支架蛋白IQGAP1对人食管癌细胞血管生成的影响及其机制的研究
发布时间：2018-08-30 07:52
【摘要】：目的:1.构建IQGAP1基因过表达和基因干扰的稳转细胞系及其对照细胞系。2.通过体内外实验分别检测IQGAP1基因对血管生成的影响。3.研究IQGAP1对血管生成影响的分子机制。方法:1.利用Lipofectamine 2000将绿色荧光蛋白(green fluorescent protein,GFP)标记的IQGAP1过表达质粒和它所对应的对照质粒分别转染到人食管癌细胞EC9706,用G418耐药单克隆细胞的筛选方法,等到稳定生长后,荧光显微镜下观察其细胞定位,并通过Western blot检测GFP-IQGAP1融合蛋白的表达,确定IQGAP1过表达的稳转细胞系是否构建成功。2.利用Lipofectamine 2000将IQGAP1干扰质粒及其对照质粒转染进人食管癌细胞KYSE150内,采用G418筛选耐药克隆,待其稳定生长后,荧光显微镜下观察KYSE150细胞GFP的表达,并通过Western blot检测对照组与干扰组IQGAP1蛋白的表达情况,确定IQGAP1干扰的稳转细胞系是否构建成功。3.分别通过体外HUVECs小管形成实验和体内的鸡胚尿囊膜实验来检测IQGAP1基因表达对肿瘤血管生成的影响。4.采用Western blot技术研究IQGAP1对食管癌细胞中VEGFA与其受体VEGFR2、p-VEGFR2血管生成相关因子表达的影响。5.通过Western blot技术研究IQGAP1对Akt和ERK活化的影响。6.在IQGAP1过表达组细胞中加入Akt和ERK抑制剂进行干预处理后,观察抑制剂对VEGFA和p-VEGFR2蛋白表达的影响及对血管生成的影响。结果:1.荧光显微镜下观察发现,在转染含GFP-IQGAP1重组质粒的EC9706细胞中观察到其为胞质定位;Western blot结果显示,在IQGAP1过表达细胞中有GFP-IQGAP1融合蛋白的表达,而在其对照组内则没有,说明IQGAP1过表达的稳转细胞系已构建成功。2.在荧光显微镜下可观察到,GFP在转染IQGAP1干扰质粒及对照质粒的KYSE150细胞中为全细胞定位;对支架蛋白IQGAP1在干扰组细胞内的表达量与其在对照组细胞内的表达量通过Western blot进行比较,发现其表达量显著降低,有统计学意义(P0.0001)。3.体外HUVECs小管形成的实验结果显示,与对照组相比较,IQGAP1过表达组小管生成数更多,而且所生成小管的管腔结构更加完整,两者差异有统计学意义(P0.0001),表明IQGAP1过表达在体外可以促进血管的生成;体内鸡胚尿囊膜的实验结果也显示,IQGAP1过表达组生成的血管数量较对照组多,并且所生成血管的形态舒展,颜色较为鲜红,经统计学分析,差异有统计学意义(P0.001),说明IQGAP1过表达可以促进体内血管的生成;体内外实验结果一致,表明IQGAP1过表达后会促进肿瘤血管的生成。4.IQGAP1干扰组采用体外HUVECs小管生成实验,结果显示,与对照组相比较,发现IQGAP1基因干扰后小管生成数较少,生成小管的管腔结构也不完整,差异有统计学意义(P0.0001),表明IQGAP1干扰后在体外可以抑制血管的生成;体内鸡胚尿囊膜实验结果显示,IQGAP1基因干扰后所生成的血管数量较对照组少,生成的血管色泽灰暗并且主血管较少,经数据统计得,两者之间的差异有统计学意义(P0.001);上述体内外实验结果一致,表明干扰IQGAP1基因表达后能够明显抑制肿瘤的血管生成。5.在IQGAP1过表达的细胞中,经Western blot检测发现,VEGFA与p-VEGFR2蛋白表达量与其在对照细胞内的表达量相比都升高,差异有统计学意义(P0.0001),而VEGFR2蛋白的表达量则在IQGAP1过表达组与其对照组细胞中无明显差异,表明IQGAP1可能通过VEGFA与VEGFR2相互作用后促进VEGFR2发生磷酸化,进而促进血管的生成。6.在IQGAP1干扰组细胞中,经Western blot检测发现,VEGFA与p-VEGFR2蛋白的表达量与其在对照细胞内相比,表达量都降低,差异有统计学方面的意义(P0.0001),而VEGFR2的表达量则在IQGAP1干扰组与其对照组中无明显差异,表明IQGAP1可能通过阻断了VEGFA与其受体VEGFR2的相互作用进而抑制血管生成的过程。7.经Western blot检测发现,IQGAP1过表达组细胞中p-Akt、p-ERK蛋白的表达量与其在对照组的结果比较,发现两者的表达量都升高,差异有统计学的意义(P0.0001);而IQGAP1干扰组细胞p-Akt、p-ERK的表达量均低于其在对照组内的表达量,差异有统计学意义(P0.0001);说明IQGAP1对肿瘤细胞血管生成的发生机制的调控可能通过活化Akt和ERK信号通路发挥作用的。8.在IQGAP1过表达组细胞中加入Akt和ERK抑制剂干预后,VEGFA和p-VEGFR2蛋白的表达量显著降低,并且经抑制剂干预后血管生成数也降低,说明IQGAP1可能通过AKT或ERK/VEGFA-VEGFR2信号通路来影响肿瘤血管生成。结论:1.成功构建了IQGAP1过表达稳转细胞系及IQGAP1干扰的稳转细胞系。2.IQGAP1过表达可以促进体内外肿瘤的血管生成,而干扰IQGAP1表达后则会有效的抑制肿瘤血管的生成。3.IQGAP1对食管癌中血管生成的作用机制可能与VEGFA与VEGFR2相互作用后活化VEGFR2有关,以及与Akt和ERK信号通路活化有关。4.IQGAP1过表达组细胞中加入Akt和ERK抑制剂干预后,可能阻断了Akt或ERK/VEGFA-VEGFR2信号通路,进而导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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
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