下调DJ-1基因对多发性骨髓瘤细胞增殖、凋亡及细胞周期的影响

发布时间：2018-06-06 18:55

本文选题：多发性骨髓瘤 + RPMI8226　；参考：《川北医学院》2017年硕士论文

【摘要】：研究目的:1.人多发性骨髓瘤细胞株中DJ-1 mRNA及蛋白质的表达情况;2.初步探明DJ-1是否对多发性骨髓瘤细胞增殖、细胞周期及凋亡有调节作用;3.初步探明DJ-1是否影响药物(硼替佐米、地塞米松)诱导的骨髓瘤细胞凋亡。研究方法:1.实时荧光定量PCR(realtime quantitative PCR,RT-qPCR)检测骨髓瘤细胞系(human myeloma cell lines,HMCLs)RPMI8226、U266及正常人外周血单个核细胞DJ-1 mRNA水平,免疫印迹杂交(Western Blot,WB)检测DJ-1蛋白表达水平;2.分别用无关序列shRNA病毒液(Control shRNA Lentiviral Particles-A)及靶向下调DJ-1的shRNA病毒液(DJ-1 shRNA Lentiviral Particles)感染RPMI8226、U266细胞,使用含适当浓度的嘌呤霉素持续筛选稳定转染细胞,运用RT-qPCR和WB检测稳定转染细胞中DJ-1的表达情况以验证转染效果;3.慢病毒转染RPMI8226和U266细胞下调DJ-1表达后,采用CCK8比色法、流式细胞术检测细胞增殖、自发凋亡、细胞周期,并与对照组细胞比较;4.转染慢病毒下调RPMI8226、U266细胞DJ-1表达后,给予硼替佐米、地塞米松处理细胞,采用CCK8比色法检测细胞增殖、流式细胞术检测细胞周期及细胞凋亡情况,并与对照组细胞比较。研究结果:1.rt-qpcr及wb检测发现,与正常人外周血单个核细胞相比,多发性骨髓瘤细胞株rpmi8226、u266中dj-1mrna和蛋白均呈高表达状态。2.dj-1shrna慢病毒干扰载体(shdj-1)转染多发性骨髓瘤细胞株rpmi8226、u266,并用含嘌呤霉素的完全培养基持续培养,筛选出稳定转染细胞。经验证,shdj-1转染的骨髓瘤细胞中,dj-1mrna和蛋白质水平明显低于空白对照组及shcontrol组,表明我们成功构建了dj-1下调的骨髓瘤细胞模型。3.转染shdj-1后,除种板48hrpmi8226两组细胞od值无明显差异(p=0.0779),其余各时间点检测发现,与shcontorl对照组相比,细胞增殖减慢(p0.05);细胞周期中g1期细胞比例减少,s期细胞比例上升,细胞阻滞于s期(p0.05);细胞早期凋亡率显著升高(p0.05)。4.分别使用终浓度为0、2.0、4.0、6.0、8.0、10.0nmol/l的硼替佐米,0、0.5、1.0、5.0、10.0、20.0μmol/l的地塞米松处理各组细胞24h,cck8法检测各组细胞od值,并计算细胞增殖抑制率,结果显示,随着药物浓度升高,细胞增殖抑制率逐渐升高。比较同一药物浓度处理下的各组细胞发现,除0.5、1.0μmol/l地塞米松处理u266各组细胞的增殖抑制率差异无统计学意义外,其余各浓度药物处理下,shdj-1组细胞增殖抑制率较shcontrol组细胞显著升高(p0.05)。5.使用终浓度为2.0nmol/l的硼替佐米、1.0μmol/l的地塞米松处理骨髓瘤细胞株rpmi8226各组细胞48h,流式细胞术检测细胞周期,结果显示,与shControl组相比,shDJ-1组G1期细胞比例降低,S期细胞比例升高,细胞阻滞于S期(P0.05)。6.分别用不同浓度的硼替佐米(终浓度为0、2.5、5.0、10.0nmol/L)、地塞米松(终浓度为0、0.1、1.0、10.0μmol/L)处理各组细胞,24h后收集细胞FITC Annexin V/PI染色,采用流式细胞术检测细胞凋亡,结果发现,随着药物浓度增加,细胞早期凋亡率升高;同一浓度药物处理下,除5.0nmol/L硼替佐米、0.1μmol/L地塞米松处理U266细胞差异无统计学意义外(P=0.0505;0.051),其余shDJ-1组细胞凋亡率较shControl组均明显升高(P0.05),差异具有统计学意义。结论:1.在两种常见的人多发性骨髓瘤细胞株RPMI8226、U266中,DJ-1mRNA及蛋白质呈异常高表达。2.DJ-1参与调节RPMI8226、U266细胞增殖、细胞周期及凋亡,抑制DJ-1增强细胞对硼替佐米和地塞米松的敏感性,但具体作用机制有待进一步研究。3.DJ-1可能是多发性骨髓瘤潜在的治疗靶点,值得进一步研究。
[Abstract]:Objective: To investigate the expression of DJ-1 mRNA and protein in 1. human myeloma cell lines; 2. preliminarily explore whether DJ-1 can regulate the proliferation, cell cycle and apoptosis of multiple myeloma cells; 3. preliminarily explore whether DJ-1 affects the apoptosis of myeloma cells induced by bortezomizomi and dexamethasone. Method: 1. real-time fluory PCR (realtime quantitative PCR, RT-qPCR) was used to detect the myeloma cell line (human myeloma cell lines, HMCLs) RPMI8226, U266 and normal human peripheral blood mononuclear cells were level, and the level of protein expression was detected by immunoblotting hybridization. 2. Icles-A) and DJ-1 shRNA Lentiviral Particles (DJ-1 shRNA Lentiviral Particles) infected RPMI8226, U266 cells, using a suitable concentration of purinamycin to continuously screen the stable transfected cells, using RT-qPCR and WB to detect the expression of DJ-1 in the stable transfected cells to verify the transfection effect. 3. After DJ-1 expression, CCK8 colorimetric method was used to detect cell proliferation, spontaneous apoptosis and cell cycle, and compared with the control group; 4. transfected with lentivirus and RPMI8226, U266 cell DJ-1 expression, the cells were treated with bortezomizomi and dexamethasone, cell proliferation was detected by CCK8 colorimetry, cell cycle was detected by flow cytometry, and cell cycle was detected by flow cytometry. The results of apoptosis were compared with those in the control group. The results of 1.rt-qpcr and WB detection showed that the multiple myeloma cell line RPMI8226, dj-1mrna and protein in U266 were higher than those of normal human peripheral blood mononuclear cells..2.dj-1shrna lentivirus vector (shdj-1) transfected with multiple myeloma cell line RPMI8226, U266, The stable transfection cells were screened using the complete medium of purinamycin, and the level of dj-1mrna and protein in shdj-1 transfected myeloma cells was obviously lower than that of the blank control group and the shcontrol group. It showed that we successfully constructed the DJ-1 down-regulated myeloma cell model.3. transfected shdj-1, except the 48hrpmi8226 two group of the seed plate. The cell OD value was not significantly different (p=0.0779). The cell proliferation slowed down (P0.05) compared with the shcontorl control group, the proportion of G1 cells in the cell cycle decreased, the proportion of cells in the S phase increased, the cells blocked in the S phase (P0.05), and the early apoptosis rate of the cells was significantly increased (P0.05).4. use final concentration was 0,2.0,4.0,6.0,8.0,10.. 0nmol/l's Boron for Zomi, 0,0.5,1.0,5.0,10.0,20.0 mol/l dexamethasone treated each group of cells 24h, CCK8 method was used to detect the OD value of each cell, and the cell proliferation inhibition rate was calculated. The results showed that the cell proliferation inhibition rate increased with the increase of drug concentration. Compared with the same drug concentration, all the cells were found in 0.5,1.0 mu mol/l. There was no significant difference in the proliferation inhibition rate of U266 cells in each group. The inhibition rate of cell proliferation in group shdj-1 was significantly higher than that in group shcontrol (P0.05).5. using bortezomizomi at the final concentration of 2.0nmol/l, and 48h in each group of myeloma cell line RPMI8226 by 1 mu mol/l. The cell cycle was detected by flow cytometry. The results showed that compared with the shControl group, the proportion of G1 phase cells in group shDJ-1 decreased, the proportion of S cells increased, and the cells blocked in S phase (P0.05).6. with different concentrations of bortel (final concentration 0,2.5,5.0,10.0nmol/L), and dexamethasone (the final concentration of 0,0.1,1.0,10.0 micron mol/L) treated each group of cells, after 24h. The cell apoptosis was detected by flow cytometry, and cell apoptosis was detected by flow cytometry. The results showed that the early apoptosis rate increased with the increase of drug concentration. Under the same concentration of drug treatment, there was no significant difference in the difference of U266 cells between 0.1 mol/L dexamethasone except 5.0nmol/L boron for Zomi and 0.1 Mu mol/L dexamethasone (P=0.0505; 0.051), and the other shDJ-1 group cells. The apoptosis rate was significantly higher than that in the shControl group (P0.05), and the difference was statistically significant. Conclusion: 1. in the two common human multiple myeloma cell lines, RPMI8226, U266, DJ-1mRNA and protein are abnormal high expression of.2.DJ-1 involved in regulating RPMI8226, U266 cell proliferation, cell cycle and apoptosis, and inhibiting the DJ-1 enhanced cells to bortezomizomi and the ground The sensitivity of amethasone, but the specific mechanism remains to be further studied..3.DJ-1 may be a potential therapeutic target for multiple myeloma and deserves further study.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3

【参考文献】