【摘要】：目的 將血管內(nèi)皮生長(zhǎng)因子基因(vascular endothelial growth factor,VEGF)轉(zhuǎn)染到人臍帶間充質(zhì)干細(xì)胞(human umbilical cord derived mesenchymal stem cell,hUCMSCs),檢測(cè)VEGF轉(zhuǎn)染后的人臍帶間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞誘導(dǎo)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(xì)胞組;不轉(zhuǎn)染質(zhì)粒但進(jìn)行轉(zhuǎn)染實(shí)驗(yàn)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(dá)載體pEGFP-N3中的VEGF基因轉(zhuǎn)染入hUCMSCs。然后用Real-time PCR檢測(cè)VEGF基因表達(dá),進(jìn)行相對(duì)表達(dá)量分析。Western blot檢測(cè)VEGF蛋白表達(dá),以GAPDH作為內(nèi)參,進(jìn)行蛋白相對(duì)表達(dá)量分析,觀察VEGF基因的表達(dá)對(duì)hUCMSCs的影響。 用β-巰基乙醇為誘導(dǎo)劑的定向誘導(dǎo)法將轉(zhuǎn)染后的hUCMSCs定向誘導(dǎo)成神經(jīng)樣細(xì)胞,并且從細(xì)胞形態(tài)學(xué)觀察和免疫組化神經(jīng)標(biāo)記物檢測(cè)(神經(jīng)元特異性標(biāo)志NSE、神經(jīng)干細(xì)胞標(biāo)記物神經(jīng)巢蛋白Nestin、星形膠質(zhì)細(xì)胞特異性標(biāo)志GFAP的檢測(cè))的方法來觀察轉(zhuǎn)染后的hUCMSCs向神經(jīng)樣細(xì)胞誘導(dǎo)能力的變化,評(píng)價(jià)VEGF基因轉(zhuǎn)染對(duì)hUCMSCs誘導(dǎo)為神經(jīng)樣細(xì)胞能力的影響。 結(jié)果 1.RT-PCR結(jié)果顯示,VEGF基因在hUCMSCs-VEGF細(xì)胞中的表達(dá)量與其他三組細(xì)胞有顯著差異(P0.01),而未轉(zhuǎn)染細(xì)胞,空轉(zhuǎn)染細(xì)胞,轉(zhuǎn)化空質(zhì)粒細(xì)胞相比較,無(wú)明顯差異。 2. Western Blot檢測(cè)結(jié)果顯示,未轉(zhuǎn)染細(xì)胞,空轉(zhuǎn)染細(xì)胞,轉(zhuǎn)化空質(zhì)粒細(xì)胞,轉(zhuǎn)染VEGF細(xì)胞中的VEGF蛋白表達(dá)量有明顯變化,未轉(zhuǎn)染細(xì)胞,空轉(zhuǎn)染細(xì)胞,轉(zhuǎn)化空質(zhì)粒細(xì)胞,轉(zhuǎn)染VEGF細(xì)胞中VEGF的表達(dá)量明顯上升(P0.01)。 3.誘導(dǎo)后細(xì)胞形態(tài)學(xué)觀察利用β-巰基乙醇為誘導(dǎo)劑的定向誘導(dǎo)法將轉(zhuǎn)染后的hUCMSCs定向誘導(dǎo)成神經(jīng)樣細(xì)胞,未誘導(dǎo)細(xì)胞呈長(zhǎng)梭形或成纖維細(xì)胞樣,誘導(dǎo)后細(xì)胞形態(tài)出現(xiàn)明顯變化,細(xì)胞收縮,逐漸向神經(jīng)細(xì)胞轉(zhuǎn)化,誘導(dǎo)6h后,多數(shù)細(xì)胞誘導(dǎo)為典型的神經(jīng)樣細(xì)胞,誘導(dǎo)24h后細(xì)胞收縮為錐形,三角形或者不規(guī)則形,折光性好,有些長(zhǎng)突起末端形成長(zhǎng)錐樣及絲狀。 4.通過免疫組化實(shí)驗(yàn),鑒定誘導(dǎo)分化出來的神經(jīng)樣細(xì)胞,分別進(jìn)行了神經(jīng)元特異性標(biāo)志NSE、神經(jīng)干細(xì)胞標(biāo)記物神經(jīng)巢蛋白Nestin和星形膠質(zhì)細(xì)胞特異性標(biāo)志GFAP的檢測(cè),發(fā)現(xiàn)NSE和Nestin染色有陽(yáng)性結(jié)果,有熒光, GFAP染色和對(duì)照組無(wú)陽(yáng)性結(jié)果,定量計(jì)數(shù)分析,NSE陽(yáng)性的細(xì)胞約為(43.25±4.1)%,Nestin陽(yáng)性的細(xì)胞約為(38.63±3.6)%。 結(jié)論 VEGF基因?qū)雋UCMSCs,可以穩(wěn)定表達(dá),誘導(dǎo)后hUCMSCs-VEGF出現(xiàn)明顯神經(jīng)樣細(xì)胞變化,并且轉(zhuǎn)染了VEGF的hUCMSCs向神經(jīng)樣細(xì)胞誘導(dǎo)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(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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