本文關(guān)鍵詞：染料木素對(duì)人胰腺癌細(xì)胞株P(guān)ANC-1的作用研究　出處：《山西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 染料木素 吉西他濱 胰腺癌 細(xì)胞凋亡 增殖

【摘要】：目的:觀察不同濃度染料木素對(duì)人胰腺癌細(xì)胞株P(guān)ANC-1作用及其抑癌基因表達(dá)的的研究,從而為臨床治療胰腺癌提供新的思路方法。方法:本實(shí)驗(yàn)共分實(shí)驗(yàn)組(80,120,160,200μmol/L)的染料木素、空白對(duì)照組(PBS)和標(biāo)準(zhǔn)對(duì)照(吉西他濱)。各組培養(yǎng)基處理細(xì)胞12h、24h、36h、48h、60h后,細(xì)胞計(jì)數(shù)試劑盒(CCK-8)法檢測(cè)人胰腺癌細(xì)胞株P(guān)ANC-1在染料木素作用下的增殖活性;流式細(xì)胞術(shù)檢測(cè)人胰腺癌細(xì)胞株P(guān)ANC-1在染料木素木素作用下的凋亡細(xì)胞百分率;用逆轉(zhuǎn)錄-聚合酶連反應(yīng)(RT-PCR)方法測(cè)定P53及P21抑癌基因的表達(dá);Western blot法檢測(cè)抑癌基因P53和P21的蛋白表達(dá)水平。實(shí)驗(yàn)結(jié)果統(tǒng)計(jì)學(xué)分析:細(xì)胞凋亡與細(xì)胞基因m RNA采用的是LSD法,細(xì)胞增殖數(shù)據(jù)采用的是配對(duì)t檢驗(yàn),P0.05比較差異有統(tǒng)計(jì)學(xué)意思。結(jié)果:1.CCK-8法檢測(cè)染料木素對(duì)胰腺癌細(xì)胞(PANC-1)的生長(zhǎng)抑制作用分別對(duì)160μmol/L與200μmol/L、120μmol/L與200μmol/L、160μmol/L與GEM組、200μmol/L與GEM組通過SPSS 13.0進(jìn)行配對(duì)樣本t檢驗(yàn),結(jié)果顯示160μmol/L對(duì)GEM組無(wú)統(tǒng)計(jì)學(xué)差異(P=0.067),而其余三組P值均小于0.05;針對(duì)36h以內(nèi)的數(shù)據(jù),統(tǒng)計(jì)發(fā)現(xiàn)120μmol/L對(duì)GEM組P值亦小于0.05,而200μmol/L與GEM組無(wú)統(tǒng)計(jì)學(xué)差異(P=0.051)。根據(jù)以上結(jié)果,我們認(rèn)為染料木素總體呈濃度依賴性和時(shí)間依賴性,其中36h內(nèi)200μmol/L效果與GEM組較為接近,但160μmol/L總體效果最好,且與GEM組最為接近。2.流式細(xì)胞儀(FCM)各組凋亡指數(shù)比較FCM結(jié)果可以看出,4個(gè)染料木素分組中,80μmol/L對(duì)細(xì)胞增殖抑制效果并不明顯,而120μmol/L和160μmol/L最接近GEM組的效果,200μmol/L晚期凋亡細(xì)胞占大部分�？梢娙玖夏舅氐淖饔糜忻黠@的濃度依賴性。3.Western bloting檢測(cè)P53、P21等相關(guān)蛋白表達(dá)Western-blot檢測(cè)蛋白水平顯示160μmol/L和200μmol/L染料木素相對(duì)于空白對(duì)照能夠明顯的增強(qiáng)抑癌基因P21和P53的蛋白水平的表達(dá),相對(duì)于標(biāo)準(zhǔn)組沒有明顯的差異。結(jié)論:1.通過FCM和CCK-8法表明染料木素作用具有濃度依賴性和時(shí)間依賴性,但是在低濃度時(shí),染料木素的作用并不理想,而當(dāng)濃度達(dá)到160μmol/L以上時(shí)其FCM結(jié)果與標(biāo)準(zhǔn)對(duì)照組吉西他濱非常接近,濃度達(dá)到200μmol/L時(shí)晚期凋亡細(xì)胞已占60%以上2.CCK-8法細(xì)胞增殖的結(jié)果顯示160μmol/L的抑制效果在48h達(dá)到最佳,整體效果最好且曲線與標(biāo)準(zhǔn)對(duì)照相近,而Western-blot結(jié)果顯示染料木素對(duì)P21、P53蛋白均有明顯上調(diào)作用,濃度依賴性小。3.我們推斷濃度為160μmol/L染料木素對(duì)胰腺癌細(xì)胞株P(guān)ANC-1的抑制作用最佳,其可能通過下調(diào)細(xì)胞增殖、細(xì)胞周期相關(guān)基因以及上調(diào)P21、P53蛋白表達(dá),使染料木素對(duì)胰腺癌細(xì)胞株P(guān)ANC-1體外增殖的抑制作用表現(xiàn)出濃度及時(shí)間依賴性,從而發(fā)揮抑制癌細(xì)胞作用。
[Abstract]:Objective: To investigate the effects of genistein on the function of PANC-1 in human pancreatic cancer cell lines and expression of tumor suppressor genes, so as to provide new ideas for clinical treatment of pancreatic cancer. Methods: the experiment was divided into experimental group (80120160200 mol/L) of genistein, blank control group (PBS) and control (standard gemcitabine) and cultured 12h cells. Each treatment, 36h, 48h, 24h, 60H, cell counting Kit (CCK-8) method for the detection of human pancreatic cancer cell line PANC-1 in genistein under the effect of proliferation; flow cytometry of human pancreatic cancer cell line PANC-1 in dye lignin under the action of apoptosis percentage; reverse transcriptase polymerase chain reaction (RT-PCR) method for the determination of the expression of P53 and P21 tumor suppressor gene; Western blot method to detect the expression of tumor suppressor gene P53 and P21 protein levels. Statistical analysis results: cell apoptosis and expression of M gene RNA Using the LSD method. The cell proliferation data using the paired t test, P0.05 difference was statistically. Results: 1.CCK-8 assay of genistein on pancreatic cancer cells (PANC-1) growth inhibition of 160 mol/L and 200 mol/L, 120 mol/L and 200 mol/L, 160 mol/L and GEM group 200 mol/L GEM group and SPSS 13 paired samples t test, the results showed that 160 mol/L had no significant difference in GEM group (P=0.067), while the remaining three group P values were less than 0.05; for 36h within the data statistics found that 120 mol/L is less than 0.05 of the GEM group P, and 200 mol/L and GEM group had no statistical difference (P=0.051). According to the above results, we believe that genistein showed a dose-dependent and time-dependent manner, the effect of mol/L 200 in 36h and GEM group is close to 160 mol/L, but the overall effect is the best, and the GEM group is most close to the.2. flow cytometry (FCM) The apoptotic index compared with FCM results, 4 genistein group, 80 mol/L on cell proliferation inhibition effect is not obvious, while the 120 mol/L and 160 mol/L closest to the effect of group GEM, 200 mol/L of late apoptotic cells accounted for the majority. The effect of concentration of genistein showed significant dependence on.3.Western bloting detection of P53, P21 and other related protein expression level of Western-blot was detected 160 mol/L and 200 mol/L genistein compared with blank control can enhance the protein expression of tumor suppressor gene P21 and P53 significantly, compared with no significant differences between the standard group. Conclusion: 1. through FCM and CCK-8 method showed that genistein had concentration and time dependent manner, but at low concentrations, genistein's effect is not ideal, and when the concentration reaches above 160 mol/L the results of FCM and gemcitabine non standard group Very close to, the concentration reached 200 mol/L in late apoptotic cells accounted for more than 60% 2.CCK-8 cell proliferation showed inhibitory effect of 160 mol/L in 48h to achieve the best overall effect, and the best curve with the standard of photography, and Western-blot results showed that genistein on P21, P53 protein were significantly up-regulated, concentration dependent we conclude that the small.3. concentration of 160 mol/L inhibitory effects of genistein on human pancreatic cancer cell line PANC-1, it may down regulate cell proliferation, cell cycle related genes and upregulation of P21, P53 protein expression, so that genistein inhibits proliferation of human pancreatic cancer cell line PANC-1 in vitro showed a concentration and time dependent, so the inhibition of cancer cells.

【學(xué)位授予單位】：山西醫(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)】：R735.9

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本文編號(hào)：1430115