ANXA2对人结直肠癌细胞行为的调节作用研究

【作者】 牟茂森；

【导师】 侯颖春；

【作者基本信息】 陕西师范大学 ， 细胞生物学， 2012， 博士

【摘要】 目的基因敲除优于基因敲低的各种方法,是因为某些蛋白可能在痕量的情况下即能完成其特定的生理功能。基因敲低不能完全去除靶蛋白的表达背景,从而得出的实验结果具有不确定性。本文采用基因完全敲除方法研究了ANXA2(膜联蛋白A2, AnnexinA2)基因在结直肠癌细胞的功能,该基因属于膜联蛋白家族的重要成员。该家族为一类具有钙离子螯合作用的磷脂蛋白,它们参与了生物体的多种生理和病理过程。目前,在人和动物体发现了有12种膜联蛋白家族成员,统称为ANXA。已有资料显示ANXA2的表达可能与结直肠癌等多种肿瘤的发病进程有关,并参与了心脑血管疾病等多种疾病的生理过程；而结直肠癌是人类十分常见的恶性肿瘤。有关ANXA2在肿瘤的发生、发展方面的研究还不够,现有资料大多是以基因表达敲低的方法进行的,这样的研究结果带有一定的不确定性。所以,为了确切地揭示ANXA2在肿瘤特别是结直肠癌的发展过程中的作用,以人结直肠癌细胞系Caco2为靶细胞,对该细胞的ANXA2基因进行了敲除(knockout),获得了ANXA2-/-Caco2细胞系,以野生型Caco2细胞(ANXA2+/+Caco2)为对照,研究了ANXA2基因的表达对癌细胞的生长、运动、凋亡的影响。方法：1.从GenBank搜索人ANXA2基因信息,通过clustalxl.83软件进行了序列比对,构建了ANXA2的基因敲除的重组子,用NotI限制性内切酶将重组子子线性化。2.用电转染方法,将靶向ANXA2的基因敲除重组子导入Caco2细胞。通过有限细胞稀释法,在96孔板中培养单个细胞,把外源性基因EGFP(绿色荧光蛋白,这里作为报告基因)、neoR(G418抗性基因,这里作为细胞筛选基因),插入到Caco2细胞基因组ANXA2基因外显子6使该基因表达完全终止,后以特异性PCR和Western blot鉴定基因敲除成功,遂建系成功。3.以MTT法检测靶向ANXA2基因敲除对Caco2细胞增殖的影响。4.以体外损伤修复法评价靶向ANXA2基因敲除对Caco2细胞运动能力的影响。5.以Hoechst33258染色、MitoViewTM633染色结合DAPI检测靶向ANXA2基因敲除对Caco2细胞凋亡的影响。结果：1.设计了以ANXA2基因外显子6为插入位点的基因敲除方法并成功的构建了ANXA2pPNT/I1/I2/I3基因敲除重组子。2.用电转染方法、有限稀释法、抗性筛选,建立了ANXA2-/-Caco2细胞系。3.以MTT法、损伤修复法检测及Hoechst33258染色、MitoViewTM633染色结合DAPI的结果显示,当ANXA2基因敲除后,Caco2细胞的增殖能力降低(p<0.05)；细胞迁移能力出现抑制(p<0.05)；细胞凋亡发生率有所增高(p<0.05)。结论：首次以ANXA2基因敲除的Caco2细胞(ANXA2-/-Caco2)研究了ANXA2在肿瘤发展过程中的作用,发现敲除ANXA2基因后显著降低Caco2细胞体外增殖和运动能力,但是对其凋亡发生有明显差异。本文结果提示ANXA2的表达在人结直肠癌的发展过程中扮演有重要角色,即促进了肿瘤的恶性发展过程。所以,敲除ANXA2基因对于肿瘤的预防、诊断和治疗具有的潜在的应用价值。更多还原

【Abstract】 Objective:Gene knockout is better than gene knockdown methods, because some protein may be in trace of circumstances, namely to complete its special physiological function. Gene knockdown cannot completely remove target protein expression background, and the experimental results obtained with uncertainty. ANXA2belongs to a super family of closely related calcium-and membrane-binding proteins. The family for a class of calcium phosphatide-binding protein, they are involved in a variety of organism physiological and pathological process. At present, Annexins have12members in human and vertebrate orthologues, collectively referred to as ANXA. Existing research data shows ANXA2’s expression was closely related to the process of colorectal cancer and others many kinds of tumor, and participated in the many kinds of disease physiological process, e.g. cardiovascular and cerebrovascular diseases. And, colorectal cancer is the human very common malignant tumor. The ANXA2in cancer occurrence, development research is not enough, the existing research materials are mostly employing RNAi technology or gene knockdown, so that the results of the study has some uncertainty. So, in order to reveal exactly ANXA2in tumor especially colorectal cancer development process’s function, the ANXA2-/-Caco2cell line was generated, with wild type Caco2cells (ANXA2+/+Caco2) for comparison, in order to invest the function the ANXA2gene expression in the tumor proliferation, motility and apoptosis.Methods:1. Based on the sequences of ANXA2searched from GenBank database, we select ANXA2gene exon6as the target using clustalx1.83software, then construct the pPNT/ANXA2/EGFP recombinant plasmids, then linearized by Not-I.2. Linearized recombinant was introduced into Caco2cells by using a Bio-Rad Xcell-Electroporator. Using limited dilution method, we cultured the single cell in96well plates. We employ the exogenous gene EGFP (enhanced green fluorescent protein, here as reporter gene), neoR (G418resistance gene, here as a cell genetic screening gene), inserted into the Caco2cell genome ANXA2gene exon6, in specific PCR and Western blot appraisal knockout success. So, the ANXA2-/-Caco2cell line was generated successfully.3. MTT assay was to investigate the influence of knockout treatment for ANXA2on cell proliferation.4. Wound healing assay was to estimate the effect of down-regulation of ANXA2on the cell motility of Caco2cells.5. Hoechst33258staining, MitoViewTM633staining and DAPI staining were performed to measure the effects of gene knockout of ANXA2on cell apoptosis.Results:1. Construct the pPNT/ANXA2/EGFP recombinant plasmids successfully, then linearized by Not1.2. Using the method of electricity transfection method, limit dilution method and resistance screening, we established a ANXA2-/-Caco2cell lines.3. In MTT, Hoechst33258staining, wound healing assay and MitoViewTM633staining and DAPI staining, the results show that ANXA2gene knockout cells’cell proliferation was lower (p<0.05); cell migration ability appeared inhibition (p<0.05); cell apoptosis rate was significantly difference (p<0.05) in ANXA2-/-Caco2cells than in ANXA2+/+Caco2cells.Conclusion:We studied the function of ANXA2in cancer development process for the first time in ANXA2gene knock out of Caco2cells (ANXA2-/-Caco2), this study suggested ANXA2’s expression in human colorectal cancer development process plays an important role, which promoted the development of malignant tumor. So, knockout of the ANXA2for tumor prevention, diagnosis and treatment has potential application value.更多还原