本文選題：ARTD10 + 大腸癌��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：背景及目的:ADP核糖基化是一種重要的翻譯后修飾,可參與多種生理和病理行為的調控,如細胞死亡、細胞周期和DNA修復。我們之前的研究發(fā)現(xiàn)單ADP核糖基化轉移酶1(clostridial toxin-like ADP-ribosyltransferases 1,ARTC1)與大腸癌的增殖、凋亡、遷移、轉移、血管形成以及其他生物學行為密切相關。單ADP核糖基轉移酶ARTD10(ADP-ribosyltransferase diphtheria toxin-like 10)是新近發(fā)現(xiàn)的一種細胞內單ADP核糖基化轉移酶,其與腫瘤性疾病的關系研究甚少。ARTD10在大腸癌組織中的表達以及其對大腸癌生物學行為的影響尚不清楚。本課題首先檢測ARTD10在人大腸癌組織中及癌旁正常大腸粘膜中的表達情況,探討ARTD10在人大腸癌組織中及癌旁正常大腸粘膜中的表達差異;并以小鼠大腸癌CT26細胞系為研究對象,通過在體和離體實驗,觀察ARTD10對大腸癌CT26細胞增殖的影響,并初步探討其可能的機制,為ARTD10作為大腸癌靶向治療的潛在靶點提供初步實驗依據(jù)。方法:1.免疫組化方法檢測51例不同分化程度的人大腸癌組織及癌旁正常大腸黏膜中ARTD10的表達,激光共聚焦顯微鏡觀察人大腸癌細胞系LOVO細胞、SW480細胞及小鼠大腸癌細胞系CT26細胞中ARTD10的表達情況。2.以高表達ARTD10的小鼠大腸癌細胞系CT26細胞為研究對象,使用ARTD10特異性抑制劑處理后,以CCK-8法檢測CT26細胞增殖能力變化。構建Balb/c小鼠腋窩皮下移植瘤模型,觀察ARTD10對CT26細胞移植瘤生長的影響。3.使用Western Blot方法檢測ARTD10特異性抑制劑處理后CT26細胞核轉位因子β-catenin的表達和功能水平及其下游信號蛋白AXIN2和c-MYB的蛋白表達水平變化。結果:1.免疫組化結果顯示,ARTD10在人大腸癌組織和癌旁正常大腸黏膜中都有表達,主要定位于胞質,偶可見于胞核;人大腸癌組織中ARTD10的陽性率(85%)明顯高于癌旁正常大腸黏膜組織(19.4%)(P㩳0.05);不同分級的大腸癌中ARTD10的陽性程度有顯著性差異(P㩳0.05),ARTD10的陽性程度與大腸癌的分級呈正相關(P㩳0.05),但在不同年齡、性別、腫瘤部位、淋巴結轉移、遠處轉移、腫瘤分期和浸潤深度的大腸癌中ARTD10的表達沒有顯著差異(P0.05)。2.激光共聚焦顯微鏡觀察結果顯示,ARTD10在分化較高的人大腸癌SW480細胞中表達水平較低,而在分化較差的小鼠大腸癌CT26細胞系和人大腸癌LOVO細胞系中表達水平明顯高于前者。3.CCK8結果顯示,使用ARTD10特異性抑制劑抑制CT26細胞內ARTD10酶活性后,CT26細胞的增殖明顯受到抑制;并且隨著抑制劑濃度的增加,抑制作用也增強,表現(xiàn)出明顯的濃度依賴性(P0.05),在ARTD10特異性抑制劑濃度為0.5μM時對CT26細胞增殖抑制作用最明顯。用0.5μM濃度的ARTD10特異性抑制劑處理CT26細胞24h,48h,72h,96h和120h后,處理時間為72h時對CT26細胞增殖抑制作用最強。4.當腹腔注射ARTD10特異性抑制劑(2mg/kg)后,Balb/c小鼠腋窩皮下移植瘤重量和體積均明顯低于對照組(P㩳0.05)。5.Western Blot結果顯示,使用特異性抑制劑抑制ARTD10酶活性后,CT26細胞全細胞和細胞核的β-catenin蛋白表達水平較對照組都明顯降低(P0.05);β-catenin下游蛋白AXIN2表達增高(P0.05),而c-MYB表達降低(P0.05)。在Balb/c小鼠腋窩皮下移植瘤中,當ARTD10被特異性抑制劑抑制,移植瘤全細胞和移植瘤細胞核中的β-catenin蛋白表達水平也較對照組顯著降低(P0.05)。結論:1.ARTD10在大腸癌組織中的表達水平明顯高于癌旁正常的大腸黏膜組織,ARTD10的陽性程度與腫瘤的惡性程度呈正相關。2.ARTD10可促進大腸癌CT26細胞的增殖。3.ARTD10促進大腸癌CT26細胞的增殖,可能與ARTD10調節(jié)核轉位蛋白β-catenin的表達和活性,進一步影響β-catenin下游增殖相關蛋白c-MYB、AXIN2的表達有關。
[Abstract]:Background and purpose: ADP ribonylation is an important post-translational modification that can participate in the regulation of a variety of physiological and pathological behaviors, such as cell death, cell cycle and DNA repair. Our previous study found that single ADP ribonucleotransferase 1 (clostridial toxin-like ADP-ribosyltransferases 1, ARTC1) and colorectal cancer proliferation, apoptosis, migration, Metastasis, angiogenesis and other biological behavior are closely related. Single ADP ribonucleotransferase ARTD10 (ADP-ribosyltransferase diphtheria toxin-like 10) is a newly discovered intracellular single ADP ribonucleotransferase, and its relationship with tumor disease is less than the expression of.ARTD10 in colorectal carcinoma and its effect on colorectal cancer The effect of biological behavior is not clear. Firstly, the expression of ARTD10 in human colorectal carcinoma tissue and normal colorectal mucosa near cancer was first detected, and the difference in expression of ARTD10 in human colorectal carcinoma tissues and normal colorectal mucosa adjacent to cancer was investigated. The CT26 cell line of large intestine cancer in mice was used as the research object, and the observation was carried out in vivo and in vitro experiments. The effect of ARTD10 on the proliferation of colorectal cancer CT26 cells was investigated and its possible mechanism was preliminarily explored to provide a preliminary experimental basis for the potential target of ARTD10 as a target therapy for colorectal cancer. Method: 1. immunohistochemical method was used to detect the expression of ARTD10 in the colorectal cancer tissues and the normal colorectal mucosa adjacent to the cancer of different degrees of differentiation, and the laser copolymerization was shown. The expression of ARTD10 in human colorectal cancer cell line LOVO cells, SW480 cells and mouse colorectal cancer cell line CT26 cells was observed by microscope.2...2. was used to study the CT26 cells of colorectal cancer cell lines with high expression of ARTD10 in mice. The proliferation ability of CT26 cells was detected by CCK-8 method. The axillary growth of Balb/c mice was constructed by the CCK-8 method. The effect of ARTD10 on the growth of CT26 cell xenografts,.3. using Western Blot method to detect the expression and function level of CT26 nuclear transposition factor beta -catenin, and the changes in the protein expression level of the downstream signal protein AXIN2 and c-MYB. Results: 1. immunohistochemical results showed, A RTD10 was expressed in the human colorectal carcinoma tissue and the normal colorectal mucosa adjacent to the carcinoma, which was mainly located in the cytoplasm and even in the nucleus. The positive rate of ARTD10 in the colorectal cancer tissue was significantly higher than that of the normal colorectal mucosa (19.4%) (P? 0.05) beside the carcinoma (P? 0.05); the positive degree of ARTD10 in different classification of colorectal cancer was significantly different (P? 0.05), and the positive of ARTD10 The degree of sex was positively correlated with the classification of colorectal cancer (P? 0.05), but there was no significant difference in the expression of ARTD10 at different age, sex, tumor site, lymph node metastasis, distant metastasis, tumor staging and infiltration depth (P0.05).2. laser confocal microscope observation results showed that ARTD10 was in the SW480 cells with higher differentiation of human colorectal cancer cells. The expression level of the CT26 cell line and the human colorectal cancer LOVO cell line in the poorly differentiated mice was significantly higher than that of the former.3.CCK8. The proliferation of CT26 cells was obviously inhibited after the use of ARTD10 specific inhibitors to inhibit the ARTD10 activity in CT26 cells, and the inhibition was inhibited with the increase of the inhibitor concentration. The effect was also enhanced, showing a significant concentration dependence (P0.05), the most obvious inhibitory effect on the proliferation of CT26 cells when the concentration of ARTD10 specific inhibitors was 0.5 M. ARTD10 specific inhibitors of ARTD10 concentration were used to treat CT26 cell 24h, 48h, 72h, 96h and 120h. After the injection of ARTD10 specific inhibitor (2mg/kg), the weight and volume of subcutaneous transplantation tumor in Balb/c mice were significantly lower than that of the control group (P? 0.05).5.Western Blot results showed that the expression level of beta -catenin protein in whole cells and nuclei of CT26 cells was significantly lower than that of the control group (P0.05) after the inhibition of ARTD10 enzyme activity by specific inhibitors (P0.05). The expression of catenin downstream protein AXIN2 increased (P0.05) and c-MYB expression decreased (P0.05). In the subcutaneous tumor of axillary subcutaneous transplantation in Balb/c mice, when ARTD10 was suppressed by specific inhibitors, the expression level of beta -catenin in the whole cell and transplanted cell nucleus of the transplanted tumor was also significantly lower than that of the control group (P0.05). Conclusion: 1.ARTD10 in colorectal cancer tissue The level of ARTD10 was significantly higher than that of the normal colorectal mucosa, and the positive degree of the tumor was positively correlated with the malignant degree of the tumor..2.ARTD10 could promote the proliferation of colorectal cancer CT26 cells and promote the proliferation of CT26 cells in colorectal cancer. It may regulate the expression and activity of the nuclear transposition protein beta -catenin with ARTD10, and further influence the downstream of the beta -catenin. The expression of proliferation related protein c-MYB, AXIN2 is related.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.34

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本文編號：2052623