本文關(guān)鍵詞： 胰腺癌 TRPM8 Western Blot 膜片鉗 CCK8　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:1.TRPM8作為臨床生物標(biāo)記物,為準(zhǔn)確判斷胰腺癌的惡性度及預(yù)后提供指導(dǎo)。2.TRPM8作為潛在的藥物作用靶點,將來可能通過下調(diào)TRPM8的表達(dá),來抑制胰腺癌細(xì)胞增殖擴(kuò)撒,從而最終可以發(fā)現(xiàn)有效的胰腺癌個性化治療的新靶點和新思路。方法:1.購買北京龍邁達(dá)斯科技開發(fā)有限公司胰腺癌組織77例,正常組織30例,用免疫組織化學(xué)技術(shù)對標(biāo)本進(jìn)行實驗,并對結(jié)果進(jìn)行統(tǒng)計分析。2.購買正常胰腺細(xì)胞株HPCY-5和胰腺癌細(xì)胞株Bx PC-3、PANC-1并進(jìn)行細(xì)胞培養(yǎng),通過QPCR技術(shù)測定TRPM8離子通道m(xù) RNA的表達(dá)情況。3.用Western bot技術(shù)檢測細(xì)胞株HPCY-5、Bx PC-3、PANC-1通道蛋白的表達(dá)情況。4.用細(xì)胞膜片鉗技術(shù)檢測轉(zhuǎn)染si RNA前后胰腺癌細(xì)胞株P(guān)ANC1細(xì)胞膜電流的改變。5.用CCK8試劑盒檢測轉(zhuǎn)染si RNA前后胰腺癌細(xì)胞株Bx PC-3、PANC-1,分析細(xì)胞增殖能力的改變結(jié)果:免疫組化實驗、Western Blot實驗都提示TRPM通道蛋白在胰腺癌細(xì)胞中成高表達(dá)狀態(tài),且免疫組化結(jié)果提示腫瘤細(xì)胞中TRPM8通道蛋白的高表達(dá)與腫瘤的分化程度具有相關(guān)性(中低分化vs高分化P0.05,具有統(tǒng)計學(xué)意義)。QPCR檢測胰腺癌腫瘤細(xì)胞株Bx PC-3、PANC-1中TRPM8 m RNA相對于正常細(xì)胞株HPCY-5呈高表達(dá),與免疫組化和Western Blot結(jié)果相一致,且P0.05具有統(tǒng)計學(xué)意義。膜片鉗技術(shù)測定薄荷醇激活的TRPM8離子通道電流明顯增大。CCK8法提示TRPM8通道基因敲除的癌細(xì)胞株增殖能力出現(xiàn)明顯減弱,證明TRPM8通道基因的表達(dá)對癌細(xì)胞的增殖具有一定影響。
[Abstract]:Objective: 1. TRPM8, as a clinical biomarker, provides guidance for accurately judging the malignancy and prognosis of pancreatic cancer. 2. TRPM8 is a potential drug target. It may inhibit proliferation and diffusion of pancreatic cancer cells by down-regulating the expression of TRPM8 in the future. In the end, we can find new targets and new ideas for effective personalized treatment of pancreatic cancer. Methods: 1. Buy 77 cases of pancreatic cancer tissue and 30 cases of normal tissue from Beijing Longmeidas Technology Development Co., Ltd. The results were statistically analyzed by immunohistochemical technique. 2. The normal pancreatic cell line HPCY-5 and the pancreatic cancer cell line Bx PC-3 PANC-1 were purchased and cultured. The expression of m RNA in TRPM8 ion channel was detected by QPCR. The expression of Western bot was detected in HPCY-5 BX PC-3PANC-1 cell line. 4. Cell patch clamp technique was used to detect the cell membrane current of PANC1 cell line before and after transfection of si RNA. CCK8 kit was used to detect the proliferation of pancreatic cancer cell line BxPC-3pPANC-1 before and after transfection of si RNA, and the results showed that the expression of TRPM channel protein in pancreatic cancer cells was high. The results of immunohistochemistry indicated that the high expression of TRPM8 channel protein in tumor cells was correlated with the degree of tumor differentiation (medium and low differentiation vs highly differentiated P0.05, and the detection of TRPM8 m RNA in pancreatic cancer cell line BxPC-3PANC-1 by QPCR was statistically significant. Compared with the normal cell line, the expression of HPCY-5 was higher than that of normal cell line. The results were consistent with the results of immunohistochemistry and Western Blot (P0.05). Patch clamp technique showed that the current of TRPM8 ion channel activated by menthol was significantly increased. CCK8 method indicated that the proliferation ability of TRPM8 channel gene knockout cancer cell line was significantly decreased. The results showed that the expression of TRPM8 channel gene had a certain effect on the proliferation of cancer cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.9

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本文編號：1511925