本文關(guān)鍵詞：鎘致HEK293細(xì)胞氧化損傷和細(xì)胞凋亡中miR-21的調(diào)控作用研究　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的:(1)通過對HEK239細(xì)胞進(jìn)行不同濃度、不同時間CdCl_2染毒,研究不同濃度、不同時間CdCl_2對細(xì)胞氧化損傷、細(xì)胞凋亡和miR-21表達(dá)的影響。(2)通過對HEK239細(xì)胞進(jìn)行CdCl_2染毒、miR-21 mimic、miR-21 inhibitor轉(zhuǎn)染和轉(zhuǎn)染后CdCl_2染毒,研究miR-21在鎘致HEK293細(xì)胞氧化損傷中的作用。(3)通過對HEK239細(xì)胞進(jìn)行CdCl_2染毒、miR-21 mimic、miR-21 inhibitor轉(zhuǎn)染和轉(zhuǎn)染后CdCl_2染毒,研究miR-21在鎘致HEK293細(xì)胞凋亡中的作用。方法:培養(yǎng)HEK293細(xì)胞,對細(xì)胞進(jìn)行不同濃度、不同時間CdCl_2染毒、miR-21mimic和inhibitor轉(zhuǎn)染和轉(zhuǎn)染后CdCl_2染毒處理,用倒置顯微鏡觀察細(xì)胞形態(tài),MTT法測定細(xì)胞生長狀況,流式細(xì)胞儀測定細(xì)胞凋亡率和細(xì)胞中ROS含量、用硫代巴比妥酸法測定細(xì)胞中MDA含量,比色法測定GSH-PX活力,水溶性四唑鹽法測定細(xì)胞中SOD活力水平的變化,qRT-PCR檢測miR-21的表達(dá)豐度。結(jié)果:(1)不同濃度的CdCl_2作用不同時間,細(xì)胞貼壁性減弱,細(xì)胞變圓,偽足消失。隨著CdCl_2濃度的增高,作用時間延長,細(xì)胞的生長受到抑制,在CdCl_2濃度為120μmol/L,染毒時間為12h時抑制作用達(dá)到最大。與0μmol/L CdCl_2比較,細(xì)胞凋亡率增加(P0.05);隨著作用時間的延長,30μmol/L和60μmol/L CdCl_2染毒組細(xì)胞凋亡率逐漸增加(Ptrend=0.001,Ptrend=0.009);當(dāng)作用時間分別為1、3、6h,隨著CdCl_2濃度的增加,凋亡率逐漸增加(Ptrend=0.003或Ptrend=0.001)。與0μmol/L CdCl_2組比較,細(xì)胞內(nèi)MDA、ROS含量隨著CdCl_2濃度的升高而增高(Ptrend0.001或Ptrend=0.001);當(dāng)CdCl_2濃度分別為30、60、120μmol/L時,隨著染毒作用時間的延長,MDA和ROS含量逐漸增高(Ptrend均0.05)。與0μmol/L CdCl_2組比較,30、60、120μmol/L組細(xì)胞中SOD和GSH-PX活力均降低(p0.05),且隨著cdcl2濃度的增加細(xì)胞中sod和gsh-px活力呈下降趨勢(ptrend0.001);當(dāng)染毒濃度分別為30、60、120μmol/l時,細(xì)胞sod和gsh-px活力隨著作用時間的延長而降低(ptrend均0.01)。當(dāng)作用時間分別為1、3、6、12h時,不同濃度的cdcl2對細(xì)胞中mir-21的表達(dá)作用有差別,差別有統(tǒng)計學(xué)意義(p0.001或p=0.006),且mir-21的表達(dá)隨著cdcl2染毒濃度的增加而增加(ptrend=0.002或ptrend=0.004);當(dāng)cdcl2濃度分別為30、60、120μmol/l時,作用不同時間,細(xì)胞中mir-21表達(dá)差別有統(tǒng)計學(xué)意義(p0.001或p=0.042),在cdcl2濃度為30μmol/l和120μmol/l時,mir-21表達(dá)隨cdcl2作用時間的延長而增高(ptrend=0.030,ptrend=0.011),在cdcl2濃度為120μmol/l時,染毒時間為12h時,mir-21表達(dá)達(dá)到最大。(2)mir-21mimic轉(zhuǎn)染后,細(xì)胞中mda和ros含量與對照組和mir-21mimicnegativecontrol相比,mda和ros含量增加(p0.05);細(xì)胞中sod和gsh-px與對照組相比,sod和gsh-px的活性降低(p0.05),與mir-21mimicnegativecontrol相比,細(xì)胞中sod活性降低(p0.05);mir-21inhibitor轉(zhuǎn)染后,細(xì)胞中mda和ros含量與對照組相比,mda和ros含量降低(p0.05),與mir-21inhibitornegativecontrol相比,ros含量降低(p0.05);細(xì)胞中sod和gsh-px與對照組和mir-21inhibitornegativecontrol相比,sod和gsh-px的活性增加(p0.05)。mir-21mimic轉(zhuǎn)染后cdcl2染毒,細(xì)胞中mda和ros含量與對照組和cdcl2組相比,含量增加(p0.05);細(xì)胞中sod和gsh-px與對照組和cdcl2組相比,活性降低(p0.05)。(3)mir-21mimic轉(zhuǎn)染后,與對照組相比,細(xì)胞凋亡率增加(p0.001),mir-21表達(dá)增加(p0.05);mir-21inhibitor轉(zhuǎn)染后,與對照組相比,細(xì)胞凋亡率降低(p0.001)。mir-21mimic轉(zhuǎn)染后cdcl2染毒,與對照組和cdcl2組相比,細(xì)胞凋亡率增加(p0.05),mir-21表達(dá)增加(p0.05);mir-21inhibitor轉(zhuǎn)染cdcl2染毒,與對照組和cdcl2組相比,細(xì)胞凋亡率降低(p0.05),mir-21表達(dá)下降(p0.05)。且細(xì)胞凋亡率與mir-21含量有相關(guān)關(guān)系(p0.001),相關(guān)系數(shù)為0.732。結(jié)論:(1)鎘可以導(dǎo)致hek293細(xì)胞氧化損傷、細(xì)胞凋亡和mir-21表達(dá)改變。(2)mir-21在鎘致hek293細(xì)胞氧化損傷中起促進(jìn)作用。(3)miR-21在鎘致HEK293細(xì)胞凋亡中起促進(jìn)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.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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