下调HMGB1表达对食管鳞状细胞癌TE-1细胞放疗敏感性的影响和机制研究

发布时间：2018-06-23 20:05

本文选题：HMGB1 + 食管鳞状细胞癌　；参考：《江苏大学》2016年硕士论文

【摘要】：【目的】探讨HMGB1表达对食管鳞状细胞癌TE-1细胞放疗敏感性的影响,并初步探讨其分子调控机制。【方法】(1)应用siRNA转染技术,通过Lipofectamine2000分别将空载体阴性对照(NCsiRNA)和HMGB1干扰载体(siHMGB1)转染至人食管鳞状细胞癌细胞株TE-1,按不同分组标记为:TE-1组(空白对照组),TE-1+NCsiRNA组(空载体阴性对照组),TE-1+siHMGB1组(HMGB1沉默组);通过实时定量PCR法检测各组TE-1细胞内HMGB1mRNA的表达变化,Westernblot法进一步验证转染TE-1细胞内HMGB1蛋白的表达改变;(2)对上述三组TE-1细胞在转染后进行不同剂量(0、2、4、6、8Gy)X射线照射培养一定的时间(24、48、72、96小时),通过MTT法检测TE-1细胞的增殖率;(3)对上述三组TE-1细胞在转染后进行上述不同剂量X射线照射后培养15天,通克隆形成实验检测TE-1细胞的克隆形成率;(4)上述转染处理24小时,给予4GyX射线照射后,流式细胞术检测各组TE-1细胞的凋亡率、活性氧簇(reactiveoxygenspecies,ROS)含量和γH2AX水平;用实时定量PCR法检测各组TE-1细胞NOX1与NOX5mRNA的表达变化;Westernblot法检测各组TE-1细胞Caspase-3、CleavedPARP、p38、p-p38、JNK、p-JNK蛋白的表达变化。【结果】(1)HMGB1沉默组TE-1细胞中HMGB1mRNA和蛋白的表达量均较空白对照组、空载体阴性对照组显著降低(均P0.001);(2)MTT法检测结果发现转染后24、48、72、96小时HMGB1沉默组TE-1细胞增殖率低于空白对照组及空载体阴性对照组(均P0.05);转染后24小时行4GyX射线照射24、48、72、96小时后,转染前后三组TE-1细胞的增殖能力均受到抑制,而HMGB1沉默组TE-1细胞增殖抑制程度较其他两组明显(均P0.05);转染后24小时经不同剂量(0、2、4、6、8Gy)的X射线照射48小时后:随着X射线照射剂量的增加,三组TE-1细胞的增殖率均降低,HMGB1沉默组细胞增殖抑制较两对照组明显(均P0.05);(3)Sigmaplot软件分析结果显示HMGB1沉默组D0、Dq、N值均低于空白对照组和空载体阴性对照组;HMGB1沉默组相对于空白对照组的放射增敏比(sensitizingenhancementratio,SER)为1.26;(4)流式细胞术检测结果显示:转染后24小时,HMGB1沉默组凋亡率、ROS含量和γH2AX水平高于两对照组;实时定量PCR检测结果显示HMGB1沉默组NOX1和NOX5mRNA的表达高于两对照组;Westernblot检测结果显示HMGB1沉默组Caspase-3、CleavedPARP、p-p38、p-JNK蛋白的表达高于两对照组(均P0.05);转染后24小时起给予4GyX射线照射,流式细胞术检测结果显示HMGB1沉默组凋亡率、ROS含量和γH2AX水平高于单纯放疗组;实时定量PCR检测结果显示HMGB1沉默组NOX1与NOX5mRNA的表达高于单纯放疗组;Westernblot检测结果显示HMGB1沉默组Caspase-3、CleavedPARP、p-p38、p-JNK蛋白的表达高于单纯放疗组(均P0.05)。【结论】通过体外细胞实验研究,发现下调HMGB1表达能抑制人食管鳞状细胞癌细胞株TE-1放疗后的增殖率,降低其克隆形成能力,即下调HMGB1表达能提高TE-1细胞对放疗的敏感性。其分子机制是下调HMGB1表达可能通过上调促细胞凋亡相关蛋白Caspase-3/CleavedPARP的表达以及上调ROS产生来影响DNA损伤,并通过JNK和p38通路激活,上调p-p38和p-JNK蛋白表达,调控放疗对TE-1细胞增殖和凋亡能力改变,进而提高其对电离辐射的敏感性。
[Abstract]:[Objective] to investigate the effect of HMGB1 expression on the radiation sensitivity of esophageal squamous cell carcinoma TE-1 cells and to explore its molecular mechanism. [Methods] (1) siRNA transfection technique was used to transfect the empty carrier negative control (NCsiRNA) and HMGB1 interfering carrier (siHMGB1) to the human esophageal squamous cell carcinoma cell line TE, respectively. -1 was labeled as group TE-1 (blank control group), group TE-1+NCsiRNA (negative control group) and group TE-1+siHMGB1 (HMGB1 silence group); the expression of HMGB1mRNA in TE-1 cells in each group was detected by real-time quantitative PCR method, and Westernblot method was used to further verify the expression change of HMGB1 protein in the transfected TE-1 cells; (2) the three groups TE-1 were TE-1. After transfection, the cells were irradiated with different doses (0,2,4,6,8Gy) X ray for a certain time (24,48,72,96 hours), and the proliferation rate of TE-1 cells was detected by MTT method. (3) the above three groups of TE-1 cells were irradiated with different doses of X rays for 15 days after the transfection, and the cloning formation rate of TE-1 cells was detected by clonogenic formation; (4) After 24 hours of transfection, the apoptosis rate of TE-1 cells, the content of reactiveoxygenspecies (reactiveoxygenspecies, ROS) and the level of gamma H2AX were detected by flow cytometry, and the expression of NOX1 and NOX5mRNA in TE-1 cells in each group was detected by real-time quantitative PCR; Westernblot method was used to detect TE-1 cell Caspase-3. The expression of p38, JNK and p-JNK protein was changed. [results] the expression of HMGB1mRNA and protein in TE-1 cells of HMGB1 silencing group were all more than that in the blank control group, and the negative control group decreased significantly (P0.001). (2) the MTT assay results showed that the proliferation rate of TE-1 cells in the 24,48,72,96 hour HMGB1 silence group was lower than that of the blank control group and the empty body negative group. In the sex control group (all P0.05), the proliferation ability of the three groups of TE-1 cells after transfection for 24,48,72,96 hours after 24 hours of transfection was inhibited, while the proliferation inhibition of TE-1 cells in the HMGB1 silent group was more obvious than that in the other two groups (P0.05), and 24 hours after the transfection, the X ray of the same dose (0,2,4,6,8Gy) was irradiated for 48 hours after the transfection: with X. The proliferation rate of the three groups of TE-1 cells decreased, and the proliferation inhibition in the HMGB1 silencing group was significantly lower than that in the two control group (P0.05). (3) the Sigmaplot software analysis showed that the HMGB1 silenced group D0, Dq, N values were lower than the blank control group and the empty carrier negative control group; the HMGB1 silencing group was compared with the blank control group with the radiosensitivity ratio (SEN). Sitizingenhancementratio, SER) was 1.26. (4) flow cytometry results showed that the apoptosis rate of HMGB1 silencing group, ROS content and gamma H2AX level were higher than that of two control groups at 24 hours after transfection; real-time quantitative PCR detection showed that the expression of NOX1 and NOX5mRNA in HMGB1 silencing group was higher than that of the two control group; Westernblot test results showed HMGB1 silence group Caspase. The expression of -3, CleavedPARP, p-p38, p-JNK protein was higher than that of the two control group (all P0.05); 4GyX ray irradiation was given after the transfection. The results of flow cytometry showed the apoptosis rate of HMGB1 silencing group. The content of ROS and the level of gamma H2AX were higher than those in the simple radiotherapy group. Real time quantitative PCR detection showed that the expression of NOX1 in HMGB1 silence group was higher than that of simple group. The results of Westernblot test showed that the expression of Caspase-3, CleavedPARP, p-p38 and p-JNK protein in the HMGB1 silencing group was higher than that in the radiotherapy group (P0.05). [Conclusion] the expression of down regulated HMGB1 could inhibit the proliferation rate of human esophageal squamous cell carcinoma cell line after TE-1 radiotherapy and reduce the clone formation ability by in vitro cell test. Down regulation of HMGB1 expression can increase the sensitivity of TE-1 cells to radiotherapy. Its molecular mechanism is that the expression of HMGB1 may be regulated by up regulation of the expression of apoptosis related protein Caspase-3/CleavedPARP and up regulation of ROS production to affect DNA damage. The expression of p-p38 and p-JNK proteins is up regulated by JNK and p38 pathway, and the regulation of radiotherapy to TE-1 cells is regulated. The sensitivity of colony and apoptosis to ionizing radiation is improved.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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