經(jīng)細胞轉(zhuǎn)化,誘導6h后,多數(shù)細胞誘導為典型的神經(jīng)樣細胞,誘導24h后細胞收縮為錐形,三角形或者不規(guī)則形,折光性好,有些長突起末端形成長錐樣及絲狀。 4.通過免疫組化實驗,鑒定誘導分化出來的神經(jīng)樣細胞,分別進行了神經(jīng)元特異性標志NSE、神經(jīng)干細胞標記物神經(jīng)巢蛋白Nestin和星形膠質(zhì)細胞特異性標志GFAP的檢測,發(fā)現(xiàn)NSE和Nestin染色有陽性結(jié)果,有熒光, GFAP染色和對照組無陽性結(jié)果,定量計數(shù)分析,NSE陽性的細胞約為(43.25±4.1)%,Nestin陽性的細胞約為(38.63±3.6)%。 結(jié)論 VEGF基因?qū)雋UCMSCs,可以穩(wěn)定表達,誘導后hUCMSCs-VEGF出現(xiàn)明顯神經(jīng)樣細胞變化,并且轉(zhuǎn)染了VEGF的hUCMSCs向神經(jīng)樣細胞誘導分化的能力未受到明顯影響。
[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 鹵4.1) nestin positive cells and (38.63 鹵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.
【學位授予單位】：遼寧醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329
本文編號：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 鹵4.1) nestin positive cells and (38.63 鹵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.
【學位授予單位】：遼寧醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329
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