HL-60细胞核干细胞因子下调对p38MAPK通路相关蛋白的影响

发布时间：2018-06-09 12:41

本文选题：NS + HL-60细胞　；参考：《郑州大学》2016年硕士论文

【摘要】：目的:通过回顾本课题组前期研究结果,发现下调p53缺失型白血病HL-60细胞核干细胞因子(Nucleostemin,NS)后,该细胞p38MAPK基因发生明显上调,本研究主要对p38MAPK通路中的相关蛋白的表达改变进行检测,分析NS与p38MAPK通路之间的相关性,初步探索NS下调后所导致该通路变化的可能原因,为进一步探索NS非依赖性p53信号通路的具体机制提供理论依据。方法:(1)本课题组前期已经构建好针对NS基因的重组慢病毒载体(NS-RNAi-GV248)和阴性空载体,直接从公司病毒库购买。(2)根据HL-60细胞的合适的MOI值及病毒滴度,将NS-RNAi-GV248慢病毒载体转染至人白血病细胞株HL-60细胞内以下调其NS的表达,通过倒置荧光显微镜观察转染的效率,采用Western Blot技术测定HL-60细胞内NS蛋白的下调情况。(3)采用Western Blot技术检测慢病毒感染后空白对照组、阴性对照组及实验组HL-60细胞中p38MAPK通路中的相关蛋白的表达情况,即p-p38MAPK/p38MAPK两个比值的变化。(4)采用Image J图像处理软件对目的蛋白条带的灰度进行比对分析并计算。采用SPSS17.0统计软件对数据结果进行处理分析,所有的数据以均数±标准差(x±s)表示,多组独立的样本之间的比较采用单因素方差分析,而进一步两两样本之间的比较则采用Bonferroni法,校正检验水平α’=0.0167,P0.05表示数据之间的差异有统计学意义。结果:(1)根据本课题组前期研究结果,NS-RNAi-GV248慢病毒载体感染人白血病细胞株HL-60细胞的最适MOI值为80。以MOI=80的条件感染HL-60细胞,倒置荧光显微镜观察显示慢病毒载体转染效率达到80%以上;Western Blot检测结果显示:慢病毒转染后实验组中HL-60细胞的NS蛋白表达量出现了明显的下调。(2)Western Blot技术的检测结果显示慢病毒转染后实验组中HL-60细胞p-p38/p38的比值(0.444±0.008)高于阴性对照组(0.286±0.016)和空白对照组(0.261±0.019)(P0.05),表示NS表达下调后p38MAPK通路中的p-p38MAPK蛋白表达量增加。结论:(1)HL-60细胞内p38MAPK通路中的p-p38蛋白的表达与NS表达下调呈负相关。(2)NS非依赖性p53的作用过程可能与激活p38MAPK通路有关,p38MAPK通路可能是NS的p53非依赖性通路之一。
[Abstract]:Objective: by reviewing our previous study results, we found that p38 MAPK gene was up-regulated after down-regulation of p53 deletion leukemia HL-60 nuclear stem cell factor Nucleosteminin NSN. In this study, the expression of related proteins in p38 MAPK pathway was detected, the correlation between NS and p38 MAPK pathway was analyzed, and the possible causes of the changes in p38 MAPK pathway after downregulation of NS were preliminarily explored. It provides theoretical basis for further exploring the specific mechanism of NS independent p53 signaling pathway. Methods: 1) the recombinant lentivirus vector NS-RNAi-GV248 for NS gene and the negative empty vector were constructed by our research group and purchased directly from the virus library of the company.) according to the appropriate moi value and virus titer of HL-60 cells, the recombinant vector NS-RNAi-GV248 and the negative empty vector were constructed by our research group. NS-RNAi-GV248 lentivirus vector was transfected into human leukemia cell line HL-60 to down-regulate the expression of NS, and the transfection efficiency was observed by inverted fluorescence microscope. The down-regulation of NS protein in HL-60 cells was detected by Western Blot. The expression of p38MAPK in HL-60 cells was detected by Western Blot. That is, the change of p-p38 MAPK / p38 MAPK ratio. (4) Image J image processing software is used to analyze and calculate the gray scale of the target protein band. SPSS 17.0 statistical software was used to process and analyze the data results. All the data were expressed as mean 卤standard deviation x 卤s. The comparison between multiple independent samples was based on single factor analysis of variance, and Bonferroni method was used for further comparison between two and two samples. The calibration test level (伪) 0.0167 (P 0.05) indicated that the difference between the data was statistically significant. Results the optimal Moi of HL-60 cells infected with NS-RNAi-GV248 lentivirus vector was 80. HL-60 cells were infected with MOIN80. Inverted fluorescence microscope observation showed that the efficiency of lentivirus vector transfection reached more than 80%. The results showed that the expression of NS protein in HL-60 cells after lentivirus transfection was significantly down-regulated by Western blot. The ratio of p-p38/p38 in HL-60 cells after lentivirus transfection was 0.444 卤0.008), which was higher than that in negative control group (0.286 卤0.016) and blank control group (0.261 卤0.019), indicating that the expression of p-p38 MAPK protein in p38 MAPK pathway increased after down-regulation of NS expression. Conclusion the expression of p-p38 protein in p38 MAPK pathway in HL-60 cells is negatively correlated with the down-regulation of NS expression. NS-independent p53 may be involved in the activation of p38 MAPK pathway, which may be one of the p53 independent pathways in NS.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7

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