CXCR7在食管癌中的表达及对其细胞增殖的影响

发布时间：2018-03-03 00:32

本文选题：CXCR7　切入点：食管癌　出处：《新乡医学院》2016年硕士论文　论文类型：学位论文

【摘要】：背景恶性肿瘤的发生、发展过程是一个多基因,多阶段调控的复杂过程,其中涉及到多种癌基因的激活或者抑癌基因的失活。由于各种原因导致的癌基因的表达升高或者抑癌基因的表达下降又会引起相应信号级联通路上其它分子水平的变化,从而进一步影响细胞的生物学行为。研究证明,趋化因子及其受体与肿瘤的发生、发展关系密切。CXCR7作为一种趋化因子受体,与相应的配体结合后能参与多种恶性肿瘤的调控。然而,目前CXCR7在食管癌中的表达及作用知之甚少。目的分析趋化因子受体CXCR7在食管癌中的表达水平及其与食管癌细胞增殖之间的关系。初步探讨CXCR7在肿瘤发生、发展进程中的作用,为发现食管癌治疗领域新的分子靶点及临床诊治提供一定的理论依据。方法1.从14例食管癌组织及相应的癌旁正常组织中提取总RNA,然后反转录生成cDNA。应用q RT-PCR技术检测食管癌组织及正常组织中CXCR7 mRNA的表达水平;2.设计合成野生型和突变型CXCR7的特异性引物,应用PCR技术扩增人CXCR7野生型和突变型cDNA全长,通过双酶切和定向克隆技术将其与p EGFP-N1载体连接,分别构建野生型和突变型CXCR7过表达质粒p EGFP-wt CXCR7和p EGFP-CXCR7ΔC;设计合成靶向CXCR7基因的短发夹RNA(sh RNA),并将其插入p Silencer4.1-CMV neo载体,构建CXCR7 sh RNA干扰质粒p Silencer4.1-sh CXCR7;3.将以上构建好的重组质粒分别转染人食管癌细胞KYSE-510,48h后观察它们对食管癌细胞生物学的影响并拍照,利用Western blot实验检测重组质粒在食管癌细胞中的表达。4.将以上构建好的重组质粒分别转染人食管癌细胞KYSE-510,MTT实验检测0h、24h、48h和72h实验组和对照组细胞光密度值,分析重组质粒表达后对细胞增殖的影响。结果1.在食管癌组织中CXCR7 mRNA相对表达量(3.024±2.910)高于癌旁正常组织(1.446±1.118),差异有统计学意义(P0.05)。2.成功构建了表达质粒p EGFP-wt CXCR7、p EGFP-CXCR7ΔC和p Silencer4.1-sh CXCR7,重组质粒转染食管癌细胞后能够在细胞内成功表达。3.MTT实验检测转染后24h、48h和72h各组细胞的OD值,野生组(0.663±0.032、1.120±0.032、1.343±0.037)和突变组(0.573±0.031、0.948±0.050、1.189±0.024)均高于对照组(0.469±0.027、0.768±0.031、0.991±0.041),野生组OD值高于突变组,差异具有统计学意义(P0.01);干扰组(0.561±0.025、1.012±0.026、1.303±0.028)高于对照组(0.449±0.031、0.748±0.033、1.039±0.037),差异具有统计学意义(P0.01)。结论1.CXCR7在食管癌中高表达,提示其与食管癌的形成关系密切。2.CXCR7表达水平上调和下调后均促进了食管癌细胞的增殖,提示其可能通过直接或者间接途径发挥对肿瘤细胞的调节作用。3.野生型CXCR7比突变型CXCR7更能促进细胞的增殖,提示CXCR7碳末端在发挥其基因功能上有着不可或缺的作用。
[Abstract]:Background the occurrence and development of malignant tumor is a complex process of multi-gene and multi-stage regulation. This involves the activation of many kinds of oncogenes or the inactivation of tumor suppressor genes. The increase of oncogene expression or the decrease of tumor suppressor gene expression due to various reasons will cause changes in other molecules along the corresponding signal level. It is proved that chemokines and their receptors are closely related to the occurrence and development of tumor. CXCR7 is a chemokine receptor. When combined with the corresponding ligand, it can be involved in the regulation of many malignant tumors. However, At present, little is known about the expression and role of CXCR7 in esophageal carcinoma. Objective to analyze the expression level of chemokine receptor CXCR7 in esophageal carcinoma and its relationship with the proliferation of esophageal cancer cells, and to explore the role of CXCR7 in the carcinogenesis and progression of esophageal cancer. Methods 1. Total RNAs were extracted from 14 cases of esophageal carcinoma and corresponding adjacent normal tissues, then reverse transcription was used to produce cDNA. Q RT-PCR technique was used. The expression level of CXCR7 mRNA in esophageal carcinoma and normal tissues was detected. 2. The specific primers for the synthesis of wild-type and mutant CXCR7 were designed and synthesized. PCR technique was used to amplify the full length of human CXCR7 wild-type and mutant cDNA and ligated with p EGFP-N1 vector by double enzyme digestion and directional cloning. Wild type and mutant CXCR7 overexpression plasmids p EGFP-wt CXCR7 and p EGFP-CXCR7 螖 C were constructed, and the short hairpin RNA(sh RNAs targeting CXCR7 gene were designed and synthesized, and inserted into p Silencer4.1-CMV neo vector. Construction of CXCR7 sh RNA interference plasmid p Silencer4.1-sh CXCR7h3. The constructed recombinant plasmid was transfected into human esophageal carcinoma cell line KYSE-51010 for 48 h, and the effects on the cell biology were observed and photographed. Western blot assay was used to detect the expression of recombinant plasmid in esophageal carcinoma cells. The constructed recombinant plasmid was transfected into human esophageal carcinoma cell line KYSE-510MTT assay to detect the optical density of the cells in the experimental group and control group for 48 h and 72 h, respectively. Results 1. The relative expression of CXCR7 mRNA in esophageal carcinoma was 3.024 卤2.910), which was significantly higher than that in adjacent normal tissues (1.446 卤1.118). The expression plasmid p EGFP-wt CXCR7p EGFP-CXCR7 螖 C and p Silencer4.1-sh were successfully constructed. After transfection of CXCR7, the recombinant plasmid was able to express successfully in esophageal carcinoma cells. 3. MTT assay was used to detect the OD value of each group of cells 24 hours after transfection, 48 hours and 72 hours after transfection. The OD value of wild group was higher than that of control group (0.663 卤0.032 卤1.120 卤0.032 卤1.343 卤0.037) and mutant group (0.573 卤0.031 0.948 卤0.050 卤1.189 卤0.024) higher than that of control group (0.469 卤0.027 卤0.768 卤0.031 卤0.991 卤0.041), and the OD value of wild group was higher than that of mutant group (P0.001), and 0.561 卤0.061 卤0.0251.012 卤0.0261.303 卤0.028) was higher than that of control group (0.449 卤0.030.748 卤0.0330.39 卤0.03737). Conclusion the expression of CXCR7 in esophageal carcinoma was significantly higher than that in control group (P < 0.01). Conclusion the expression of CXCR7 in esophageal carcinoma is significantly higher than that in control group (P < 0.01). 2. The up-regulation and down-regulation of CXCR7 promoted the proliferation of esophageal cancer cells. The results suggest that CXCR7 can promote cell proliferation more directly or indirectly than mutant CXCR7, suggesting that the carbon terminal of CXCR7 plays an indispensable role in its gene function.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

【相似文献】