论文部分内容阅读
目的:研究宫颈感染HPV后是否能通过干扰SHP-2从而负调控IFN-β信号通路,最终导致宫颈癌的发生。方法:通过SiHa细胞与THP-1细胞共培养,利用SHP-2 siRNA干扰片段瞬时转染THP-1细胞,干扰SHP-2基因的表达。将SHP-2封闭肽以0、0.5、2.5μg/ml浓度和2、24 h培养THP-1细胞,收集培养后的细胞进行Western Blotting实验,进而检测SHP-2的表达,确定封闭的最佳时间和浓度。收集正常和不同处理因素的THP-1细胞,Western Blotting法检测THP-1细胞中SHP-2蛋白表达。将THP-1细胞及不同处理因素处理好的THP-1细胞分别与SiHa细胞共培养后,在选定的时间和浓度点测其OD值。收集正常THP-1细胞的培养液上清、THP-1细胞给予SHP-2封闭肽处理的培养液上清和THP-1细胞给予SHP-2 siRNA干扰的培养液上清进行ELISA实验。结果:给予SHP-2的SiRNA后SHP-2蛋白表达明显降低,在干扰后48 h、200 pmol时干扰效果最好。给予SHP-2的封闭抗体后SHP-2蛋白表达明显降低,在封闭后24 h、2.5μg/ml时抑制效果最好。THP-1细胞与SiHa细胞相互作用,从两个细胞作用时间来看,THP-1细胞对SiHa细胞杀伤作用随着时间的增长而增强,从24 h始就起明显作用。给予siRNA干扰的THP-1细胞对SiHa细胞的杀伤作用最强(OD值最低),给予封闭肽的次之,无处理因素的最弱(OD值最高)。SiRNA干扰组的浓度值最高,其次是封闭肽组的浓度值,无处理因素组(仅培养THP-1的细胞组)的浓度值最低,SiRNA干扰组与无处理因素组之间差异有统计学意义(P<0.05),封闭肽组与无处理因素组之间差异有统计学意义(P<0.05),SiRNA干扰组与封闭肽组之间差异无统计学意义(P>0.05)。结论:抑制THP-1细胞中SHP-2的表达后,THP-1细胞分泌的IFN-β明显增多,导致它对SiHa细胞的杀伤作用明显增强。SHP-2在宫颈癌的发生中起着重要作用,它发挥作用的机制是通过抑制IFN-β的信号转导,减少I型干扰素IFN-β的生成,使宫颈癌发生成为可能。
Objective: To investigate whether cervical HPV infection can negatively regulate the IFN-β signaling pathway by interfering with SHP-2 and eventually lead to cervical cancer. METHODS: SiHa cells were co-cultured with THP-1 cells, transiently transfected THP-1 cells with SHP-2 siRNA to interfere the expression of SHP-2 gene. The SHP-2 blocking peptide was cultured at 0,0.5,2.5μg / ml for 2 and 24 h, then THP-1 cells were cultured. The cultured cells were collected and subjected to Western Blotting assay to detect the expression of SHP-2. Time and concentration. THP-1 cells were collected from normal and different treatment factors, and SHP-2 protein expression in THP-1 cells was detected by Western Blotting. THP-1 cells and different treatment factors treated THP-1 cells were co-cultured with SiHa cells at the selected time and concentration point OD value measured. The culture supernatant of normal THP-1 cells was collected, the supernatant of THP-1 cells treated with SHP-2 blocking peptide and the supernatant of THP-1 cells exposed to SHP-2 siRNA were used for ELISA. Results: The expression of SHP-2 protein was significantly decreased after administration of SiRNA in SHP-2 cells. The interference effect was best at 200 pmol at 48 h after interference. After SHP-2 blocking antibody was given, the expression of SHP-2 protein was significantly decreased, and at 24 h after blocking, the inhibition effect was best at 2.5 μg / ml. THP-1 cells and SiHa cells interaction, from the two cell action time point of view, THP-1 cells on SiHa cell cytotoxicity increased with time, from 24 h began to play a significant role. The siRNA-interfering THP-1 cells had the strongest killing effect on SiHa cells (the lowest OD value), followed by the blocking peptide, and the non-treatment factor was the weakest (the OD value was the highest). The concentration of SiRNA interference group was the highest, followed by the concentration of the closed peptide group, the lowest concentration in the untreated group (only THP-1-cultured cell group), the difference between the SiRNA interference group and the untreated group was statistically (P <0.05). There was a significant difference between the group of blocking peptide and the untreated group (P <0.05), and the difference between SiRNA interference group and blocking peptide group was not statistically significant (P> 0.05). CONCLUSION: THP-1 cells inhibit the expression of SHP-2, THP-1 cells secreted IFN-β significantly increased, resulting in its killing effect on SiHa cells was significantly enhanced. SHP-2 plays an important role in the development of cervical cancer. The mechanism by which SHP-2 plays a role is to make cervical cancer possible by inhibiting the signal transduction of IFN-β and decreasing the production of IFN-β.