【摘要】：目的觀察新型PI3K小分子抑制劑BYL-93對(duì)人胰腺癌細(xì)胞株CFPAC-1和Capan-2的作用,從細(xì)胞水平上驗(yàn)證BYL-93的抗胰腺癌活性。為拓展PI3K抑制劑的應(yīng)用提供實(shí)驗(yàn)依據(jù),同時(shí)也為開發(fā)新的小分子靶向治療藥物提供科學(xué)依據(jù)。方法培養(yǎng)CFPAC-1和Capan-2細(xì)胞,采用CCK-8法檢測(cè)細(xì)胞增殖;DAPI染色觀察細(xì)胞核;流式細(xì)胞術(shù)測(cè)定細(xì)胞凋亡及其周期;JC-1法檢測(cè)線粒體跨膜電位變化;Western blot法檢測(cè)PI3K-AKT信號(hào)通路的蛋白表達(dá)和磷酸化水平。對(duì)CFPAC-1細(xì)胞進(jìn)行劃痕實(shí)驗(yàn)、Transwell小室體外侵襲實(shí)驗(yàn),實(shí)時(shí)熒光定量PCR法測(cè)定E-cadherin、Snail基因轉(zhuǎn)錄水平。結(jié)果BYL-93對(duì)CFPAC-1和Capan-2的增殖都產(chǎn)生了抑制效應(yīng),CFPAC-1和Capan-2第 24、48 小時(shí)的 IC50 值分別為 22.7μM、5.3μM 和 9.0μM、2.4μM;5、10、20μM BYL-93均能誘導(dǎo)CFPAC-1細(xì)胞凋亡,2.5、5、1OμM BYL-93均能誘導(dǎo)Capan-2細(xì)胞凋亡;不同濃度BYL-93作用后,可將CFPAC-1和Capan-2明顯阻滯于細(xì)胞G1期;BYL-93在CFPAC-1和Capan-2兩株細(xì)胞上呈現(xiàn)顯著下降線粒體膜電位的效果(p0 001);BYL-93作用于CFPAC-1和Capan-2后,AKT、mTOR、70S6、S6蛋白的磷酸化水平降低,PARP發(fā)生裂解,且BYL-93對(duì)CFPAC-1細(xì)胞的作用略強(qiáng);隨著BYL-93濃度的升高,CFPAC-1細(xì)胞的遷移能力逐漸降低,其抑制遷移能力呈濃度梯度依賴性;BYL-93能明顯抑制胰腺癌細(xì)胞的侵襲轉(zhuǎn)移,上調(diào)E-cadherin mRNA表達(dá),下調(diào)Snail mRNA表達(dá)。結(jié)論BYL-93具有抑制CFPAC-1和Capan-2腫瘤細(xì)胞的增殖、轉(zhuǎn)移的作用,驗(yàn)證BYL-93的抗胰腺癌活性。
[Abstract]:Aim to observe the effect of BYL-93, a novel small molecule inhibitor of PI3K, on human pancreatic cancer cell lines CFPAC-1 and Capan-2, and to verify the anti-pancreatic cancer activity of BYL-93 at the cell level. It can provide experimental basis for expanding the application of PI3K inhibitors and provide scientific basis for the development of new small molecular targeted therapeutic drugs. Methods CFPAC-1 and Capan-2 cells were cultured, cell proliferation was detected by CCK-8 method, cell nucleus was observed by DAPI staining, apoptosis and cell cycle were measured by flow cytometry, mitochondrial transmembrane potential (mtDNA) was measured by JC-1 method. The protein expression and phosphorylation level of PI3K-AKT signaling pathway were detected by Western blot. Scratches of CFPAC-1 cells, invasion of Transwell cells in vitro, and transcription of E-cadherin and snail genes were measured by real-time fluorescence quantitative PCR. Results BYL-93 inhibited the proliferation of CFPAC-1 and Capan-2. The IC50 values of CFPAC-1 and Capan- 224h were 22.7 渭 M, 5.3 渭 M and 9.0 渭 M, 2.4 渭 M, respectively. 5, 10 and 20 渭 M BYL-93 could induce apoptosis of CFPAC-1 cells, and 2.5, 5 and 1 渭 M BYL-93 could induce apoptosis of Capan-2 cells. After different concentrations of BYL-93, CFPAC-1 and Capan-2 could be blocked obviously in G1 phase of cells. BYL-93 significantly decreased the mitochondrial membrane potential in CFPAC-1 and Capan-2 cell lines (p0.001). When CFPAC-1 and Capan-2 were treated with BYL-93, the phosphorylation level of AKT,mTOR,70S6,S6 protein was decreased and PARP was lysed, and the effect of BYL-93 on CFPAC-1 cells was slightly stronger. With the increase of BYL-93 concentration, the migration ability of CFPAC-1 cells decreased gradually, and its ability to inhibit migration showed a concentration-gradient dependent manner. BYL-93 could significantly inhibit the invasion and metastasis of pancreatic cancer cells, upregulate the expression of E-cadherin mRNA and down-regulate the expression of Snail mRNA. Conclusion BYL-93 can inhibit the proliferation and metastasis of CFPAC-1 and Capan-2 tumor cells, and verify the anti-pancreatic cancer activity of BYL-93.
【學(xué)位授予單位】：浙江中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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