本文選題：胃癌 + IFITM3�。� 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：[背景]胃癌(Gastric cancer, GC)是全世界發(fā)病率和死亡率最高的惡性腫瘤之一據(jù)統(tǒng)計(jì),全世界每年有超過(guò)750,000例新確診病例并且五年生存率不足25%,早期發(fā)現(xiàn)、早期診斷和早期治療是提高胃癌生存率的重要措施。因此尋找新的胃癌治療靶點(diǎn)和早期診斷標(biāo)志物是控制其發(fā)生發(fā)展的關(guān)鍵。干擾素誘導(dǎo)的跨膜蛋白3(IFITM3),也稱為1-8U,是干擾素誘導(dǎo)跨膜蛋白家族的成員之一。它是由兩個(gè)短的具有高度相似但不同N-和C-末端的核心序列的跨膜結(jié)構(gòu)域蛋白(5-18 kDa)所組成。IFITM3在不同的細(xì)胞過(guò)程中起到了重要的作用,包括細(xì)胞粘附,免疫細(xì)胞調(diào)節(jié),生殖細(xì)胞歸巢和成熟和骨礦化。[目的]本研究旨在明確IFITM3在48例胃癌組織標(biāo)本及細(xì)胞中的的表達(dá)水平,通過(guò)體外實(shí)驗(yàn)重點(diǎn)探究其對(duì)胃癌細(xì)胞株遷移,侵襲,增殖和細(xì)胞周期的影響及相關(guān)的分子學(xué)機(jī)制,從而更好的探索和揭示IFITM3預(yù)防和治療胃癌的分子機(jī)理。[方法]1.通過(guò)熒光實(shí)時(shí)定量PCR (qRT-PCR)方法檢測(cè)收集的48例胃癌手術(shù)標(biāo)本(癌組織及其相應(yīng)的癌旁組織)中的IFITM3的mRNA的表達(dá)量,并進(jìn)一步分析其與臨床病例資料的關(guān)系；利用qRT-PCR和蛋白免疫印跡法(western blot)分別檢測(cè)出胃癌細(xì)胞AGS、SGC-7901、BGC-823、MKN45、MKN28和正常胃粘膜上皮細(xì)胞GES-1中IFITM3的表達(dá)；使用免疫組織化學(xué)技術(shù)檢測(cè)不同病理分期的胃癌組織中IFITM3的蛋白表達(dá)。2.利用慢病毒轉(zhuǎn)染技術(shù),建立IFITM3沉默的胃癌穩(wěn)定轉(zhuǎn)染細(xì)胞株,運(yùn)用細(xì)胞劃痕實(shí)驗(yàn)、Transwell跨膜實(shí)驗(yàn)、CCK8、流式細(xì)胞技術(shù)來(lái)觀察細(xì)胞的遷移、侵襲和增殖的能力。3.倒置顯微鏡下觀察IFITM3干擾前后穩(wěn)轉(zhuǎn)細(xì)胞株的形態(tài)學(xué)變化；使用熒光實(shí)時(shí)定量PCR和Western blot技術(shù)分別在RNA和蛋白水平上檢測(cè)沉默組、對(duì)照組以及正常細(xì)胞株中EMT (Epithelial-Mesenchymal Transitions,上皮間質(zhì)轉(zhuǎn)化)相關(guān)蛋白(E-Cadherin和Vimentin)的表達(dá)。4.使用熒光實(shí)時(shí)定量PCR、Western blot檢測(cè)MMP-2和MMP-9在IFITM3沉默胃癌細(xì)胞株和正常細(xì)胞中的不同表達(dá)。5.用VWnt/B-catenin通路特異性抑制劑XAV939作用于不同濃度的胃癌細(xì)胞株,qRT-PCR和Western blot檢測(cè)B-catenin和IFITM3表達(dá)的變化。[結(jié)果]結(jié)果1.我們發(fā)現(xiàn)IFITM3在胃癌組織中的表達(dá)量明顯高于癌旁組織,兩者差異具有統(tǒng)計(jì)學(xué)意義,通過(guò)臨床病理資料分析結(jié)果顯示：IFITM3的表達(dá)量與TNM分期、區(qū)域淋巴結(jié)的轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移相關(guān)；與胃正常粘膜上皮細(xì)胞GES-1相比,胃癌細(xì)胞株SGC-7901、BGC-823.AGS中IFITM3表達(dá)量顯著增高,其中,在SGC-7901細(xì)胞株的表達(dá)量最高,具有統(tǒng)計(jì)學(xué)差異,而MKN28、MKN45中的表達(dá)量無(wú)統(tǒng)計(jì)學(xué)意義。2.成功構(gòu)建IFITM3的穩(wěn)轉(zhuǎn)干擾胃癌細(xì)胞株SGC-7901和BGC-823, CCK8實(shí)驗(yàn)證明干擾IFITM3能夠抑制細(xì)胞的增值能力。流式周期實(shí)驗(yàn)結(jié)果表明IFITM3干擾株能夠使胃癌細(xì)胞的細(xì)胞周期停滯于G0/G1期并減少S期的細(xì)胞數(shù)。3.細(xì)胞劃痕實(shí)驗(yàn),Transwell跨膜實(shí)驗(yàn)表明IFITM3可以促進(jìn)胃癌細(xì)胞的遷移、侵襲能力。4.倒置電子顯微鏡觀察干擾IFITM3表達(dá)的穩(wěn)轉(zhuǎn)胃癌細(xì)胞株,細(xì)胞形態(tài)由正常的梭形轉(zhuǎn)變成卵圓形,且偽足變少,有逆轉(zhuǎn)EMT的趨勢(shì)；熒光實(shí)時(shí)定量PCR, Western blot結(jié)果示：在RNA和蛋白水平上,IFITM3的穩(wěn)轉(zhuǎn)干擾胃癌細(xì)胞株的E-Cadherin表達(dá)量增加,Vimentin表達(dá)量減少。5.使用熒光實(shí)時(shí)定量PCR、Western blot檢測(cè)到MMP-2和MMP-9在IFITM3沉默細(xì)胞株中顯著低表達(dá)。6.使用Wnt/β-catenin通路特異性抑制劑XAV939之后,通過(guò)熒光實(shí)時(shí)定量PCR、Western blot檢測(cè)相對(duì)于未處理組,IFITM3表達(dá)量隨β-catenin均顯著減少,差異有統(tǒng)計(jì)學(xué)意義。[結(jié)論]在胃癌組織中,IFITM3的高表達(dá)與腫瘤分期、淋巴結(jié)轉(zhuǎn)移及遠(yuǎn)處轉(zhuǎn)移密切相關(guān),IFITM3可作為一種腫瘤誘導(dǎo)因子直接影響胃癌細(xì)胞的遷移、侵襲及增殖等生物學(xué)特性。IFITM3有希望成為診斷和治療胃癌的新的分子標(biāo)記和靶點(diǎn)。
[Abstract]:[background] Gastric cancer (GC) is one of the most malignant tumors in the world. According to the statistics, there are more than 750000 newly diagnosed cases in the world and the five year survival rate is less than 25%. Early detection, early diagnosis and early treatment are important measures to improve the survival rate of gastric cancer. Therefore, to find new target for the treatment of gastric cancer. Point and early diagnostic markers are the key to control their development. Interferon induced transmembrane protein 3 (IFITM3), also known as 1-8U, is one of the members of the interferon induced transmembrane protein family. It is made up of two short transmembrane domain proteins (5-18 kDa) with highly similar but different N- and C- terminal sequences. The same cell process plays an important role, including cell adhesion, immune cell regulation, reproductive cell homing and maturation and bone mineralization. [Objective] the aim of this study was to clarify the expression level of IFITM3 in 48 specimens of gastric cancer tissues and cells, and to focus on the migration, invasion, proliferation and cell cycle of gastric cancer cell lines through in vitro experiments. The molecular mechanism of IFITM3 prevention and treatment of gastric cancer is better explored and revealed by the influence of the phase and the related molecular mechanism. [method]1.) was used to detect the mRNA expression of IFITM3 in the 48 cases of gastric cancer surgical specimens (cancer tissue and its corresponding cancerous tissue) collected by fluorescence real-time quantitative PCR (qRT-PCR) method, and to further analyze the expression of IFITM3. The expression of IFITM3 in gastric cancer cells AGS, SGC-7901, BGC-823, MKN45, MKN28 and normal gastric mucosal epithelial cells was detected by qRT-PCR and Western blot, and IFITM3 protein expression in gastric cancer tissues with different disease stages was detected by immunohistochemistry. Lentivirus transfection technique was used to establish IFITM3 silent transfected cell line for gastric cancer. The cell line scratch test, Transwell transmembrane experiment, CCK8, flow cytometry were used to observe the cell migration, invasion and proliferation. The morphological changes of the stable cell lines before and after IFITM3 interference were observed under the.3. inversion microscope; real-time quantitative PCR was used by fluorescence. The expression of EMT (Epithelial-Mesenchymal Transitions, epithelial mesenchymal transition) related protein (E-Cadherin and Vimentin) in the control group and the normal cell line (E-Cadherin and Vimentin) in the control group and the normal cell line (E-Cadherin and Vimentin) in the control group and the normal cell line were detected by Western blot technique respectively. The different expression of.5. in normal cells used VWnt/B-catenin pathway specific inhibitor XAV939 in different concentrations of gastric cancer cell lines. QRT-PCR and Western blot were used to detect the changes in the expression of B-catenin and IFITM3. [results] 1. we found that the expression of IFITM3 in gastric cancer tissues was significantly higher than that in the paracancerous tissues, and the difference was statistically significant. The significance, through the analysis of clinicopathological data, showed that the expression of IFITM3 was related to TNM staging, regional lymph node metastasis and distant metastasis; compared with GES-1 of normal gastric mucosa epithelial cells, the expression of IFITM3 in gastric cancer cell line, SGC-7901 and BGC-823.AGS increased significantly, and the expression of SGC-7901 cell lines was the highest, and the statistics were statistically significant. There was no significant difference in the expression of MKN28 and MKN45..2. was successfully constructed by the successful construction of IFITM3, which interfered with the gastric cancer cell line SGC-7901 and BGC-823. The CCK8 experiment showed that the interference of IFITM3 could inhibit the proliferation of cells. The results of the flow cycle experiment showed that the IFITM3 interfering strain could make the cell cycle of gastric cancer cells stagnate in G0/G1 and reduce S. The cell number.3. cell scratch test, the Transwell transmembrane experiment showed that IFITM3 could promote the migration of gastric cancer cells. The invasion ability.4. inverted electron microscope was used to observe the stable gastric cancer cell line that interfered with IFITM3 expression. The cell morphology changed from normal shuttle form to oval, and the pseudo foot was less, there was a tendency to reverse EMT, and real-time quantitative PCR of fluorescence PCR was found. The results of Western blot show that on the level of RNA and protein, the stability of IFITM3 interferes with the increase of E-Cadherin expression in gastric cancer cell lines, the expression of Vimentin is reduced by.5. using real-time quantitative PCR, and Western blot is used to detect the significant low expression of MMP-2 and MMP-9 in the silent cell lines. After the fluorescence real-time quantitative PCR and Western blot detection relative to the untreated group, the expression of IFITM3 decreased significantly with the beta -catenin, and the difference was statistically significant. [Conclusion] the high expression of IFITM3 is closely related to the tumor stage, lymph node metastasis and distant metastasis, and IFITM3 can be directly affected as a tumor inducer. Gastric cancer cell migration, invasion and proliferation and other biological characteristics.IFITM3 has the hope of becoming a new molecular marker and target for diagnosis and treatment of gastric cancer.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2
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本文編號(hào)：1942834