HMBA和桂皮酸对鼻咽癌细胞CNE2增殖抑制和凋亡的作用及其机制初探

发布时间：2018-06-01 11:51

本文选题：HMBA + 桂皮酸　；参考：《桂林医学院》2012年硕士论文

【摘要】：目的：从细胞及分子水平初步探讨诱导分化剂HMBA和桂皮酸对鼻咽癌细胞CNE2增殖抑制和凋亡的作用及其可能机制。方法：应用诱导分化剂HMBA处理鼻咽癌细胞CNE2,倒置相差显微镜观察处理前后鼻咽癌细胞的生长状况和细胞形态；MTT实验及流式细胞术检测细胞生长及细胞周期；Hoechst33258染色、吖啶橙染色观察细胞凋亡：Annexin V-FITC/PI法检测HMBA对CNE2细胞早期凋亡的影响。RT-PCR检测KLF6基因mRNA的表达。免疫细胞化学方法检测桂皮酸处理前后CNE2细胞中KLF6、p53、cyclin D1、c-Jun蛋白表达的变化。结果：HMBA处理后贴壁细胞的数量随药物浓度增加明显减少；MTT实验证实HMBA对鼻咽癌细胞的生长抑制作用呈浓度-时间效应关系,随着诱导剂浓度的增大、作用时间的延长,其抑制效应逐渐增加。处理后鼻咽癌细胞形状由原来的椭圆形或多边形变成不规则形状或多角形；细胞质减少,核浆比例变大。根据IC50,我们选用5mmol/L作为最佳作用浓度进行后续实验。流式细胞术检测结果显示,培养24h时对照组细胞G1期细胞比例为59.5%,S期细胞比例27.5%,而G2期细胞比例为12.9%,HMBA处理24h后,G1期细胞比例为64.9%；S期细胞为28.3%,G2期细胞为6.7%,细胞周期发生明显变化(P0.05); HMBA作用48h后G1期细胞增加为67.5%,而S期细胞为28.5%,G2期细胞也减少为4.04%,细胞明显被阻滞于G1期(P0.05)；处理72h后,细胞亦显著被阻滞于G1期(P0.05),处理96h后,G1期细胞比例为60.2%,而S期细胞比例为32.2%,G2期细胞为7.63%,细胞周期G1期阻滞效应更明显。我们采用三种方法观察细胞凋亡,Hoechst33258染色后可见呈高亮蓝色的凋亡细胞,在部分细胞内可以看到核碎片；吖啶橙染色可以看到凋亡小体的出现。Annexin V-FITC/PI法检测凋亡显示,在HMBA处理12h后即出现凋亡细胞,与对照组相比有统计学意义(p0.05)。RT-PCR结果显示HMBA处理CNE2细胞后第3天,KLF6mRNA表达水平显著上调(p0.05)。免疫细胞化学结果显示桂皮酸处理CNE2细胞后KLF6和p53蛋白表达上调(p0.05),而cyclin D1和c-Jun则表达下调(p0.05)。结论：诱导分化剂处理后,鼻咽癌细胞CNE2生长明显受到抑制,出现细胞分化形态,细胞周期受阻,诱导细胞凋亡。其作用机理一方面可能是通过上调KLF6基因的表达,上调p53,从而通过p53通路诱导细胞凋亡；另一方面,KLF6可能通过解除c-Jun对p21的抑制作用,下调cyclin D1,抑制Cdk4/6和Cyclin D1复合物活性,将细胞阻滞于G0/G1期,并促进细胞的凋亡。
[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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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