【作者】 廖刚；

【导师】 王子卫；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 博士

【摘要】 目的:证实EGFR在胃癌组织中的表达强度与胃癌进展存在联系,构建人EGFR显性负性突变体真核表达载体pEGFPN1-DNEGFR,并进行鉴定,瞬时转染COS-7细胞后检测DNEGFR-EGFP蛋白表达及进行亚细胞结构定位;稳定转染人胃癌细胞株,探讨DNEGFR-EGFP负调控EGFR功能的机制,以及检测其对胃癌细胞恶性表型的作用,并明确分子机制。方法:(1)应用免疫组织化学PV法,检测60例胃癌组织中EGFR的表达情况,并且分析其与临床病理特征的关系。(2)将RT-PCR方法扩增得到的编码EGFR信号肽段、胞外区和跨膜区的cDNA,定向克隆至空载体pEGFP-N1中,构建真核表达载体pEGFPN1-DNEGFR。经PCR扩增鉴定、双酶切鉴定、核苷酸序列测定以及生物信息学分析证明pEGFPN1-DNEGFR构建成功后,脂质体介导下瞬时转染体外培养的COS-7细胞,Western blotting检测DNEGFR-EGFP蛋白的表达,应用光谱激光扫描共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测。(3)应用pEGFPN1-DNEGFR在脂质体介导下稳定转染人胃癌细胞株SGC-7901、NCI-N87,应用Western blotting检测DNEGFR-EGFP蛋白的表达,光谱激光扫描共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测,并且RT-PCR、Western blotting检测DNEGFR-EGFP对内源性EGFR mRNA水平、蛋白及磷酸化水平的影响。(4)应用MTT法、流式细胞术、TUNEL法、划痕实验、细胞粘附实验、体外侵袭实验、HUVEC管腔结构形成实验、裸鼠皮下移植瘤模型、微血管计数检测DNEGFR-EGFP对胃癌细胞恶性表型的作用,Western blotting、ELISA检测相关蛋白明确分子机制。结果:(1)胃癌组织中EGFR阳性表达率为48.3%,其阳性表达与浸润深度、淋巴结转移、TNM分期相关。(2)PCR扩增鉴定、双酶切鉴定、核苷酸序列测定以及生物信息学分析证实pEGFPN1-DNEGFR构建成功,并且Western blotting检测DNEGFR-EGFP蛋白的表达,光谱激光扫描共聚焦显微镜观察显示DNEGFR-EGFP蛋白主要定位于细胞膜。(3)Western blotting检测到胃癌细胞中DNEGFR-EGFP蛋白的表达,光谱激光扫描共聚焦显微镜观察显示DNEGFR-EGFP蛋白主要定位于细胞膜,DNEGFR-EGFP降低内源性EGFR蛋白磷酸化水平,而对内源性EGFR mRNA水平及蛋白水平无影响。(4)DNEGFR-EGFP通过下调CDK2、Cyclin D1、pGSK-3β(ser 9),上调p21、p27导致G0/G1期阻滞和诱导凋亡,进一步达到抑制体外胃癌细胞生长的效应,通过下调MMP-2、MMP-9、VEGF的分泌,达到抑制细胞侵袭及血管形成能力的结果,并且抑制细胞粘附能力及运动能力,表明DNEGFR-EGFP对胃癌细胞恶性表型具有部分逆转作用。结论:免疫组织化学检测结果证明EGFR参与胃癌进展,为靶向EGFR生物治疗制剂在胃癌生物治疗中的应用提供了一定的理论依据;阐明DNEGFR-EGFP通过降低内源性EGFR蛋白磷酸化水平负调控内源性EGFR功能的分子机制,证实其能够部分逆转胃癌细胞恶性表型,并明确其分子机制,为“靶向EGFR显性负性策略”(我们将此阻断EGFR信号转导通路的方法命名为“靶向EGFR显性负性策略”)在胃癌生物治疗中进一步深入研究打下了基础。更多还原

【Abstract】 Objective: We aim at confirming that the expression intensity of epidermal growth factor receptor (EGFR) in gastric cancer tissues is correlated with gastric cancer progression. Then we construct and identify the eukaryotic expression vector(pEGFPN1-DNEGFR) carrying human dominant negative epidermal growth factor receptor(DNEGFR), for the purpose of detecting the expression and the sub-cellular localization of dominant negative epidermal growth factor receptor- enhanced green fluorescence protein (DNEGFR-EGFP) in COS-7 cells transfected transiently with pEGFPN1-DNEGFR.After stable transfection of human gastric cancer cells with pEGFPN1-DNEGFR,we propose to explore the mechanism of the inhibitory effect of DNEGFR-EGFP on endogenous EGFR function, to detect the effect on malignant phenotype of human gastric cancer cells, and to elucidate molecular mechanisms.Methods:(1) Immunohistochemistry PV method was used to detect the expression of EGFR in 60 cases of gastric cancer, and the relationship between protein expression and clinic pathological features was analyzed.(2) The cDNA coding signal peptide, extracellular ligand-binding domain and membrane-spanning region of epidermal growth factor receptor(EGFR) precursor was obtained by reverse transcription-polymerase chain reaction(RT-PCR),then directionally cloned into the empty vector(pEGFP-N1) to construct pEGFPN1-DNEGFR.After being confirmed by PCR amplification assay, double enzyme digestion, DNA sequencing and bioinformatics analysis of nucleotide sequence,pEGFPN1-DNEGFR was transfected transiently into COS-7 cells, mediated by Lipofectamine 2000. The expression of and the sub-cellular localization of DNEGFR-EGFP in COS-7 cells were detected by Western blotting and Laser Scanning Spectral Confocal Microscope, respectively.(3) The pEGFPN1-DNEGFR was transfected stably into human gastric cancer cells (SGC-7901 and NCI-N87), mediated by Lipofectamine 2000. The expression of and the sub-cellular localization of DNEGFR-EGFP in human gastric cancer cells were detected by Western blotting and Laser Scanning Spectral Confocal Microscope, respectively. The influence of DNEGFR-EGFP on endogenous EGFR mRNA level, EGFR protein and its phosphorylation level was detected by RT-PCR and Western blotting, respectively.(4) MTT(methyl thiazolyl tetrazolium) assay, flow cytometry, TUNEL(terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling) test, wound healing assay, cell adhesion assay, in vitro invasion assay, HUVEC(human umbilical vein endothelial cell) tube formation assay, subcutaneous xenograft model of nude mouse were used to test the effects of DNEGFR-EGFP on malignant phenotype of human gastric cancer cells, Western blotting、ELISA(enzyme linked immunosorbent assay) were used to test the expressions of relative proteins for the purpose of elucidating molecular mechanisms.Results:(1) The positive expression rate of EGFR was 48.3%(29/60) in gastric cancer tissues, and the positive expression was correlated with depth of invasion, lymph node metastasis,and TNM stage.(2)The pEGFPN1-DNEGFR was constructed successfully, which was confirmed by PCR amplification assay, double enzyme digestion, DNA sequencing and bioinformatics analysis of nucleotide sequence. The expression of DNEGFR-EGFP was verified by Western blotting, and its predominant localization on the cell membrane of COS-7 cells was identified by Laser Scanning Spectral Confocal Microscope.(3) The expression of DNEGFR-EGFP was verified by Western blotting, and its predominant localization on the cell membrane of gastric cancer cells was identified by Laser Scanning Spectral Confocal Microscope. DNEGFR-EGFP decreased the phosphorylation level of EGFR protein, but didn’t change EGFR mRNA level and protein level. (4) DNEGFR-EGFP led to G0/G1 arrest and induced apoptosis by down-regulating CDK2,Cyclin D1,pGSK-3β(ser 9) and up-regulating p21,p27,inhibited in vitro growth of human gastric cancer cells in the end; it repressed invasion and angiogenesis of SGC-7901 cells by inhibiting them from secreting MMP-2, MMP-9 and VEGF, and also inhibited adhesion ability and motility of cells. These results indicate that DNEGFR-EGFP reversed malignant phenotype of human gastric cancer cells in part.Conclusion: Immunohistochemistry results indicate EGFR takes part in gastric cancer progression, which lays a theoretical foundation on the application of biologicals targeting EGFR in gastric cancer biotherapy. The mechanisms that DNEGFR-EGFP inhibits endogenous EGFR function by down-regulating endogenous EGFR protein phosphorylation level are elucidated. It is verified that it reverses malignant phenotype of human gastric cancer cells in part, and the molecular mechanisms are elucidated, which lays a solid foundation for further research of“dominant negative strategy targeting EGFR”(we name the EGFR signal pathway blockade strategy as“dominant negative strategy targeting EGFR”) in gastric cancer biotherapy.更多还原