CerS2基因过表达对甲状腺乳头状癌BCPAP细胞生长、增殖的影响

发布时间：2018-06-05 15:16

本文选题：CerS2基因 + BCPAP　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:探讨CerS2基因过表达对甲状腺乳头状癌BCPAP细胞生长、增殖的影响。方法:1)采用pAdxsi重组腺病毒骨架载体构建人源重组CerS2腺病毒表达载体,并确认pAdxsi-CerS2-GFP腺病毒表达载体对BCPAP细胞株感染的最佳感染复数(MOI)、过表达高峰时间;2)将pAdxsi-GFP、pAdxsi-CerS2-GFP腺病毒表达载体分别感染BCPAP细胞,培养特定时间后,分别提取空白组(blank group)、空载组(pAdxsi-GFP)、实验组(pAdxsi-CerS2-GFP)细胞总RNA与总蛋白,实时荧光定量聚合酶链反应(Q-PCR)、蛋白质印迹法(WB)检测各组细胞中CerS2 mRNA、蛋白表达水平;3)噻唑蓝比色法(MTT)检测CerS2基因不同作用时间对各组细胞体外增殖的影响;4)流式细胞术PI法分析各组细胞周期变化;5)Annexin V-APC/7-AAD双染法检测各组细胞凋亡。结果:1)经酶切、测序鉴定,克隆片段大小、序列与NM_022075一致,其感染BCPAP细胞最佳MOI值为240,最佳作用时间48h;2)Q-PCR、WB结果提示实验组细胞CerS2 mRNA在24h、48h、72h相对表达量依次为空白组/空载组的134.57倍、774.26倍、347.29倍(均P0.001),其蛋白48h表达水平也较空白组、空载组明显增加(P0.01),空白组与空载组CerS2 mRNA相对表达量、蛋白表达水平均无明显统计学差异(P0.05);3)MTT法检测生长曲线发现:24h、48h实验组相对空载组、空白组,细胞OD值明显降低(均P0.001),空白组与空载组比较细胞OD值无明显统计学差异(P0.05);4)流式细胞术检测三组细胞周期,结果提示:空白组与空载组细胞周期时相分布无统计学差异(P0.05);G0/G1期、S期实验组细胞数分别为(40.38±3.43)%、(18.83±1.18)%,空载组细胞数分别为(56.63±1.95)%、(27.28±0.52)%,实验组较空载组G0/G1期、S期细胞数下降(均P0.01),实验组G2/M期细胞数(40.79±4.50)%较空载组(16.09±1.44)%明显增高(P0.001);5)流式细胞术检测空载组细胞凋亡率相对空白组无明显统计学差异(P0.05),实验组细胞凋亡率(29.64±0.20)%较空白组(5.70±0.19)%、空载组(6.07±0.17)%明显增高(均P0.001)。结论:本研究采用pAdxsi重组腺病毒骨架载体构建pAdxsi-CerS2-GFP人源重组腺病毒表达载体,成功感染甲状腺癌BCPAP细胞,并分别从mRNA、蛋白层次验证BCPAP细胞CerS2基因过表达成功。研究结果显示CerS2基因过表达对BCPAP细胞增殖有抑制作用,其主要机制可能与导致BCPAP细胞G2/M期阻滞,进而诱导细胞凋亡发生有关,继续深入研究其作用机制,可望为甲状腺癌的临床诊治及基础研究提供依据。
[Abstract]:Aim: to investigate the effect of CerS2 gene overexpression on the growth and proliferation of BCPAP cells in papillary thyroid carcinoma. Methods pAdxsi recombinant adenovirus skeleton vector was used to construct human recombinant CerS2 adenovirus expression vector. It was also confirmed that pAdxsi-CerS2-GFP adenovirus expression vector could infect BCPAP cell line with the best number of infection plural moi (over peak expression time: 2) pAdxsi-GFPFP-pAdxsi-CerS2-GFP adenovirus expression vector was respectively infected with BCPAP cell line, and cultured for a specific time, and the expression vector of pAdxsi-GFPFP-pAdxsi-CerS2-GFP adenovirus expression vector was infected with BCPAP cell line respectively. Total RNA and total protein were extracted from blank group, pAdxsi-GFPN, pAdxsi-CerS2-GFPFP, respectively. Detection of CerS2 mRNAs and protein expression levels of CerS2 mRNAs by Real-time fluorescence quantitative Polymerase chain reaction (PCR) and Western blot (WB) Detection of the effect of CerS2 Gene on Cell Proliferation in Vitro Cell cycle changes in each group were analyzed by Pi method. Apoptosis was detected by Annexin V-APC-7-AAD double staining. Results the cloned fragment size was identical to that of NM022075 by restriction endonuclease digestion and sequencing. The optimal moi of BCPAP cells infected with BCPAP was 240. The results of the optimal time of 48h treatment showed that the relative expression of CerS2 mRNA in the experimental group was 134.57 times, 774.26 times and 347.29 times as much as that in the blank group / no-load group, and the protein expression level at 48 hours was also higher than that in the blank group, and the relative expression of CerS2 mRNA in the control group was 134.57 times, 774.26 times and 347.29 times as much as that in the blank group. The relative expression of CerS2 mRNA and protein expression in the blank group and the no-load group were not significantly different. The growth curve was detected by MTT assay and the control group was compared with the no-load group at 48 h after 24 hours of growth. The results showed that there was no significant difference in the expression of CerS2 mRNA and protein between the blank group and the blank group, and there was no significant difference between the control group and the blank group. The OD value of cells decreased significantly (all P 0.001g, compared with the blank group and the no-load group, there was no significant difference in the OD value between the blank group and the no-load group). Flow cytometry was used to detect the cell cycle in the three groups. The results showed that there was no significant difference in cell phase distribution between the blank group and the no-load group. The number of cells in the experimental group was 40.38 卤3.43 and 18.83 卤1.18 respectively in G _ 0 / G _ 0 / G _ 1 phase, while the cell number in the no-load group was 27.28 卤0.522. The number of cells in the G _ 0 / G _ 1 phase in the experimental group was significantly lower than that in the no-load group (all P 0.01), and the number of the cells in the no-load group was 27.28 卤0.52. The number of cells in G _ 2 / M phase in the test group was 40.79 卤4.50%, which was significantly higher than that in the no-load group (16.09 卤1.44%). There was no significant difference in the cell apoptosis rate between the no-load group and the blank group by flow cytometry. The apoptotic rate of the experimental group was 29.64 卤0.20% higher than that of the blank group (5.70 卤0.19%), and that of the no-load group was 6.07 卤0.17% (all P 0.001). Conclusion: in this study, the recombinant adenovirus vector pAdxsi-CerS2-GFP was constructed by using pAdxsi recombinant adenovirus skeleton vector to successfully infect thyroid carcinoma BCPAP cells, and the overexpression of CerS2 gene in BCPAP cells was verified by mRNA-protein level. The results show that the overexpression of CerS2 gene can inhibit the proliferation of BCPAP cells, and its main mechanism may be related to the G _ 2 / M phase arrest of BCPAP cells, and then induce apoptosis. It is expected to provide basis for clinical diagnosis, treatment and basic research of thyroid carcinoma.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R736.1

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