本文選題：神經(jīng)干細胞 + HIF-1α��； 參考：《佳木斯大學》2009年碩士論文

【摘要】： 目的:研究NF-κB在HIF-1α上調(diào)VEGF表達通路中的作用。 方法:取新生24小時內(nèi)子鼠海馬組織,經(jīng)機械消化后,在無血清、含生長因子和細胞輔助劑以及低細胞密度的生長環(huán)境中培養(yǎng)并傳代,單細胞克隆純化細胞并進行NSCs鑒定。培養(yǎng)HEK293細胞,擴增AdHIF-1α--GFP及空載體Ad-GFP,并進行熒光鑒定、病毒滴度及感染復數(shù)測定。通過腺病毒轉(zhuǎn)染法將HIF-1α基因轉(zhuǎn)染到神經(jīng)干細胞中,并分組為NSCs組,基因轉(zhuǎn)染后NSCs組,空載體轉(zhuǎn)染后NSCs組。免疫熒光觀察及免疫組化染色檢測及Western blotting法檢測轉(zhuǎn)染后NSCs基因表達情況。免疫組化染色檢測及Western blotting法檢測基因轉(zhuǎn)染后NSCs中VEGF和NF-κB的表達情況,并檢測給予梯濃度NF-κB特異性抑制劑PDTC后AdHIF-1α--GFP修飾后NSCs中VEGF的表達情況,并就其結(jié)果進行統(tǒng)計學分析。 主要結(jié)果: 1,從子鼠海馬分離出的細胞經(jīng)原代和傳代培養(yǎng)獲得高純度的神經(jīng)干細胞,可形成細胞克隆,nestin染色呈陽性,證實為具有體外增殖和多向性分化潛能的神經(jīng)干細胞,并改良NSCs傳代時消化方法。 2,AdHIF-1α--GFP及Ad-GFP以HEK293細胞為載體擴增后,熒光顯微鏡觀察大部分細胞有明亮綠色熒光,熒光陽性細胞計數(shù)及MTT法測定重組腺病毒滴度為1×108pfu/ml,確定感染復數(shù)(MOI)值為50∶1 ,當MOI值超過50∶1時,轉(zhuǎn)染效率升高不明顯,與75:1和100:1比較基本達到高峰(P0.01)。 3,AdHIF-1α--GFP及Ad-GFP轉(zhuǎn)染NSCs后熒光顯微鏡下可直接觀察到細胞發(fā)出綠色熒光;熒光表達持續(xù)30天以上,表明AdHIF-1α--GFP及Ad-GFP有效轉(zhuǎn)染神經(jīng)干細胞,并能持續(xù)表達熒光,NSCs是外源基因的良好載體,并得出基因表達強弱與MOI及轉(zhuǎn)染時間有關(guān)。免疫組化、Western blotting顯示轉(zhuǎn)染AdHIF-1α--GFP后的NSCs中HIF-1α顯著表達,正常NSCs組及空載體組無表達,表明轉(zhuǎn)染成功。與未轉(zhuǎn)染組相比,轉(zhuǎn)染AdHIF-1α--GFP基因的神經(jīng)干細胞活力增強,且易分化。 4,通過免疫組化、Western blotting顯示轉(zhuǎn)染AdHIF-1α--GFP后的神經(jīng)干細胞中VEGF和NF-κB的表達顯著高于正常NSCs組及空載體組(P0.05),且兩者表達呈正相關(guān);給予梯濃度NF-κB特異性抑制劑PDTC后□AdHIF-1α--GFP修飾的神經(jīng)干細胞中VEGF的表達呈濃度依賴性下調(diào),各濃度組之間VEGF表達有顯著差異(P0.05)。 結(jié)論:1、對NSCs傳代時細胞消化方法的改良,能增強細胞活力,有利于細胞增殖。 2、HIF-1α基因轉(zhuǎn)染后NSCs中HIF-1α、NF-κB及VEGF表達均上調(diào),三者正相關(guān)。 3、給予HIF-1α修飾的NSCs梯濃度NF-κB特異性抑制劑PDTC后,VEGF表達呈梯濃度依賴性下調(diào)。 4、NF-κB在HIF-1α上調(diào)VEGF表達的信號通路上并起橋接作用。
[Abstract]:Aim: to investigate the role of NF- 魏 B in up-regulation of VEGF expression pathway by HIF-1 偽. Methods: the hippocampal tissue of newborn rat within 24 hours was cultured and subcultured in serum-free environment containing growth factor and cell adjuvant and low cell density after mechanical digestion. The cells were cloned and purified by single cell clone and identified by NSCs. HEK293 cells were cultured, AdHIF-1 偽 -GFP and empty vector Ad-GFPwere amplified. HIF-1 偽 gene was transfected into neural stem cells by adenovirus transfection and divided into NSCs group, NSCs group after gene transfection and NSCs group after empty vector transfection. The expression of NSCs gene after transfection was detected by immunofluorescence, immunohistochemical staining and Western blotting assay. The expression of VEGF and NF- 魏 B in NSCs after transfection was detected by immunohistochemical staining and Western blotting method. The expression of VEGF and NF- 魏 B in NSCs treated with ladder concentration of NF- 魏 B specific inhibitor PDTC was detected, and the expression of VEGF in NSCs modified by AdHIF-1 偽 -GFP was analyzed statistically. The main results were as follows: 1. The high purity neural stem cells were obtained by primary and subculture of the cells isolated from the hippocampus of the offsprings, and the clones were positive for nestin staining. It was proved that NSCs have the potential of proliferation and multidirectional differentiation in vitro, and modified digestion method of NSCs. 2AdHIF-1 偽 -GFP and Ad-GFP were amplified by HEK293 cells. Fluorescence microscopy showed that most of the cells had bright green fluorescence. The fluorescent positive cell count and MTT assay showed that the titer of recombinant adenovirus was 1 脳 10 8 pFu / ml, and the complex number of infection (moi) was 50:1. When the moi value exceeded 50:1, the transfection efficiency was not significantly increased. Compared with 75:1 and 100: 1, the peak was reached (P0.01). After transfection of NSCs with AdHIF-1 偽 -GFP and Ad-GFP, green fluorescence could be observed directly under fluorescence microscope, and the fluorescence expression lasted more than 30 days, indicating that AdHIF-1 偽 -GFP and Ad-GFP could effectively transfect neural stem cells. The expression of NSCs was a good vector of exogenous gene, and the expression of NSCs was related to moi and transfection time. The expression of HIF-1 偽 in NSCs transfected with AdHIF-1 偽-GFP was significantly higher than that in normal NSCs and empty vector group, which indicated that the transfection was successful. The activity of neural stem cells transfected with AdHIF-1 偽 -GFP gene was increased compared with the control group. The expression of VEGF and NF- 魏 B in neural stem cells transfected with AdHIF-1 偽-GFP was significantly higher than that in normal NSCs and empty vector groups (P0.05), and there was a positive correlation between the expression of VEGF and NF- 魏 B in the neural stem cells transfected with AdHIF-1 偽-GFP (P0.05). The expression of VEGF in neural stem cells modified by -AdHIF-1 偽 -GFP was down-regulated in a concentration-dependent manner after treatment with NF- 魏 B specific inhibitor PDTC, and the expression of VEGF was significantly different among different concentration groups (P0.05). Conclusion the modified digestion method for NSCs can enhance cell viability and promote cell proliferation. 2HIF-1 偽 -NF- 魏 B and VEGF expression in NSCs were up-regulated after transfection of HIF-1 偽 gene. The expression of VEGF was down-regulated in a concentration-dependent manner after treatment with HIF-1 偽 modified NF- 魏 B specific inhibitor PDTC. 4NF-kappa B played a bridging role in the signal pathway of HIF-1 偽 upregulation of VEGF expression.
【學位授予單位】：佳木斯大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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相關(guān)期刊論文 前1條

1 ;PROLIFERATION AND DIFFERENTIATION OF NEURAL STEMCELLS IN ADULT RATS AFTER CEREBRAL INFARCTION[J];Chinese Medical Sciences Journal;2004年02期

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本文編號：2051333