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  本文關(guān)鍵詞： HIF-2α VEGF 免疫組化 786-0 OS-RC-2細(xì)胞 shRNA 腎癌　出處：《蘭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究低氧誘導(dǎo)因子2a (hypoxia-inducible factors-2α, HIF-2α)和血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)在腎細(xì)胞癌(renal cell carcinoma, RCC)中的表達(dá)及臨床意義。構(gòu)建在哺乳動物細(xì)胞中表達(dá)的低氧誘導(dǎo)因子-2的短發(fā)夾RNA的表達(dá)質(zhì)粒,在體外模擬腫瘤常氧和低氧狀態(tài)下研究對VEGF表達(dá)的影響。探討在786-0和OS-RC-2細(xì)胞株中HIF-2a對VEGF的表達(dá)影響,HIF-2α可能為腎透明細(xì)胞癌(clear cell renal cell carcinoma, CCRCC)潛在的新效靶。 方法：選用免疫組織化學(xué)方法,檢測HIF-2a蛋白和VEGF蛋白的表達(dá),其中76例RCC組織,12例癌旁組織,研究與RCC的相關(guān)性。構(gòu)建HIF-2a的重組載體質(zhì)粒psiHIV-U6, shRNA-1、2、3、4為重組質(zhì)粒。轉(zhuǎn)染至786-0細(xì)胞,常氧條件培養(yǎng)48h后提取總RNA逆轉(zhuǎn)錄cDNA后Real time-PCR篩選沉默效率最強(qiáng)的shRNA載體質(zhì)粒。該質(zhì)粒psiHIV-U6送公司測序鑒定。實(shí)驗(yàn)設(shè)空白、陰性對照組和實(shí)驗(yàn)組即重組質(zhì)粒組,每組均設(shè)常氧組和低氧組,共6組,空白對照組不做任何處理,陰性對照組轉(zhuǎn)染空質(zhì)粒,實(shí)驗(yàn)組轉(zhuǎn)染重組質(zhì)粒psiHIV-U6,細(xì)胞缺氧微環(huán)境通過含150umol/L氯化鈷(Cocl2)的完全培養(yǎng)基模擬。實(shí)時定量PCR分析HIF-2a shRNA重組質(zhì)粒轉(zhuǎn)染786-0和OS-RC-2細(xì)胞后常氧及低氧狀態(tài)下細(xì)胞的HIF-2a mRNA表達(dá),流式細(xì)胞儀分析凋亡變化,蛋白免疫印跡方法測定VEGF蛋白的變化。實(shí)驗(yàn)數(shù)據(jù)進(jìn)行了spssl8.2統(tǒng)計學(xué)軟件處理,計算資料以均值±標(biāo)準(zhǔn)差表示,臨床特點(diǎn)指標(biāo)采用x2檢驗(yàn)和秩和檢驗(yàn),Spearman用來等級相關(guān)分析,組間比較應(yīng)用方差分析,以P0.05有顯著性差異。 結(jié)果:HIF-2α和VEGF在正常腎組織中不表達(dá),而在RCC組織中表達(dá)較強(qiáng)。HIF-2α與腫瘤組織的病理類型,病理分級及臨床分期有顯著相關(guān)性,VEGF與腫瘤組織病理分級及臨床分期有顯著相關(guān)性。DNA測序分析證實(shí)了重組質(zhì)粒psiHIV-U6構(gòu)建成功,在786-0細(xì)胞株中shRNA-4沉默HIF-2a mRNA的效率最強(qiáng),shRNA-4可用于后續(xù)實(shí)驗(yàn)。HIF-2a重組質(zhì)粒shRNA-4成功轉(zhuǎn)染786-0和OS-RC-2細(xì)胞,轉(zhuǎn)染成功的細(xì)胞發(fā)出綠色熒光,與對照組的細(xì)胞形態(tài)不同。RT-PCR分析顯示：786-0細(xì)胞和OS-RC-2細(xì)胞中,在陰性對照組中,HIF-2a mRNA的表達(dá)低氧環(huán)境較高。實(shí)驗(yàn)組在低氧微環(huán)境下HIF-2α mRNA的表達(dá)量較高。低氧環(huán)境下較常氧環(huán)境下HIF-2α mRNA的表達(dá)量無明顯變化。轉(zhuǎn)染HIF-2α shRNA-4重組質(zhì)粒在低氧環(huán)境下能誘導(dǎo)786-0和OS-RC-2細(xì)胞凋亡增加。采用Quantity one軟件進(jìn)行灰度值分析,陰性對照組低氧組VEGF蛋白表達(dá)高于常氧組,實(shí)驗(yàn)組VEGF蛋白表達(dá)量同時下降,且常氧較低氧環(huán)境下VEGF蛋白下降顯著。 結(jié)論:HIF-2α和VEGF與腎透明細(xì)胞癌的生長密切相關(guān),HIF-2α的研究可能成為治療CCRCC患者潛在的效靶,為下一步研究提供了基礎(chǔ)。
[Abstract]:Objective: to study the expression and clinical significance of hypoxia-inducible factors-2 偽 (HIF-2 偽) and vascular endothelial growth factor vascular endothelial growth factor (VEGF) in renal cell carcinoma (RCC). The expression plasmid of hairpin RNA, The effect of HIF-2a on the expression of VEGF in 786-0 and OS-RC-2 cell lines was studied in vitro under simulated hypoxic and normoxic conditions. To explore the effect of HIF-2 偽 on VEGF expression in 786-0 and OS-RC-2 cell lines, HIF-2 偽 may be a potential new target for clear cell renal cell carcinoma (CCRCCs). Methods: immunohistochemical method was used to detect the expression of HIF-2a protein and VEGF protein, including 76 cases of RCC tissues and 12 cases of adjacent tissues, to study the correlation with RCC. The recombinant plasmid psiHIV-U6 of HIF-2a and shRNA-1m2m3O4 were constructed and transfected into 786-0 cells. After the total RNA reverse transcription cDNA was extracted under normoxic condition for 48 h, Real time-PCR was used to screen the shRNA vector plasmid with the best silencing efficiency. The plasmid psiHIV-U6 was sent to the company for sequencing. The experiment was blank, the negative control group and the experimental group were the recombinant plasmid group. Each group was divided into normoxic group and hypoxic group, six groups, blank control group did not do any treatment, negative control group transfected empty plasmid, In the experimental group, the recombinant plasmid psiHIV-1 U6 was transfected, and the cell anoxic microenvironment was simulated by a complete medium containing 150umoll / L cobalt chloride Cocl2.Real-time quantitative PCR was used to analyze the expression of HIF-2a mRNA in the cells transfected with HIF-2a shRNA recombinant plasmid 786-0 and OS-RC-2 cells in normoxic and hypoxic state. The changes of apoptosis were analyzed by flow cytometry and the changes of VEGF protein were measured by Western blot. The experimental data were processed by spssl8.2 software and the calculated data were expressed as mean 鹵standard deviation. The clinical characteristics were analyzed by Spearman and x2 test and rank sum test respectively. ANOVA was used in the comparison between groups. There was significant difference between the two groups (P0.05). Results there was no expression of HIF-2 偽 and VEGF in normal renal tissues, but strong expression of .HIF-2 偽 in RCC tissues and pathological types of tumor tissues. There was a significant correlation between the pathological grade and clinical stage. DNA sequencing confirmed the successful construction of recombinant plasmid psiHIV-U6. In 786-0 cell line, shRNA-4 silencing HIF-2a mRNA was the most efficient, which could be used in further experiment. HIF-2a recombinant plasmid shRNA-4 was successfully transfected into 786-0 and OS-RC-2 cells. The cell morphology was different from that in the control group. RT-PCR analysis showed that, in the OS-RC-2 cells and the cell lines, In the negative control group, the expression of HIF-2a mRNA was higher in hypoxic environment, the expression of HIF-2 偽 mRNA in experimental group was higher in hypoxic microenvironment, but the expression of HIF-2 偽 mRNA in hypoxic environment had no significant change. The recombinant plasmid transfected with HIF-2 偽 shRNA-4 was transfected with HIF-2a. The apoptosis of 786-0 and OS-RC-2 cells was increased in hypoxic environment. The gray value of 786-0 and OS-RC-2 cells was analyzed by Quantity one software. The expression of VEGF protein in hypoxic group was higher than that in normoxic group. The expression of VEGF protein in experimental group was decreased at the same time, and the VEGF protein in normoxic group was significantly lower than that in hypoxic group. Conclusion the study on the relationship between the growth of clear cell carcinoma of kidney and the growth of HIF-2 偽 and VEGF may be a potential target for the treatment of CCRCC patients, which may provide a basis for further research.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11

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相關(guān)期刊論文 前1條

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本文編號：1494125