本文選題：EphrinB　切入點(diǎn)：EphB　出處：《中風(fēng)與神經(jīng)疾病雜志》2017年06期

【摘要】：目的將攜帶Ephrin B2基因慢病毒轉(zhuǎn)染至骨髓間充質(zhì)干細(xì)胞(BMSCs),檢測(cè)其過(guò)表達(dá)水平及細(xì)胞形態(tài)學(xué)變化,并探討Eph B4/Ephrin B2是否具有促進(jìn)大鼠BMSCs體外遷移活性的作用。方法單純貼壁法分離大鼠BMSCs,倒置顯微鏡觀察細(xì)胞生長(zhǎng)形態(tài)。攜帶Ephrin B2慢病毒以感染復(fù)數(shù)3和10感染BMSCs分為低濃度轉(zhuǎn)染組(MOI=3)和高濃度轉(zhuǎn)染組(MOI=10),對(duì)照組采用未轉(zhuǎn)染組、陰性轉(zhuǎn)染組,利用q PCR檢測(cè)Ephrin B2基因表達(dá)及Western blot檢測(cè)Ephrin B2蛋白水平。觀察Ephrin B2基因轉(zhuǎn)染后BMSCs形態(tài)學(xué)變化。15 d后通過(guò)免疫熒光檢測(cè)MAP2表達(dá)了解細(xì)胞分化。進(jìn)一步Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力來(lái)了解Eph B4/Ephrin B2在調(diào)節(jié)干細(xì)胞遷移的作用。結(jié)果培養(yǎng)BMSCs為多角形或棱形呈漩渦狀排列生長(zhǎng)。Ephrin B2-BMSCs經(jīng)q PCR和Western blot檢測(cè)證實(shí)有外源性Ephrin B2表達(dá)。Ephrin B2基因轉(zhuǎn)染后BMSCs 24 h,BMSCs胞體開(kāi)始收縮細(xì)胞有突起伸出,72 h后可見(jiàn)典型的神經(jīng)樣細(xì)胞形態(tài)改變。15 d后,BMSCs分化成為MAP2神經(jīng)元,與陰性轉(zhuǎn)染組相比,低濃度轉(zhuǎn)染組和高濃度轉(zhuǎn)染組MAP2表達(dá)率上升(P0.05)。Transwell實(shí)驗(yàn)結(jié)果顯示:低濃度轉(zhuǎn)染組與高濃度轉(zhuǎn)染組較未轉(zhuǎn)染組及陰性轉(zhuǎn)染組穿膜細(xì)胞數(shù)量明顯增加(P0.05)。結(jié)論慢病毒介導(dǎo)Ephrin B2能高效感染BMSCs,并隨著時(shí)間延長(zhǎng)分化成神經(jīng)樣細(xì)胞,同時(shí)Eph B4/Ephrin B2在調(diào)節(jié)BMSCs遷移具有重要作用。
[Abstract]:Objective to transfect lentivirus carrying Ephrin B2 gene into bone marrow mesenchymal stem cells (BMSCs) to detect its overexpression level and cell morphology, and to investigate whether Eph B4/Ephrin B2 can promote the migration of rat BMSCs in vitro.Methods BMSCs were isolated from rats by simple adherent method, and cell growth morphology was observed by inverted microscope.Ephrin B2 lentivirus was divided into low concentration transfection group and high concentration transfection group by infecting BMSCs with complex number 3 and 10. The control group was used to detect the expression of Ephrin B2 gene by Q PCR and the Western blot to detect Ephrin B2 protein level by using untransfected group and negative transfection group.The morphological changes of BMSCs were observed after Ephrin B2 gene transfection. After 15 days, the expression of MAP2 was detected by immunofluorescence to understand the cell differentiation.Further Transwell assay was used to investigate the role of Eph B4/Ephrin B2 in regulating stem cell migration.Results the cultured BMSCs were polygonal or prism in swirl shape. Ephrin B2-BMSCs was confirmed by Q PCR and Western blot to express Ephrin B2. Ephrin B2 gene was transfected into the cell body of BMSCs 24 h after transfection, the cell body began to contract. 72 h later, typical cells could be seen.After 15 days, BMSCs differentiated into MAP2 neurons.Compared with the negative transfection group, the expression rate of MAP2 in the low concentration transfection group and the high concentration transfection group was higher than that in the low concentration transfection group and the high concentration transfection group. The results showed that the number of the perforating cells in the low concentration transfection group and the high concentration transfection group was significantly higher than that in the non-transfection group and the negative transfection group.Conclusion Lentivirus-mediated Ephrin B2 can efficiently infect BMSCs and differentiate into neuron-like cells over time. Meanwhile, Eph B4/Ephrin B2 plays an important role in regulating the migration of BMSCs.
【作者單位】： 南京醫(yī)科大學(xué)附屬兒童醫(yī)院康復(fù)科;南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院神經(jīng)內(nèi)科;
【基金】：國(guó)家自然科學(xué)基金青年基金(No.81401864) 南京醫(yī)科大學(xué)科學(xué)基金重點(diǎn)項(xiàng)目(No.2013NJMU097)
【分類號(hào)】：R329.2

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