【摘要】：目的:胃癌為最常見的消化道腫瘤之一,發(fā)病率及死亡率均高居消化道腫瘤之首。胃癌侵襲及遷移的生物學(xué)特征是導(dǎo)致胃癌患者死亡及預(yù)后差的主要原因。而腫瘤細胞侵襲、遷移的特性與腫瘤酸性微環(huán)境之間有著密不可分的關(guān)系。TSPAN9為四跨膜蛋白超家族中的一員,在胃癌中研究甚少,本研究擬探討TSPAN9對胃癌SGC7901細胞在腫瘤酸性微環(huán)境下侵襲、遷移的影響。方法:用合成的TSPAN9基因連接至pIRES2-ZsGreen1載體從而獲得重組質(zhì)粒,然后轉(zhuǎn)入TOP10克隆菌株中進行大規(guī)�；驍U增,轉(zhuǎn)染至胃癌SGC7901細胞中從而構(gòu)建TSPAN9過表達轉(zhuǎn)染組,即SGC7901/TSPAN9(轉(zhuǎn)染組)。同時培養(yǎng)SGC7901(空白組)及SGC7901/NC(對照組)。后用3%的HCL調(diào)節(jié)細胞培養(yǎng)基PH值為7.4及6.5分別培養(yǎng)72小時,形成7.4-SGC7901、6.5-SGC7901、7.4-SGC7901/NC、6.5-SGC7901/NC、7.4-SGC7901/TSPAN9、6.5-SGC7901/TSPAN9。應(yīng)用實時熒光定量PCR(q RT-PCR)和Western Blot分別在RNA、蛋白質(zhì)水平驗證TSPAN9過表達。通過劃痕實驗、Transwell侵襲及遷移實驗觀察腫瘤酸性微環(huán)境對胃癌細胞侵襲、遷移的影響及TSPAN9在其中所發(fā)揮的作用。應(yīng)用Western blot檢測TSPAN9在腫瘤酸性微環(huán)境下對胃癌SGC7901細胞中u PA、MMP-9表達情況的影響。結(jié)果:SGC7901/TSPAN9轉(zhuǎn)染組在PH=6.5即處于腫瘤酸性微環(huán)境下TSPAN9在RNA及蛋白質(zhì)水平均高表達。通過劃痕實驗、Transwell實驗,6.5-SGC7901較7.4-SGC7901可明顯促進胃癌SGC7901細胞的侵襲及遷移能力,P0.05。而6.5-SGC7901、6.5-SGC7901/NC和6.5-SGC7901/TSPAN9組相比,轉(zhuǎn)染組可明顯促進u PA、MMP-9的表達,同時促進細胞的侵襲及遷移,P0.05。結(jié)論:腫瘤酸性微環(huán)境促進TSPAN9在RNA及蛋白質(zhì)水平上的表達,腫瘤酸性微環(huán)境促進胃癌細胞的侵襲及轉(zhuǎn)移。腫瘤酸性微環(huán)境通過上調(diào)TSPAN9促進胃癌SGC7901細胞的侵襲、遷移能力。將為胃癌在腫瘤酸性微環(huán)境下的治療提供新靶點及新思路。
[Abstract]:Objective: gastric cancer is one of the most common digestive tract tumors. The biological characteristics of invasion and migration of gastric cancer are the main causes of death and poor prognosis of gastric cancer patients. However, the characteristics of invasion and migration of tumor cells are closely related to the acidic microenvironment of tumor. TSPAN9 is a member of the four-transmembrane protein superfamily. The purpose of this study was to investigate the effect of TSPAN9 on invasion and migration of gastric cancer SGC7901 cells in acidic microenvironment. Methods: the recombinant plasmid was obtained by ligating the synthesized TSPAN9 gene into the pIRES2-ZsGreen1 vector and then transferred into the TOP10 clone strain to amplify the gene. The recombinant plasmid was transfected into gastric cancer SGC7901 cells to construct the transfection group of TSPAN9 overexpression, that is, SGC7901/TSPAN9 (transfection group). SGC7901 (blank group) and SGC7901/NC (control group) were cultured at the same time. The cell culture medium PH value of 3% HCL was 7.4 and 6.5 for 72 hours, respectively, forming 7.4-SGC7901 6.5-SGC7901 + 7.4-SGC7901 / NC6.5-SGC7901 / SGC7901 / SGC7901 / TSPAN96.5-SGC7901 / TSPAN9. Real-time fluorescence quantitative PCR (q RT-PCR) and Western Blot were used to verify the overexpression of TSPAN9 at RNA, protein level, respectively. The effects of tumor acidic microenvironment on the invasion and migration of gastric cancer cells and the role of TSPAN9 in the invasion and migration of gastric cancer cells were observed by the scratch test and Transwell invasion and migration experiments. Western blot was used to detect the effect of TSPAN9 on the expression of u PA,MMP-9 in gastric cancer SGC7901 cells in acidic microenvironment. Results the expression of TSPAN9 in RNA and protein levels in the PH=6.5 group was significantly higher than that in the TSPAN9 / SGC7901 / TSPAN9 transfection group in the presence of tumor acidic microenvironment. Compared with 7.4-SGC7901, 6.5-SGC7901 significantly promoted the invasion and migration of gastric cancer SGC7901 cells (P0.05). Compared with 6.5-SGC7901/TSPAN9 group, 6.5-SGC7901 + 6.5-SGC7901 / NC could significantly promote the expression of u PA,MMP-9 and promote cell invasion and migration (P0.05). Conclusion: tumor acidic microenvironment promotes the expression of TSPAN9 at the level of RNA and protein, and tumor acidic microenvironment promotes the invasion and metastasis of gastric cancer cells. Tumor acidic microenvironment promotes the invasion and migration of gastric cancer SGC7901 cells by upregulating TSPAN9. It will provide new targets and new ideas for the treatment of gastric cancer in acidic microenvironment.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2

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