發(fā)布時間：2018-03-03 00:32

  本文選題：CXCR7　切入點：食管癌　出處：《新鄉(xiāng)醫(yī)學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：背景惡性腫瘤的發(fā)生、發(fā)展過程是一個多基因,多階段調(diào)控的復(fù)雜過程,其中涉及到多種癌基因的激活或者抑癌基因的失活。由于各種原因?qū)е碌陌┗虻谋磉_升高或者抑癌基因的表達下降又會引起相應(yīng)信號級聯(lián)通路上其它分子水平的變化,從而進一步影響細胞的生物學(xué)行為。研究證明,趨化因子及其受體與腫瘤的發(fā)生、發(fā)展關(guān)系密切。CXCR7作為一種趨化因子受體,與相應(yīng)的配體結(jié)合后能參與多種惡性腫瘤的調(diào)控。然而,目前CXCR7在食管癌中的表達及作用知之甚少。目的分析趨化因子受體CXCR7在食管癌中的表達水平及其與食管癌細胞增殖之間的關(guān)系。初步探討CXCR7在腫瘤發(fā)生、發(fā)展進程中的作用,為發(fā)現(xiàn)食管癌治療領(lǐng)域新的分子靶點及臨床診治提供一定的理論依據(jù)。方法1.從14例食管癌組織及相應(yīng)的癌旁正常組織中提取總RNA,然后反轉(zhuǎn)錄生成cDNA。應(yīng)用q RT-PCR技術(shù)檢測食管癌組織及正常組織中CXCR7 mRNA的表達水平;2.設(shè)計合成野生型和突變型CXCR7的特異性引物,應(yīng)用PCR技術(shù)擴增人CXCR7野生型和突變型cDNA全長,通過雙酶切和定向克隆技術(shù)將其與p EGFP-N1載體連接,分別構(gòu)建野生型和突變型CXCR7過表達質(zhì)粒p EGFP-wt CXCR7和p EGFP-CXCR7ΔC;設(shè)計合成靶向CXCR7基因的短發(fā)夾RNA(sh RNA),并將其插入p Silencer4.1-CMV neo載體,構(gòu)建CXCR7 sh RNA干擾質(zhì)粒p Silencer4.1-sh CXCR7;3.將以上構(gòu)建好的重組質(zhì)粒分別轉(zhuǎn)染人食管癌細胞KYSE-510,48h后觀察它們對食管癌細胞生物學(xué)的影響并拍照,利用Western blot實驗檢測重組質(zhì)粒在食管癌細胞中的表達。4.將以上構(gòu)建好的重組質(zhì)粒分別轉(zhuǎn)染人食管癌細胞KYSE-510,MTT實驗檢測0h、24h、48h和72h實驗組和對照組細胞光密度值,分析重組質(zhì)粒表達后對細胞增殖的影響。結(jié)果1.在食管癌組織中CXCR7 mRNA相對表達量(3.024±2.910)高于癌旁正常組織(1.446±1.118),差異有統(tǒng)計學(xué)意義(P0.05)。2.成功構(gòu)建了表達質(zhì)粒p EGFP-wt CXCR7、p EGFP-CXCR7ΔC和p Silencer4.1-sh CXCR7,重組質(zhì)粒轉(zhuǎn)染食管癌細胞后能夠在細胞內(nèi)成功表達。3.MTT實驗檢測轉(zhuǎn)染后24h、48h和72h各組細胞的OD值,野生組(0.663±0.032、1.120±0.032、1.343±0.037)和突變組(0.573±0.031、0.948±0.050、1.189±0.024)均高于對照組(0.469±0.027、0.768±0.031、0.991±0.041),野生組OD值高于突變組,差異具有統(tǒng)計學(xué)意義(P0.01);干擾組(0.561±0.025、1.012±0.026、1.303±0.028)高于對照組(0.449±0.031、0.748±0.033、1.039±0.037),差異具有統(tǒng)計學(xué)意義(P0.01)。結(jié)論1.CXCR7在食管癌中高表達,提示其與食管癌的形成關(guān)系密切。2.CXCR7表達水平上調(diào)和下調(diào)后均促進了食管癌細胞的增殖,提示其可能通過直接或者間接途徑發(fā)揮對腫瘤細胞的調(diào)節(jié)作用。3.野生型CXCR7比突變型CXCR7更能促進細胞的增殖,提示CXCR7碳末端在發(fā)揮其基因功能上有著不可或缺的作用。
[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 鹵2.910), which was significantly higher than that in adjacent normal tissues (1.446 鹵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 鹵0.032 鹵1.120 鹵0.032 鹵1.343 鹵0.037) and mutant group (0.573 鹵0.031 0.948 鹵0.050 鹵1.189 鹵0.024) higher than that of control group (0.469 鹵0.027 鹵0.768 鹵0.031 鹵0.991 鹵0.041), and the OD value of wild group was higher than that of mutant group (P0.001), and 0.561 鹵0.061 鹵0.0251.012 鹵0.0261.303 鹵0.028) was higher than that of control group (0.449 鹵0.030.748 鹵0.0330.39 鹵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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.1

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