腺病毒介导的hsp70对大鼠肝细胞氧化应激的保护作用

发布时间：2018-05-09 10:40

  本文选题：Hsp70 + BRL-3A　； 参考：《黑龙江八一农垦大学》2012年硕士论文

【摘要】：动物在冷应激过程中，肝脏因发生氧化应激而受损。Hsp70是一种能够提高机体应激耐受的蛋白，通过腺病毒载体感染可以将外源的hsp70引入细胞中并表达。基于此，本试验采用WST-1、原位染色、实时定量反转录PCR和western blotting等方法，针对腺病毒介导的hsp70对大鼠肝细胞氧化应激的保护作用及机制展开研究：（一）利用不同浓度H_2O_2作用BRL-3A细胞，根据细胞存活率筛选最佳的H_2O_2作用浓度和时间。然后以此浓度处理细胞，检测蛋白质羰基含量，SOD、GSH-Px和CAT酶活力，并与大鼠冷应激试验中肝脏氧化损伤指标相对比，探讨建立动物冷应激过程中肝脏氧化应激损伤的细胞模型的可行性。（二）以带有标签基因的腺病毒感染BRL-3A细胞，根据感染效率初筛病毒感染的最适浓度和作用时间。然后用带有目的基因hsp70的腺病毒以初筛出的最适浓度和时间感染BRL-3A细胞，，并检测hsp70的mRNA丰度，最终确定能使hsp70的mRNA丰度最高的感染浓度和作用时间。之后在蛋白水平上验证BRL-3A细胞Hsp70过表达模型是否建立成功。（三）将感染带有hsp70腺病毒的Hsp70过表达细胞、感染空载体腺病毒的细胞和未感染病毒的细胞每一组都分为应激组和无应激组，给予相应的处理后，检测各组细胞的增殖情况，SOD、CAT和GSH-Px酶活力，LDH漏出率，GSH、MDA和蛋白质羰基含量，hsp70mRNA丰度及Hsp70表达量。以此探究重组腺病毒介导的hsp70感染细胞的方法对大鼠肝细胞氧化应激的保护作用，并希望能为Hsp70在动物抗冷应激相关领域的研究奠定基础。 研究结果如下：（一）与对照组相比，500μmol/L H_2O_2作用于BRL-3A细胞3h，可导致细胞存活率下降（P＜0.01），蛋白质羰基含量升高（P＜0.01），SOD（P＜0.05）、GSH-Px（P＜0.05）和CAT（P＜0.01）酶活力均降低。结果表明，500μmol/L H_2O_2作用于BRL-3A细胞可导致氧化应激的发生，其相关指标与大鼠冷暴露后肝脏受损指标改变趋势相符。（二）通过筛选，浓度为1×107PFU/mL的病毒Ad-CMV-hsp70按MOI=20感染BRL-3A细胞48h，比其他浓度和时间组的细胞形态保持良好，感染效率高，hsp70的mRNA丰度高（P＜0.01）。检测该组细胞和病毒Ad-CMV-Null处理的细胞以及无病毒处理的对照组细胞蛋白表达量，结果显示Ad-CMV-hsp70组细胞Hsp70表达量最高，与其他两组相比均差异极显著（P＜0.01）。（三）与无病毒/应激组细胞相比，Ad-CMV-hsp70/应激组的增殖率高（P＜0.01），LDH漏出率低（P＜0.01），CAT活力高（P＜0.01），GSH-Px活力低（P＜0.05），GSH含量高（P0.05），SOD活力高（P0.05），蛋白质羰基含量高（P＜0.01），hsp70mRNA丰度及Hsp70蛋白表达量均高（P＜0.01）。 结论：（一）500μmol/L H_2O_2作用于BRL-3A细胞3h可导致氧化应激的发生，并基本模拟了大鼠冷应激过程中肝脏所受的氧化应激损伤程度。（二）以浓度为1×107PFU/mL的病毒Ad-CMV-hsp70按MOI=20感染BRL-3A细胞48h，能够建立过表达Hsp70的肝细胞模型。（三）腺病毒介导的hsp70对大鼠肝细胞氧化应激有一定的保护作用。
[Abstract]:During cold stress, the liver is damaged by oxidative stress. Hsp70 is a protein that can enhance stress tolerance. Exogenous hsp70 can be introduced into cells and expressed by adenovirus vector infection. Based on this, WST-1, in situ staining, real-time quantitative reverse transcription PCR and western blotting were used to study the protective effect and mechanism of adenovirus-mediated hsp70 on oxidative stress of rat hepatocytes. The effects of different concentrations of H_2O_2 on BRL-3A cells were studied. The optimal concentration and time of H_2O_2 were selected according to the cell survival rate. Then, the activity of GSH-Px and CAT enzyme in protein carbonyl group was measured by this concentration, and compared with the oxidative damage index of liver in the cold stress test of rats. To explore the feasibility of establishing a cell model of liver oxidative stress during cold stress in animals. (2) BRL-3A cells were infected with adenovirus with tagged gene, and the optimal concentration and time of infection were screened according to the infection efficiency. Then the adenovirus with the target gene hsp70 was used to infect BRL-3A cells with the optimal concentration and time of initial screening, and the mRNA abundance of hsp70 was detected to determine the infection concentration and time of action which could make the mRNA abundance of hsp70 the highest. Then the Hsp70 overexpression model of BRL-3A cells was successfully established at the protein level. (3) the Hsp70 overexpression cells infected with hsp70 adenovirus, the cells infected with empty vector adenovirus and the cells without infection were divided into stress group and non-stress group. The activity of cat and GSH-Px and the leakage rate of LDH were measured. The contents of GSH-MDA and protein carbonyl were determined. The mRNA abundance and Hsp70 expression of Hsp70 were measured. The purpose of this study was to explore the protective effect of recombinant adenovirus mediated hsp70 infection on oxidative stress of rat hepatocytes, and to lay a foundation for the study of Hsp70 in the field of anti-cold stress in animals. The results were as follows: (1) compared with the control group, the cell viability of BRL-3A cells was decreased after treated with 渭 mol/L H_2O_2 for 3 h, and the protein carbonyl content increased (P < 0.01) and the activity of GSH-PxP < 0.05 and CAT(P < 0.01) decreased. The results showed that 500 渭 mol/L H_2O_2 could induce oxidative stress in BRL-3A cells, and the related indexes were consistent with the changes of liver damage indexes after cold exposure in rats. (2) after screening, 1 脳 107PFU/mL virus Ad-CMV-hsp70 infected BRL-3A cells with MOI=20 for 48h, which was better than that of other concentration and time groups, and the mRNA abundance of HSP70 with high infection efficiency was higher than that of other groups (P < 0.01). The protein expression of cells treated with virus Ad-CMV-Null and control group without virus was detected. The results showed that the expression of Hsp70 in Ad-CMV-hsp70 group was the highest, compared with the other two groups, the difference was significant (P < 0.01). (3) the proliferation rate of Ad-CMV-hsp70 / stress group was higher than that of virus-free / stress-free group (P < 0.01) and the leakage rate of LDH was lower than that of control group (P < 0.01). The activity of GSH-Px, GSH-Px, GSH-Px, the activity of P0.05SOD, the content of protein carbonyl, the abundance of hsp70 mRNA and the expression of Hsp70 protein were all high (P < 0.011). Conclusion the oxidative stress in BRL-3A cells could be induced by the action of 1: 500 渭 mol/L H_2O_2 for 3 h, and the degree of oxidative stress in the liver during cold stress in rats was simulated. (2) Hepatocyte model expressing Hsp70 could be established by infecting BRL-3A cells with 1 脳 107PFU/mL virus Ad-CMV-hsp70 for 48 h. (3) Adenovirus-mediated hsp70 can protect rat hepatocytes from oxidative stress.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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