镉致HEK293细胞氧化损伤和细胞凋亡中miR-21的调控作用研究

发布时间：2018-01-15 12:35

本文关键词：镉致HEK293细胞氧化损伤和细胞凋亡中miR-21的调控作用研究　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

更多相关文章：镉 HEK293细胞 凋亡 氧化应激 miR-21

【摘要】：目的:(1)通过对HEK239细胞进行不同浓度、不同时间CdCl_2染毒,研究不同浓度、不同时间CdCl_2对细胞氧化损伤、细胞凋亡和miR-21表达的影响。(2)通过对HEK239细胞进行CdCl_2染毒、miR-21 mimic、miR-21 inhibitor转染和转染后CdCl_2染毒,研究miR-21在镉致HEK293细胞氧化损伤中的作用。(3)通过对HEK239细胞进行CdCl_2染毒、miR-21 mimic、miR-21 inhibitor转染和转染后CdCl_2染毒,研究miR-21在镉致HEK293细胞凋亡中的作用。方法:培养HEK293细胞,对细胞进行不同浓度、不同时间CdCl_2染毒、miR-21mimic和inhibitor转染和转染后CdCl_2染毒处理,用倒置显微镜观察细胞形态,MTT法测定细胞生长状况,流式细胞仪测定细胞凋亡率和细胞中ROS含量、用硫代巴比妥酸法测定细胞中MDA含量,比色法测定GSH-PX活力,水溶性四唑盐法测定细胞中SOD活力水平的变化,qRT-PCR检测miR-21的表达丰度。结果:(1)不同浓度的CdCl_2作用不同时间,细胞贴壁性减弱,细胞变圆,伪足消失。随着CdCl_2浓度的增高,作用时间延长,细胞的生长受到抑制,在CdCl_2浓度为120μmol/L,染毒时间为12h时抑制作用达到最大。与0μmol/L CdCl_2比较,细胞凋亡率增加(P0.05);随着作用时间的延长,30μmol/L和60μmol/L CdCl_2染毒组细胞凋亡率逐渐增加(Ptrend=0.001,Ptrend=0.009);当作用时间分别为1、3、6h,随着CdCl_2浓度的增加,凋亡率逐渐增加(Ptrend=0.003或Ptrend=0.001)。与0μmol/L CdCl_2组比较,细胞内MDA、ROS含量随着CdCl_2浓度的升高而增高(Ptrend0.001或Ptrend=0.001);当CdCl_2浓度分别为30、60、120μmol/L时,随着染毒作用时间的延长,MDA和ROS含量逐渐增高(Ptrend均0.05)。与0μmol/L CdCl_2组比较,30、60、120μmol/L组细胞中SOD和GSH-PX活力均降低(p0.05),且随着cdcl2浓度的增加细胞中sod和gsh-px活力呈下降趋势(ptrend0.001);当染毒浓度分别为30、60、120μmol/l时,细胞sod和gsh-px活力随着作用时间的延长而降低(ptrend均0.01)。当作用时间分别为1、3、6、12h时,不同浓度的cdcl2对细胞中mir-21的表达作用有差别,差别有统计学意义(p0.001或p=0.006),且mir-21的表达随着cdcl2染毒浓度的增加而增加(ptrend=0.002或ptrend=0.004);当cdcl2浓度分别为30、60、120μmol/l时,作用不同时间,细胞中mir-21表达差别有统计学意义(p0.001或p=0.042),在cdcl2浓度为30μmol/l和120μmol/l时,mir-21表达随cdcl2作用时间的延长而增高(ptrend=0.030,ptrend=0.011),在cdcl2浓度为120μmol/l时,染毒时间为12h时,mir-21表达达到最大。(2)mir-21mimic转染后,细胞中mda和ros含量与对照组和mir-21mimicnegativecontrol相比,mda和ros含量增加(p0.05);细胞中sod和gsh-px与对照组相比,sod和gsh-px的活性降低(p0.05),与mir-21mimicnegativecontrol相比,细胞中sod活性降低(p0.05);mir-21inhibitor转染后,细胞中mda和ros含量与对照组相比,mda和ros含量降低(p0.05),与mir-21inhibitornegativecontrol相比,ros含量降低(p0.05);细胞中sod和gsh-px与对照组和mir-21inhibitornegativecontrol相比,sod和gsh-px的活性增加(p0.05)。mir-21mimic转染后cdcl2染毒,细胞中mda和ros含量与对照组和cdcl2组相比,含量增加(p0.05);细胞中sod和gsh-px与对照组和cdcl2组相比,活性降低(p0.05)。(3)mir-21mimic转染后,与对照组相比,细胞凋亡率增加(p0.001),mir-21表达增加(p0.05);mir-21inhibitor转染后,与对照组相比,细胞凋亡率降低(p0.001)。mir-21mimic转染后cdcl2染毒,与对照组和cdcl2组相比,细胞凋亡率增加(p0.05),mir-21表达增加(p0.05);mir-21inhibitor转染cdcl2染毒,与对照组和cdcl2组相比,细胞凋亡率降低(p0.05),mir-21表达下降(p0.05)。且细胞凋亡率与mir-21含量有相关关系(p0.001),相关系数为0.732。结论:(1)镉可以导致hek293细胞氧化损伤、细胞凋亡和mir-21表达改变。(2)mir-21在镉致hek293细胞氧化损伤中起促进作用。(3)miR-21在镉致HEK293细胞凋亡中起促进作用。
[Abstract]:Objective: (1) by different concentration of HEK239 cells in different time of exposure to CdCl_2, to study the effects of different concentrations, different time of CdCl_2 on cell oxidative damage effect on apoptosis and the expression of miR-21. (2) by CdCl_2 miR-21 mimic on HEK239 cells were transfected with inhibitor, miR-21 and CdCl_2 after transfection were studied by miR-21 the oxidative damage of HEK293 cells in CD. (3) by CdCl_2 miR-21 mimic on HEK239 cells were transfected with inhibitor, miR-21 and CdCl_2 after transfection of HEK293 cells induced by miR-21 exposure, apoptosis in cadmium. Methods: HEK293 cells were cultured with different concentration of cells, different time of exposure to CdCl_2 and miR-21mimic. Inhibitor and CdCl_2 were transfected after transfection, cell morphology was observed by inverted microscope, cell growth was determined by MTT method, the determination of the content of ROS and cell apoptosis rate by flow cytometry The amount, determination of MDA content in cells using thiobarbituric acid method, the activity of GSH-PX was determined with colorimetric method, determination of the activity of SOD cells in the level of change of water soluble four tetrazolium method. The expression abundance of qRT-PCR miR-21 detection. Results: (1) different concentrations of CdCl_2 for different time, cell adherent cells became decreased. Round, pseudopod disappeared. With the increasing of CdCl_2 concentration, the reaction time prolonged, cell growth was inhibited when the CdCl_2 concentration is 120 mol/L, exposure time is 12h. The maximum inhibition compared with 0 mol/L CdCl_2, the cell apoptosis rate increased (P0.05); with the prolongation of time, and 30 mol/L 60 mol/L CdCl_2 group cell apoptosis rate increased gradually (Ptrend=0.001, Ptrend=0.009); when the time was 1,3,6h, with the increase of CdCl_2 concentration, the apoptosis rate increased gradually (Ptrend=0.003 or Ptrend=0.001). Compared with the 0 mol/L CdCl_2 group, MDA cells The content of ROS, and increased with the increasing concentration of CdCl_2 (Ptrend0.001 or Ptrend=0.001); when CdCl_2 concentration was 30,60120 mol/L, with the extension of exposure time, MDA and ROS content increased gradually (Ptrend < 0.05). Compared with the 0 mol/L group CdCl_2, SOD and GSH-PX activity of 30,60120 mol/ in L cells (P0.05), and decreased with the increase of CdCl2 concentration and SOD activity of GSH-Px cells decreased (ptrend0.001); when the exposure concentration was 30,60120 ~ mol/l, SOD cells and the activity of GSH-Px decreased with the prolongation of time (ptrend < 0.01). When the time was 1,3,6,12h, the effect of different expression the concentration of CdCl2 in the cells of miR-21 are different, the difference was statistically significant (p0.001 or p=0.006), and the expression of miR-21 increased with the increase of CdCl2 concentrations (ptrend=0.002 or ptrend= 0.004); when the concentration of CdCl2 was 30 60120, mol/l, different time, the expression of miR-21 in cells was statistically significant difference (p0.001 or p=0.042), the concentration of CdCl2 was 30 mol/l and 120 mol/l, while the expression of miR-21 increased with the prolongation of CdCl2 action time (ptrend=0.030, ptrend=0.011), the CdCl2 concentration is 120 mol/l, exposure time is 12h, the expression of miR-21 reached the maximum (2 mir-21mimic). After transfection, cells in MDA and ROS were compared with control group and mir-21mimicnegativecontrol, increased MDA and ROS content (P0.05); SOD and GSH-Px compared with the control group of cells, decreased SOD and GSH-Px activity (P0.05), compared with mir-21mimicnegativecontrol, decreased the activity of SOD cells (P0.05); mir-21inhibitor after transfection, MDA and ROS were compared with the control group of cells, decreased MDA and ROS content (P0.05), compared with mir-21inhibitornegativecontrol, ROS decreased (P0.05); SOD and GSH-Px cells Compared with the control group and mir-21inhibitornegativecontrol, SOD increased and the activity of GSH-Px (P0.05).Mir-21mimic transfected CdCl2 cells were MDA and ROS were compared with the control group and CdCl2 group increased (P0.05); SOD and GSH-Px cells compared with control group and CdCl2 group, decreased the activity of (P0.05) (3). Mir-21mimic after transfection, compared with the control group, the apoptosis rate increased (p0.001), increase the expression of miR-21 (P0.05); mir-21inhibitor after transfection, compared with the control group, the apoptosis rate decreased after transfection of CdCl2.Mir-21mimic (p0.001) were compared with control group and CdCl2 group, the apoptosis rate increased, the expression of miR-21 (P0.05) increased (P0.05); mir-21inhibitor transfected with CdCl2 were compared with control group and CdCl2 group, the apoptosis rate decreased (P0.05), decreasing the expression of miR-21 (P0.05) and the cell apoptosis rate and miR-21 content are related (p0.001), the correlation coefficient is 0.732 Conclusion: (1) cadmium can induce oxidative damage, apoptosis and miR-21 expression in HEK293 cells. (2) miR-21 plays an important role in the oxidative damage of HEK293 cells induced by cadmium. (3) miR-21 plays an important role in the apoptosis of HEK293 cells induced by cadmium.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

【参考文献】