【摘要】： 目的:以核因子κB (NF-κB) p65基因特異的RNA干擾(RNAi)腺病毒表達(dá)載體作為研究工具,初步探討NF-κB信號(hào)通路對(duì)波動(dòng)性高糖(含5.5或30.5 mM D-葡萄糖培養(yǎng)液,每日交替)下人臍靜脈內(nèi)皮細(xì)胞(Human Umbilical Vein Endothelial Cell, HUVEC)增殖的影響,同時(shí)檢測(cè)細(xì)胞凋亡相關(guān)分子bcl-2表達(dá)情況。 方法:利用體外同源重組技術(shù)構(gòu)建NF-κB p65基因特異的siRNA腺病毒表達(dá)載體,在HEK293A細(xì)胞中包裝并擴(kuò)增病毒、空斑實(shí)驗(yàn)法測(cè)定病毒滴度;p65特異的siRNA腺病毒感染HUVEC,同時(shí)予波動(dòng)性高糖干預(yù),Western印記法測(cè)定p65蛋白表達(dá)和NF-κB核轉(zhuǎn)錄的影響;以AlamarBlue法檢測(cè)細(xì)胞增殖;Western印記法檢測(cè)bcl-2表達(dá)。 結(jié)果:1.成功構(gòu)建了針對(duì)NF-κBp65特異的RNAi腺病毒表達(dá)載體;2.NF-κBp65特異的RNAi腺病毒感染HUVEC后可使p65表達(dá)量明顯下降; 3.波動(dòng)性高糖下,RNAi腺病毒感染HUVEC后,亦可檢測(cè)到p65表達(dá)量下降,且隨時(shí)間延長(zhǎng)p65表達(dá)量進(jìn)一步減少,抑制效應(yīng)至少持續(xù)至干預(yù)后第七天;4.NF-κBp65特異腺病毒感染HUVEC,可以明顯抑制波動(dòng)性高糖刺激的NF-κBp65核蛋白轉(zhuǎn)錄,使核內(nèi)p65蛋白表達(dá)處于基礎(chǔ)水平; 5.AlamarBlue結(jié)果分析,NF-κBp65特異腺病毒感染HUVEC可以使波動(dòng)性高糖誘導(dǎo)的細(xì)胞增殖抑制下降(P0.01),但比正常葡萄糖濃度(5.5 mmol/L)下的細(xì)胞增殖率低(P0.01); 6.NF-κBp65特異腺病毒可以上調(diào)波動(dòng)性高糖下HUVEC bcl-2的表達(dá),有可能減少細(xì)胞凋亡。 結(jié)論:構(gòu)建NF-κB p65特異的RNAi腺病毒表達(dá)載體能有效抑制HUVEC p65基因的表達(dá);腺病毒感染HUVEC,可以明顯抑制波動(dòng)性高糖刺激的NF-κB p65核轉(zhuǎn)錄,從而保護(hù)波動(dòng)性高糖下的HUVEC增殖抑制,且有可能減少波動(dòng)性高糖誘導(dǎo)的HUVEC凋亡。
[Abstract]:Aim: to investigate the effect of nuclear factor kappa B (NF- kappa B) p65 gene specific RNA interfering (RNAi) adenoviral expression vector on the proliferation of human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cell, HUVEC) in fluctuating high glucose (containing 5.5 or 30.5 mM D-glucose medium, alternately daily), and to detect the expression of apoptosis related molecule bcl-2 in human umbilical vein endothelial cells (umbilical vein endothelial cells). Methods: NF- 魏 B p65 gene specific siRNA adenoviral expression vector was constructed by homologous recombination technique in vitro, and the virus was packaged and amplified in HEK293A cells. The virus titer was determined by plaque assay. P65 specific siRNA adenovirus infected HUVEC, was interfered with volatile high glucose, Western printing was used to detect the expression of p65 protein and NF- kappa B nuclear transcription, AlamarBlue assay was used to detect cell proliferation, and Western printing method was used to detect the expression of bcl-2. Result: 1. The RNAi adenoviral expression vector specific to NF- 魏 Bp65 was successfully constructed. 2. The expression of p65 was significantly decreased after infection with RNAi adenoviruses. Under fluctuating high glucose, p65 expression decreased after HUVEC infection with RNAi adenoviral infection, and the expression of p65 decreased further with the prolongation of time, and the inhibitory effect lasted at least until the seventh day after intervention. 4. NF-魏 Bp65 specific adenoviral infection HUVEC, could significantly inhibit the transcription of NF- 魏 Bp65 nucleoprotein stimulated by fluctuating high glucose, making the expression of p65 protein in the nucleus at the basic level. 5.AlamarBlue results showed that HUVEC infection with HUVEC could decrease the cell proliferation inhibition induced by fluctuating high glucose (P01), but lower the cell proliferation rate (P01) than that at normal glucose concentration (5.5Bp65). 6. NF-魏 Bp65 specific adenovirus could up-regulate the expression of HUVEC bcl-2 under fluctuating high glucose, which might reduce apoptosis. Conclusion: the construction of NF- 魏 B p65 specific RNAi adenoviral expression vector can effectively inhibit the expression of HUVEC p65 gene, and adenoviral infection with HUVEC, can significantly inhibit the nuclear transcription of NF- 魏 B p65 stimulated by volatile high glucose, thus protecting the proliferation inhibition of HUVEC under volatile high glucose, and may reduce the apoptosis of HUVEC induced by volatile high glucose.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R363

【共引文獻(xiàn)】

中國(guó)碩士學(xué)位論文全文數(shù)據(jù)庫(kù) 前2條

1 喬玉芳;NF-κB特異的RNAi腺病毒載體的構(gòu)建及其對(duì)血管內(nèi)皮細(xì)胞增殖凋亡的影響[D];福建醫(yī)科大學(xué);2007年

2 孔永;腺病毒介導(dǎo)CTLA4Ig和CD40Ig基因轉(zhuǎn)染對(duì)人皮膚成纖維細(xì)胞免疫性能影響的體外研究[D];西南大學(xué);2008年

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本文編號(hào)：2507167