發(fā)布時(shí)間：2018-06-01 11:51

  本文選題：HMBA + 桂皮酸��； 參考：《桂林醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：從細(xì)胞及分子水平初步探討誘導(dǎo)分化劑HMBA和桂皮酸對(duì)鼻咽癌細(xì)胞CNE2增殖抑制和凋亡的作用及其可能機(jī)制。 方法：應(yīng)用誘導(dǎo)分化劑HMBA處理鼻咽癌細(xì)胞CNE2,倒置相差顯微鏡觀察處理前后鼻咽癌細(xì)胞的生長狀況和細(xì)胞形態(tài)；MTT實(shí)驗(yàn)及流式細(xì)胞術(shù)檢測(cè)細(xì)胞生長及細(xì)胞周期；Hoechst33258染色、吖啶橙染色觀察細(xì)胞凋亡：Annexin V-FITC/PI法檢測(cè)HMBA對(duì)CNE2細(xì)胞早期凋亡的影響。RT-PCR檢測(cè)KLF6基因mRNA的表達(dá)。免疫細(xì)胞化學(xué)方法檢測(cè)桂皮酸處理前后CNE2細(xì)胞中KLF6、p53、cyclin D1、c-Jun蛋白表達(dá)的變化。 結(jié)果：HMBA處理后貼壁細(xì)胞的數(shù)量隨藥物濃度增加明顯減少；MTT實(shí)驗(yàn)證實(shí)HMBA對(duì)鼻咽癌細(xì)胞的生長抑制作用呈濃度-時(shí)間效應(yīng)關(guān)系,隨著誘導(dǎo)劑濃度的增大、作用時(shí)間的延長,其抑制效應(yīng)逐漸增加。處理后鼻咽癌細(xì)胞形狀由原來的橢圓形或多邊形變成不規(guī)則形狀或多角形；細(xì)胞質(zhì)減少,核漿比例變大。根據(jù)IC50,我們選用5mmol/L作為最佳作用濃度進(jìn)行后續(xù)實(shí)驗(yàn)。流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示,培養(yǎng)24h時(shí)對(duì)照組細(xì)胞G1期細(xì)胞比例為59.5%,S期細(xì)胞比例27.5%,而G2期細(xì)胞比例為12.9%,HMBA處理24h后,G1期細(xì)胞比例為64.9%；S期細(xì)胞為28.3%,G2期細(xì)胞為6.7%,細(xì)胞周期發(fā)生明顯變化(P0.05); HMBA作用48h后G1期細(xì)胞增加為67.5%,而S期細(xì)胞為28.5%,G2期細(xì)胞也減少為4.04%,細(xì)胞明顯被阻滯于G1期(P0.05)；處理72h后,細(xì)胞亦顯著被阻滯于G1期(P0.05),處理96h后,G1期細(xì)胞比例為60.2%,而S期細(xì)胞比例為32.2%,G2期細(xì)胞為7.63%,細(xì)胞周期G1期阻滯效應(yīng)更明顯。我們采用三種方法觀察細(xì)胞凋亡,Hoechst33258染色后可見呈高亮藍(lán)色的凋亡細(xì)胞,在部分細(xì)胞內(nèi)可以看到核碎片；吖啶橙染色可以看到凋亡小體的出現(xiàn)。Annexin V-FITC/PI法檢測(cè)凋亡顯示,在HMBA處理12h后即出現(xiàn)凋亡細(xì)胞,與對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(p0.05)。RT-PCR結(jié)果顯示HMBA處理CNE2細(xì)胞后第3天,KLF6mRNA表達(dá)水平顯著上調(diào)(p0.05)。免疫細(xì)胞化學(xué)結(jié)果顯示桂皮酸處理CNE2細(xì)胞后KLF6和p53蛋白表達(dá)上調(diào)(p0.05),而cyclin D1和c-Jun則表達(dá)下調(diào)(p0.05)。 結(jié)論：誘導(dǎo)分化劑處理后,鼻咽癌細(xì)胞CNE2生長明顯受到抑制,出現(xiàn)細(xì)胞分化形態(tài),細(xì)胞周期受阻,誘導(dǎo)細(xì)胞凋亡。其作用機(jī)理一方面可能是通過上調(diào)KLF6基因的表達(dá),上調(diào)p53,從而通過p53通路誘導(dǎo)細(xì)胞凋亡；另一方面,KLF6可能通過解除c-Jun對(duì)p21的抑制作用,下調(diào)cyclin D1,抑制Cdk4/6和Cyclin D1復(fù)合物活性,將細(xì)胞阻滯于G0/G1期,并促進(jìn)細(xì)胞的凋亡。
[Abstract]:Aim: to investigate the effects of HMBA and cinnamic acid on the proliferation and apoptosis of nasopharyngeal carcinoma (NPC) cells at cellular and molecular levels. Methods: nasopharyngeal carcinoma cell line CNE2 was treated with HMBA. The growth and morphology of nasopharyngeal carcinoma cells were observed by inverted phase contrast microscope. Cell growth and Hoechst33258 staining were detected by flow cytometry. The effect of HMBA on the early apoptosis of CNE2 cells was detected by V-FITC/PI assay with acridine orange staining. RT-PCR was used to detect the expression of KLF6 gene mRNA. Immunocytochemistry was used to detect the expression of c-Jun protein in CNE2 cells before and after cinnamic acid treatment. Results the number of adherent cells decreased with the increase of drug concentration. The results showed that the inhibitory effect of HMBA on the growth of nasopharyngeal carcinoma cells showed a concentration-time effect relationship, and with the increase of the concentration of inducer, the time of action was prolonged. Its inhibitory effect increased gradually. After treatment, the shape of nasopharyngeal carcinoma cells changed from ellipse or polygon to irregular shape or polygonal shape. According to IC50, we selected 5mmol/L as the optimal concentration for follow-up experiments. Flow cytometry showed that, The ratio of G 1 phase cells to G 1 phase cells in the control group was 59.5% and that of the G 2 phase cells was 12.9%. After 24h treatment, the proportion of G 1 phase cells in the G 1 phase was 64.9%. The percentage of cells in G 2 phase was 28.3%. The cell cycle changed significantly (P0.05); after 48 h HMBA treatment, the ratio of G 1 phase cells to G 2 phase cells was 6.70%, and the cell cycle changed significantly (P0.05) after treatment with HMBA for 48 h. The cells in G1 phase increased to 67.5%, while in S phase cells decreased from 28.5G 2 phase to 4.04, and the cells were obviously blocked in G 1 phase P0.05G, and after 72 h of treatment, the cells were significantly blocked in G 1 phase. Cells were also significantly blocked in G _ 1 phase P _ (0.05). After 96 h treatment, the proportion of cells in G _ 1 phase was 60.2%, while that in S phase was 32.2G _ 2 phase cells was 7.63%, and cell cycle G _ 1 phase arrest effect was more obvious. We observed apoptosis by Hoechst33258 staining and nuclear fragments in some cells, the apoptotic bodies were observed by acridine orange staining. Annexin V-FITC/PI method was used to detect apoptosis. Apoptotic cells appeared 12 hours after HMBA treatment. Compared with the control group, the results of RT-PCR showed that the expression of KLF6 mRNA in CNE2 cells treated with HMBA was significantly up-regulated on the 3rd day. Immunocytochemistry showed that the expression of KLF6 and p53 protein was up-regulated in CNE2 cells treated with cinnamic acid, while the expression of cyclin D1 and c-Jun was down-regulated. Conclusion: the CNE2 growth of nasopharyngeal carcinoma cells was inhibited, cell differentiation morphology was observed, cell cycle was blocked and apoptosis was induced after treated with differentiation agent. On the one hand, the mechanism may be to up-regulate the expression of KLF6 gene and the expression of p53, thus inducing apoptosis through p53 pathway; on the other hand, KLF6 may inhibit the activity of Cdk4/6 and Cyclin D1 complex by removing the inhibitory effect of c-Jun on p21, down-regulating cyclin D1, and inhibiting the activity of Cdk4/6 and Cyclin D1 complex. The cells were blocked in G0/G1 phase and promoted apoptosis.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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