本文選題：表皮干細(xì)胞　切入點(diǎn)：增殖　出處：《暨南大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：(1)構(gòu)建人血小板源性生長因子hPDGF-BB基因腺病毒表達(dá)載體； (2)觀察hPDGF-BB腺病毒表達(dá)載體修飾對hESCs增殖的影響。 方法：(1)通過PCR方法合成PDGF-BB,與pAD-track-CMV質(zhì)粒連接后構(gòu)建穿梭質(zhì)粒pAD-PDGF-BB, pAD-PDGF-BB進(jìn)行測序,檢測PDGF-BB序列；取骨架質(zhì)粒pAD-easy I和穿梭質(zhì)粒pAD-PDGF-BB在BJ5183細(xì)菌中進(jìn)行同源重組獲得陽性重組子rad-PDGF, Pac I限制性內(nèi)切酶切鑒定rad-PDGF,0.8%瓊脂糖凝膠電泳檢測；用lipofectaminTM 2000脂質(zhì)體將rad-PDGF導(dǎo)入HEK293細(xì)胞,細(xì)胞出毒后將細(xì)胞反復(fù)凍融3次,高速離心收集上清液作為第一代病毒,檢測其病毒滴度,PCR鑒定rad-PDGF攜帶目的片段PDGF-BB后進(jìn)行病毒擴(kuò)增與純化。 (2)取健康青年人包皮來源的表皮干細(xì)胞(hESCs)作為靶細(xì)胞,體外分離和純化hESCs,用免疫組織化學(xué)法檢測表皮干細(xì)胞表達(dá)K19情況；用hPDGF-BB腺病毒表達(dá)載體(rad-PDGF)修飾表皮干細(xì)胞后,熒光顯微鏡觀察修飾后的hESCs熒光蛋白表達(dá)情況,Western blot分析PDGF-BB的表達(dá),用MTT法檢測細(xì)胞增殖情況,并進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果：(1)PDGF-BB cDNA成功連接到穿梭載體pAD-track-CMV上,DNA測序結(jié)果與Genebank上PDGF-BB基因CDS區(qū)完全一致；rad-PDGF經(jīng)Pac I限制性內(nèi)切酶切可分離出一條為4.5kbp特異性大小的基因片段；包裝成功的腺病毒顆粒,其病毒滴度可達(dá)到1×109pfu/ml,通過PCR方法可以獲得大小為744bp大小的基因片段。 (2)分離后的表皮干細(xì)胞表達(dá)K19；在熒光顯微鏡下觀察約85%的rad-PDGF修飾后的hESCs有綠色熒光蛋白表達(dá)；rad-PDGF修飾的hESCs可表達(dá)hPDGF-BB蛋白；與對照組相比較,rad-PDGF修飾促進(jìn)了hESCs增殖(P0.05)。 結(jié)論：(1)通過細(xì)菌BJ5183同源重組的方法成功構(gòu)建PDGF-BB重組腺病毒表達(dá)載體rad-PDGF; (2) rad-PDGF能高效的導(dǎo)入hESCs中,并能在hESCs表達(dá)hPDGF-BB蛋白,rad-PDGF修飾促進(jìn)了hESCs的增殖。
[Abstract]:Objective to construct adenovirus expression vector of human platelet-derived growth factor (hPDGF-BB) gene. (2) to observe the effect of hPDGF-BB adenovirus expression vector modification on the proliferation of hESCs. Methods PCR method was used to synthesize PDGF-BB. the shuttle plasmid pAD-PDGF-BBB was constructed after being ligated with pAD-track-CMV plasmid, and pAD-PDGF-BB was sequenced to detect the PDGF-BB sequence. The recombinant plasmid rad-PDGF was obtained by homologous recombination of the skeleton plasmid pAD-easy I and the shuttle plasmid pAD-PDGF-BB in BJ5183 bacteria, and the Pac I restriction endonuclease digestion was performed to identify rad-PDGFI 0.8% agarose gel electrophoresis. Rad-PDGF was introduced into HEK293 cells by lipofectaminTM 2000 liposome. The supernatant was collected as the first generation virus by high speed centrifugation. The virus titer was detected by PCR to identify the PDGF-BB fragment carried by rad-PDGF and then the virus was amplified and purified. (2) Epidermal stem cells derived from prepuce of healthy young people were used as target cells, hESCs were isolated and purified in vitro, the expression of K19 in epidermal stem cells was detected by immunohistochemical method, and epidermal stem cells were modified with hPDGF-BB adenovirus expression vector. The expression of hESCs fluorescence protein was observed by fluorescence microscope. The expression of PDGF-BB was detected by Western blot, and the proliferation of cells was detected by MTT assay. Results the DNA sequence of PDGF-BB cDNA ligated to the shuttle vector pAD-track-CMV was identical to that of the CDS region of PDGF-BB gene on Genebank. A 4.5kbp specific gene fragment was isolated by Pac I restriction endonuclease digestion, and the adenovirus particles were successfully packaged. The titer of the virus can reach 1 脳 10 9 pFu / ml, and a 744 BP gene fragment can be obtained by PCR method. (2) the isolated epidermal stem cells expressed K19, the hESCs modified with about 85% rad-PDGF was observed to express hPDGF-BB protein by green fluorescent protein (GFP), and compared with the control group, the proliferation of hESCs was promoted by P0.05. Conclusion the recombinant adenovirus expression vector rad-PDGF of PDGF-BB was successfully constructed by bacterial BJ5183 homologous recombination. 2) rad-PDGF can be efficiently introduced into hESCs, and the expression of hPDGF-BB protein rad-PDGF in hESCs can promote the proliferation of hESCs.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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本文編號：1586462