【摘要】：目的:1.構(gòu)建IQGAP1基因過(guò)表達(dá)和基因干擾的穩(wěn)轉(zhuǎn)細(xì)胞系及其對(duì)照細(xì)胞系。2.通過(guò)體內(nèi)外實(shí)驗(yàn)分別檢測(cè)IQGAP1基因?qū)ρ苌傻挠绊憽?.研究IQGAP1對(duì)血管生成影響的分子機(jī)制。方法:1.利用Lipofectamine 2000將綠色熒光蛋白(green fluorescent protein,GFP)標(biāo)記的IQGAP1過(guò)表達(dá)質(zhì)粒和它所對(duì)應(yīng)的對(duì)照質(zhì)粒分別轉(zhuǎn)染到人食管癌細(xì)胞EC9706,用G418耐藥單克隆細(xì)胞的篩選方法,等到穩(wěn)定生長(zhǎng)后,熒光顯微鏡下觀(guān)察其細(xì)胞定位,并通過(guò)Western blot檢測(cè)GFP-IQGAP1融合蛋白的表達(dá),確定IQGAP1過(guò)表達(dá)的穩(wěn)轉(zhuǎn)細(xì)胞系是否構(gòu)建成功。2.利用Lipofectamine 2000將IQGAP1干擾質(zhì)粒及其對(duì)照質(zhì)粒轉(zhuǎn)染進(jìn)人食管癌細(xì)胞KYSE150內(nèi),采用G418篩選耐藥克隆,待其穩(wěn)定生長(zhǎng)后,熒光顯微鏡下觀(guān)察KYSE150細(xì)胞GFP的表達(dá),并通過(guò)Western blot檢測(cè)對(duì)照組與干擾組IQGAP1蛋白的表達(dá)情況,確定IQGAP1干擾的穩(wěn)轉(zhuǎn)細(xì)胞系是否構(gòu)建成功。3.分別通過(guò)體外HUVECs小管形成實(shí)驗(yàn)和體內(nèi)的雞胚尿囊膜實(shí)驗(yàn)來(lái)檢測(cè)IQGAP1基因表達(dá)對(duì)腫瘤血管生成的影響。4.采用Western blot技術(shù)研究IQGAP1對(duì)食管癌細(xì)胞中VEGFA與其受體VEGFR2、p-VEGFR2血管生成相關(guān)因子表達(dá)的影響。5.通過(guò)Western blot技術(shù)研究IQGAP1對(duì)Akt和ERK活化的影響。6.在IQGAP1過(guò)表達(dá)組細(xì)胞中加入Akt和ERK抑制劑進(jìn)行干預(yù)處理后,觀(guān)察抑制劑對(duì)VEGFA和p-VEGFR2蛋白表達(dá)的影響及對(duì)血管生成的影響。結(jié)果:1.熒光顯微鏡下觀(guān)察發(fā)現(xiàn),在轉(zhuǎn)染含GFP-IQGAP1重組質(zhì)粒的EC9706細(xì)胞中觀(guān)察到其為胞質(zhì)定位;Western blot結(jié)果顯示,在IQGAP1過(guò)表達(dá)細(xì)胞中有GFP-IQGAP1融合蛋白的表達(dá),而在其對(duì)照組內(nèi)則沒(méi)有,說(shuō)明IQGAP1過(guò)表達(dá)的穩(wěn)轉(zhuǎn)細(xì)胞系已構(gòu)建成功。2.在熒光顯微鏡下可觀(guān)察到,GFP在轉(zhuǎn)染IQGAP1干擾質(zhì)粒及對(duì)照質(zhì)粒的KYSE150細(xì)胞中為全細(xì)胞定位;對(duì)支架蛋白IQGAP1在干擾組細(xì)胞內(nèi)的表達(dá)量與其在對(duì)照組細(xì)胞內(nèi)的表達(dá)量通過(guò)Western blot進(jìn)行比較,發(fā)現(xiàn)其表達(dá)量顯著降低,有統(tǒng)計(jì)學(xué)意義(P0.0001)。3.體外HUVECs小管形成的實(shí)驗(yàn)結(jié)果顯示,與對(duì)照組相比較,IQGAP1過(guò)表達(dá)組小管生成數(shù)更多,而且所生成小管的管腔結(jié)構(gòu)更加完整,兩者差異有統(tǒng)計(jì)學(xué)意義(P0.0001),表明IQGAP1過(guò)表達(dá)在體外可以促進(jìn)血管的生成;體內(nèi)雞胚尿囊膜的實(shí)驗(yàn)結(jié)果也顯示,IQGAP1過(guò)表達(dá)組生成的血管數(shù)量較對(duì)照組多,并且所生成血管的形態(tài)舒展,顏色較為鮮紅,經(jīng)統(tǒng)計(jì)學(xué)分析,差異有統(tǒng)計(jì)學(xué)意義(P0.001),說(shuō)明IQGAP1過(guò)表達(dá)可以促進(jìn)體內(nèi)血管的生成;體內(nèi)外實(shí)驗(yàn)結(jié)果一致,表明IQGAP1過(guò)表達(dá)后會(huì)促進(jìn)腫瘤血管的生成。4.IQGAP1干擾組采用體外HUVECs小管生成實(shí)驗(yàn),結(jié)果顯示,與對(duì)照組相比較,發(fā)現(xiàn)IQGAP1基因干擾后小管生成數(shù)較少,生成小管的管腔結(jié)構(gòu)也不完整,差異有統(tǒng)計(jì)學(xué)意義(P0.0001),表明IQGAP1干擾后在體外可以抑制血管的生成;體內(nèi)雞胚尿囊膜實(shí)驗(yàn)結(jié)果顯示,IQGAP1基因干擾后所生成的血管數(shù)量較對(duì)照組少,生成的血管色澤灰暗并且主血管較少,經(jīng)數(shù)據(jù)統(tǒng)計(jì)得,兩者之間的差異有統(tǒng)計(jì)學(xué)意義(P0.001);上述體內(nèi)外實(shí)驗(yàn)結(jié)果一致,表明干擾IQGAP1基因表達(dá)后能夠明顯抑制腫瘤的血管生成。5.在IQGAP1過(guò)表達(dá)的細(xì)胞中,經(jīng)Western blot檢測(cè)發(fā)現(xiàn),VEGFA與p-VEGFR2蛋白表達(dá)量與其在對(duì)照細(xì)胞內(nèi)的表達(dá)量相比都升高,差異有統(tǒng)計(jì)學(xué)意義(P0.0001),而VEGFR2蛋白的表達(dá)量則在IQGAP1過(guò)表達(dá)組與其對(duì)照組細(xì)胞中無(wú)明顯差異,表明IQGAP1可能通過(guò)VEGFA與VEGFR2相互作用后促進(jìn)VEGFR2發(fā)生磷酸化,進(jìn)而促進(jìn)血管的生成。6.在IQGAP1干擾組細(xì)胞中,經(jīng)Western blot檢測(cè)發(fā)現(xiàn),VEGFA與p-VEGFR2蛋白的表達(dá)量與其在對(duì)照細(xì)胞內(nèi)相比,表達(dá)量都降低,差異有統(tǒng)計(jì)學(xué)方面的意義(P0.0001),而VEGFR2的表達(dá)量則在IQGAP1干擾組與其對(duì)照組中無(wú)明顯差異,表明IQGAP1可能通過(guò)阻斷了VEGFA與其受體VEGFR2的相互作用進(jìn)而抑制血管生成的過(guò)程。7.經(jīng)Western blot檢測(cè)發(fā)現(xiàn),IQGAP1過(guò)表達(dá)組細(xì)胞中p-Akt、p-ERK蛋白的表達(dá)量與其在對(duì)照組的結(jié)果比較,發(fā)現(xiàn)兩者的表達(dá)量都升高,差異有統(tǒng)計(jì)學(xué)的意義(P0.0001);而IQGAP1干擾組細(xì)胞p-Akt、p-ERK的表達(dá)量均低于其在對(duì)照組內(nèi)的表達(dá)量,差異有統(tǒng)計(jì)學(xué)意義(P0.0001);說(shuō)明IQGAP1對(duì)腫瘤細(xì)胞血管生成的發(fā)生機(jī)制的調(diào)控可能通過(guò)活化Akt和ERK信號(hào)通路發(fā)揮作用的。8.在IQGAP1過(guò)表達(dá)組細(xì)胞中加入Akt和ERK抑制劑干預(yù)后,VEGFA和p-VEGFR2蛋白的表達(dá)量顯著降低,并且經(jīng)抑制劑干預(yù)后血管生成數(shù)也降低,說(shuō)明IQGAP1可能通過(guò)AKT或ERK/VEGFA-VEGFR2信號(hào)通路來(lái)影響腫瘤血管生成。結(jié)論:1.成功構(gòu)建了IQGAP1過(guò)表達(dá)穩(wěn)轉(zhuǎn)細(xì)胞系及IQGAP1干擾的穩(wěn)轉(zhuǎn)細(xì)胞系。2.IQGAP1過(guò)表達(dá)可以促進(jìn)體內(nèi)外腫瘤的血管生成,而干擾IQGAP1表達(dá)后則會(huì)有效的抑制腫瘤血管的生成。3.IQGAP1對(duì)食管癌中血管生成的作用機(jī)制可能與VEGFA與VEGFR2相互作用后活化VEGFR2有關(guān),以及與Akt和ERK信號(hào)通路活化有關(guān)。4.IQGAP1過(guò)表達(dá)組細(xì)胞中加入Akt和ERK抑制劑干預(yù)后,可能阻斷了Akt或ERK/VEGFA-VEGFR2信號(hào)通路,進(jìn)而導(dǎo)致血管生成作用被有效抑制。
[Abstract]:AIM: 1. To construct stable cell lines with IQGAP1 gene overexpression and gene interference and control cell lines. 2. To detect the effect of IQGAP1 gene on angiogenesis in vitro and in vivo. 3. To study the molecular mechanism of IQGAP1 on angiogenesis. Methods: 1. Green fluorescent protein (GFP) was prepared by Lipofectamine 2000. The labeled over-expression plasmid of IQGAP1 and its corresponding control plasmid were transfected into human esophageal carcinoma cell line EC9706, and the G418-resistant monoclonal cells were screened. After stable growth, the localization of the cells was observed under fluorescence microscope, and the expression of the fusion protein of GFP-IQGAP1 was detected by Western blot to confirm the steady thinning of the over-expression of IQGAP1. Cell lines were successfully constructed. 2. The interfering plasmid of IQGAP1 and its control plasmid were transfected into human esophageal carcinoma cell line KYSE150 by Lipofectamine 2000. The resistant clones were screened by G418. After stable growth, the expression of GFP in KYSE150 cells was observed by fluorescence microscope, and the expression of IQGAP1 protein in control group and interfering group was detected by Western blot. The effect of IQGAP1 gene expression on tumor angiogenesis was detected by HUVECs tubule formation test in vitro and chicken embryo allantoic membrane test in vivo. 4. The angiogenesis of VEGF A and its receptors, VEGF R2, p-VEGFR2, in esophageal cancer cells was studied by Western blot. The effects of IQGAP1 on Akt and ERK activation were studied by Western blot. 6. After intervention with Akt and ERK inhibitors, the effects of inhibitors on the expression of VEGFA and p-VEGFR2 and on angiogenesis were observed in IQGAP1 overexpression cells. Cytoplasmic localization was observed in EC9706 cells transfected with GFP-IQGAP1 recombinant plasmid; Western blot analysis showed that GFP-IQGAP1 fusion protein was expressed in IQGAP1 overexpressed cells, but not in the control group, indicating that stable transfected cell lines with IQGAP1 overexpression were successfully constructed. 2. GFP transfection was observed under fluorescence microscope. The expression of scaffold protein IQGAP1 in KYSE150 cells infected with the interfering plasmid and the control plasmid was compared with that in the interfering group by Western blot. The expression of scaffold protein IQGAP1 in KYSE150 cells was significantly lower than that in the control group (P 0.0001). 3. The experimental results of HUVECs tubule formation in vitro were statistically significant (P 0.0001). Compared with the control group, IQGAP1 overexpression group had more tubules and more intact lumen structure. The difference was statistically significant (P 0.0001), indicating that IQGAP1 overexpression could promote angiogenesis in vitro; chicken embryo allantoic membrane experiment in vivo also showed that IQGAP1 overexpression group had more vessels. The amount of IQGAP1 was more than that of the control group, and the angiogenesis was more relaxed and the color was bright red. The difference was statistically significant (P 0.001). The results of S-tubule formation test showed that IQGAP1 gene interference resulted in less tubule formation and incomplete tubule formation compared with the control group (P 0.0001), indicating that IQGAP1 gene interference could inhibit angiogenesis in vitro; chicken embryo allantoic membrane test in vivo showed that IQGAP1 gene interference could inhibit angiogenesis. Compared with the control group, the number of blood vessels was less, the color of blood vessels was gray and the main blood vessels were less. The difference between the two groups was statistically significant (P 0.001). Western blot analysis showed that the expression of VEGF A and p-VEGF R2 protein was significantly higher than that of the control cells (P 0.0001), while the expression of VEGF R2 protein was not significantly different between the IQGAP1 overexpression group and the control cells, suggesting that IQGAP1 may promote the expression of VEGF R2 through the interaction of VEGF A and VEGF R2. In the IQGAP1 interfering group, Western blot analysis showed that the expression of VEGF A and p-VEGFR2 protein was significantly lower than that in the control group (P 0.0001), while the expression of VEGF R2 was not significant in the IQGAP1 interfering group and the control group. The difference indicated that IQGAP1 might inhibit angiogenesis by blocking the interaction between VEGFA and its receptor, VEGFR2. 7. Western blot analysis showed that the expression of p-Akt and p-ERK protein in IQGAP1 overexpression group was higher than that in control group, and the difference was statistically significant (P 0.00). The expression of p-Akt and p-ERK in IQGAP1-treated cells was significantly lower than that in the control group (P 0.0001), indicating that IQGAP1 may play a role in the regulation of angiogenesis by activating Akt and ERK signaling pathways. 8. Inhibitors of Akt and ERK were added to IQGAP1-treated cells. After intervention, the expression of VEGFA and p-VEGFR2 protein was significantly decreased, and the number of angiogenesis was also decreased after intervention by inhibitors, indicating that IQGAP1 may affect tumor angiogenesis through AKT or ERK/VEGFA-VEGFR2 signaling pathway. Conclusion: 1. IQGAP1 overexpression stable transduction cell line and IQGAP1 interference stable transduction cell line were successfully constructed. IQGAP1 can promote angiogenesis in vitro and in vivo, and interfere with the expression of IQGAP1 can effectively inhibit tumor angiogenesis. 3. The mechanism of IQGAP1 on angiogenesis in esophageal cancer may be related to the activation of VEGF R2 after the interaction between VEGF A and VEGF R2, and the activation of Akt and ERK signaling pathways. After the intervention of KT and ERK inhibitors, Akt or ERK/VEGFA-VEGFR2 signaling pathway may be blocked, which leads to the inhibition of angiogenesis.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.1

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