發(fā)布時(shí)間：2018-03-21 11:19

  本文選題：缺氧誘導(dǎo)因子-1α　切入點(diǎn)：血管內(nèi)皮生長(zhǎng)因子　出處：《華中科技大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:研究缺氧誘導(dǎo)因子-1α(HIF-1α)、血管內(nèi)皮生長(zhǎng)因子(VEGF)在鱗狀細(xì)胞癌細(xì)胞中的表達(dá)情況,及基因干擾HIF-1α后對(duì)鱗狀細(xì)胞癌細(xì)胞的影響。 方法:應(yīng)用逆轉(zhuǎn)錄PCR( RT-PCR)、蛋白免疫印記(Western Blot)技術(shù)比較鱗狀細(xì)胞癌細(xì)胞系(A431細(xì)胞)和永生化角質(zhì)細(xì)胞系(hacat細(xì)胞)mRNA和蛋白水平表達(dá);及基因敲除HIF-1α基因后HIF-1α、VEGF mRNA和蛋白表達(dá)情況;應(yīng)用流式細(xì)胞儀技術(shù)和CCK8細(xì)胞增殖檢測(cè)法檢測(cè)基因敲除HIF-1α基因后A431細(xì)胞凋亡和增殖情況。 結(jié)果:RT-PCR技術(shù)檢測(cè)A431細(xì)胞與hacat細(xì)胞相比HIF-1α、VEGF mRNA表達(dá)增加(P 0.05),敲除HIF-1α基因的A431細(xì)胞與未敲除HIF-1α基因的A431細(xì)胞相比HIF-1α、VEGF mRNA表達(dá)降低(P 0.05);Western Blot技術(shù)檢測(cè)A431細(xì)胞與hacat細(xì)胞相比蛋白表達(dá)增加(P 0.05),敲除HIF-1α基因的A431細(xì)胞與未敲除HIF-1α基因的A431細(xì)胞相比HIF-1α、VEGF蛋白表達(dá)降低(P 0.05);流式細(xì)胞儀技術(shù)檢測(cè)敲除HIF-1α基因的A431細(xì)胞與未敲除HIF-1α基因的A431細(xì)胞相比,凋亡細(xì)胞數(shù)增加、凋亡細(xì)胞比例增高且差異性有意義(P 0.05);CCK8細(xì)胞增殖檢測(cè)法檢測(cè)敲除HIF-1α基因的A431細(xì)胞與未敲除HIF-1α基因的A431細(xì)胞相比,細(xì)胞增殖速度慢,存在差異性表達(dá)(P 0.05)。 結(jié)論:HIF-1α、VEGF在鱗狀細(xì)胞癌細(xì)胞系A(chǔ)431中的表達(dá)明顯高于永生化的角質(zhì)細(xì)胞系hacat細(xì)胞中的表達(dá)。敲除HIF-1α基因后,A431細(xì)胞中HIF-1α、VEGF的表達(dá)均下降,且凋亡細(xì)胞數(shù)增加,增殖速度降低。這些均可說(shuō)明基因敲出HIF-1α后,可抑制VEGF的表達(dá)和功能,抑制鱗癌細(xì)胞的增殖并促進(jìn)其凋亡,表明HIF-1α基因靶向治療可做為治療鱗狀細(xì)胞癌的新型手段之一。
[Abstract]:Aim: to study the expression of hypoxia inducible factor-1 偽 (HIF-1 偽) and vascular endothelial growth factor (VEGF) in squamous cell carcinoma (SCC) and the effect of gene interference with HIF-1 偽 on squamous cell carcinoma (SCC). Methods: reverse transcriptase PCR (RT-PCR) technique was used to compare the expression of HIF-1 mRNA and protein in the squamous cell carcinoma cell line (Con A431 cell line) and immortalized keratinocyte cell line (Hhacat cell line), and the expression of HIF-1 偽 mRNA and protein after gene knockout (HIF-1 偽 gene knockout). Apoptosis and proliferation of A431 cells after HIF-1 偽 gene knockout were detected by flow cytometry and CCK8 cell proliferation assay. Results the expression of HIF-1 偽 -VEGF mRNA in A431 cells was higher than that in hacat cells. The expression of HIF-1 偽 -VEGF mRNA in A431 cells with and without HIF-1 偽 gene knockout was lower than that in A431 cells without HIF-1 偽 gene knockout. Western Blot technique was used to detect the expression of HIF-1 偽 -VEGF mRNA in A431 cells compared with hacat cells. The expression of HIF-1 偽 in A431 cells with HIF-1 偽 knockout was significantly lower than that in A431 cells without HIF-1 偽 gene knockout, and the expression of HIF-1 偽 -VEGF protein in A431 cells with HIF-1 偽 knockout gene was detected by flow cytometry compared with A431 cells without HIF-1 偽 gene knockout, and the expression of HIF-1 偽 protein in A431 cells with HIF-1 偽 gene knockout was significantly lower than that of A431 cells without HIF-1 偽 gene knockout by flow cytometry. When the number of apoptotic cells increased, the proportion of apoptotic cells increased and the difference was significant. The proliferation of A431 cells with HIF-1 偽 knockout was slower than that of A431 cells without HIF-1 偽 gene knockout by P0.05CCK8 cell proliferation assay. Conclusion the expression of HIF-1 偽 -VEGF in squamous cell carcinoma cell line A431 was significantly higher than that in immortalized keratinocyte cell line A431. The expression of HIF-1 偽 -VEGF was decreased and the number of apoptotic cells increased after knockout of HIF-1 偽 gene. These results suggest that HIF-1 偽 gene knockout can inhibit the expression and function of VEGF, inhibit the proliferation and promote the apoptosis of squamous cell carcinoma cells, suggesting that the targeted therapy of HIF-1 偽 gene can be used as one of the new methods for the treatment of squamous cell carcinoma.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R739.5

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