IFITM3在胃癌中的表达及其机制的研究

发布时间：2018-05-27 16:06

  本文选题：胃癌 + IFITM3　； 参考：《南京医科大学》2015年硕士论文

【摘要】：[背景]胃癌(Gastric cancer, GC)是全世界发病率和死亡率最高的恶性肿瘤之一据统计,全世界每年有超过750,000例新确诊病例并且五年生存率不足25%,早期发现、早期诊断和早期治疗是提高胃癌生存率的重要措施。因此寻找新的胃癌治疗靶点和早期诊断标志物是控制其发生发展的关键。干扰素诱导的跨膜蛋白3(IFITM3),也称为1-8U,是干扰素诱导跨膜蛋白家族的成员之一。它是由两个短的具有高度相似但不同N-和C-末端的核心序列的跨膜结构域蛋白(5-18 kDa)所组成。IFITM3在不同的细胞过程中起到了重要的作用,包括细胞粘附,免疫细胞调节,生殖细胞归巢和成熟和骨矿化。[目的]本研究旨在明确IFITM3在48例胃癌组织标本及细胞中的的表达水平,通过体外实验重点探究其对胃癌细胞株迁移,侵袭,增殖和细胞周期的影响及相关的分子学机制,从而更好的探索和揭示IFITM3预防和治疗胃癌的分子机理。[方法]1.通过荧光实时定量PCR (qRT-PCR)方法检测收集的48例胃癌手术标本(癌组织及其相应的癌旁组织)中的IFITM3的mRNA的表达量,并进一步分析其与临床病例资料的关系；利用qRT-PCR和蛋白免疫印迹法(western blot)分别检测出胃癌细胞AGS、SGC-7901、BGC-823、MKN45、MKN28和正常胃粘膜上皮细胞GES-1中IFITM3的表达；使用免疫组织化学技术检测不同病理分期的胃癌组织中IFITM3的蛋白表达。2.利用慢病毒转染技术,建立IFITM3沉默的胃癌稳定转染细胞株,运用细胞划痕实验、Transwell跨膜实验、CCK8、流式细胞技术来观察细胞的迁移、侵袭和增殖的能力。3.倒置显微镜下观察IFITM3干扰前后稳转细胞株的形态学变化；使用荧光实时定量PCR和Western blot技术分别在RNA和蛋白水平上检测沉默组、对照组以及正常细胞株中EMT (Epithelial-Mesenchymal Transitions,上皮间质转化)相关蛋白(E-Cadherin和Vimentin)的表达。4.使用荧光实时定量PCR、Western blot检测MMP-2和MMP-9在IFITM3沉默胃癌细胞株和正常细胞中的不同表达。5.用VWnt/B-catenin通路特异性抑制剂XAV939作用于不同浓度的胃癌细胞株,qRT-PCR和Western blot检测B-catenin和IFITM3表达的变化。[结果]结果1.我们发现IFITM3在胃癌组织中的表达量明显高于癌旁组织,两者差异具有统计学意义,通过临床病理资料分析结果显示：IFITM3的表达量与TNM分期、区域淋巴结的转移和远处转移相关；与胃正常粘膜上皮细胞GES-1相比,胃癌细胞株SGC-7901、BGC-823.AGS中IFITM3表达量显著增高,其中,在SGC-7901细胞株的表达量最高,具有统计学差异,而MKN28、MKN45中的表达量无统计学意义。2.成功构建IFITM3的稳转干扰胃癌细胞株SGC-7901和BGC-823, CCK8实验证明干扰IFITM3能够抑制细胞的增值能力。流式周期实验结果表明IFITM3干扰株能够使胃癌细胞的细胞周期停滞于G0/G1期并减少S期的细胞数。3.细胞划痕实验,Transwell跨膜实验表明IFITM3可以促进胃癌细胞的迁移、侵袭能力。4.倒置电子显微镜观察干扰IFITM3表达的稳转胃癌细胞株,细胞形态由正常的梭形转变成卵圆形,且伪足变少,有逆转EMT的趋势；荧光实时定量PCR, Western blot结果示：在RNA和蛋白水平上,IFITM3的稳转干扰胃癌细胞株的E-Cadherin表达量增加,Vimentin表达量减少。5.使用荧光实时定量PCR、Western blot检测到MMP-2和MMP-9在IFITM3沉默细胞株中显著低表达。6.使用Wnt/β-catenin通路特异性抑制剂XAV939之后,通过荧光实时定量PCR、Western blot检测相对于未处理组,IFITM3表达量随β-catenin均显著减少,差异有统计学意义。[结论]在胃癌组织中,IFITM3的高表达与肿瘤分期、淋巴结转移及远处转移密切相关,IFITM3可作为一种肿瘤诱导因子直接影响胃癌细胞的迁移、侵袭及增殖等生物学特性。IFITM3有希望成为诊断和治疗胃癌的新的分子标记和靶点。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2
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本文编号：1942834