【作者】 臧琳；

【导师】 杨焕民；

【作者基本信息】 黑龙江八一农垦大学 ， 基础兽医学， 2012， 硕士

【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H₂O₂作用BRL-3A细胞，根据细胞存活率筛选最佳的H₂O₂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 H₂O₂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 H₂O₂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（P>0.05），SOD活力高（P>0.05），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 H₂O₂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。更多还原

【Abstract】 Animal’s liver can be injured due to oxidative stress during the cold stress. Hsp70is a protein which canimprove the body’s tolerance for stressors. An exogenous hsp70can be introduced and expressed in the cellsusing an adenoviral vector. Based on this, the effect and mechanism of the protection by adenovirus-mediatedtransfer of hsp70in rat liver cells against oxidative stress were studied by using the techniques of WST-1, insitu staining, qRT-PCR and western blotting, etc. The research consisted of3parts:（I） Determining the optimalconcentration and treatment duration of H₂O₂according to the cells’ survival rates. Then treated the cells withthis concentration, detected the protein carbonyl content, SOD, GSH-Px and CAT activities, and thencompared with the indicators from cold stress rat experiments, in order to investigate the feasibility ofmimicking oxidative liver damages in vitro.（II） Transfecting the BRL-3A cells using the adenovirus vectorwith gene tags, then proceed with a preliminary screening of the optimal concentration and treatment durationfor virus transfection according to the transfection efficiency. Then the BRL-3A cells with adenovirus carryinghsp70under those concentration and treatment duration were measured the mRNA abundance of hsp70, anddetermined the optimal transfection concentration and exposure time which enabled the maximum expressionof hsp70. Finally, verified the successful establishment of the overexpression model of Hsp70in the BRL-3Acells at the protein level.（III） Dividing the Ad-CMV-hsp70cells group, the Ad-CMV-Null cells group and thenon-transfected cells group into each one another2groups: stressed and non-stressed, then treated themseparately, and detected, in each of these6groups, the proliferation of cells, the LDH leakage rate, the SOD,CAT and GSH-Px activities, the GSH, MDA and protein carbonyl contents, the hsp70mRNA abundance andthe Hsp70protein expression level. This was to study the protective effects of hsp70gene transfer using anadenoviral vector on rat liver cells against oxidative stress, and thus we hoped to lay a foundation for thefurther researches on Hsp70in animals’ resistance to cold stress.The results were as follows:（I） Compared with the control group, H₂O₂of500μmol/L which acted on theBRL-3A cells for3h lead to a decrease of survival rate （P<0.01）, an increase of protein carbonyl content（P<0.01） and reductions of activities of SOD （P<0.05）, GSH-Px （P<0.05） and CAT （P<0.01）. These resultsshowed that the H₂O₂of500μmol/L acting on BRL-3A cells could generate oxidative stress, and the trendsof change on indicators were similar to those of a cold exposed and damaged rat liver.（II） It was screened outthat transfecting the BRL-3A cells with the Ad-CMV-hsp70virus at the concentration of1×107PFU/mL,MOI=20, could better keep the cell morphology than with the other concentrations and action times, and it alsobrought a higher transfection efficiency, and a higher hsp70mRNA abundance （P<0.01）. Then it was found,with statistically high significance （P<0.01）, that the cells in Ad-CMV-hsp70group had the highest expressionof Hsp70than the Ad-CMV-Null group and the non-transfected control group.（III） Compared with thenon-transfected/stressed group, cells in the Ad-CMV-hsp70/stressed group showed higher proliferation rate（P<0.01）, lower LDH leakage rate （P<0.01）, higher CAT activity （P<0.01）, lower GSH-Px activity （P<0.01）,higher GSH content （P>0.05）, higher SOD activity （P>0.05）, higher protein carbonyl content （P<0.01）, higherhsp70mRNA abundance and the protein expression level （P<0.01）.The conclusions are as follows:（A） An oxidative stress can be generated when H₂O₂is acting on BRL-3A cells for3h, and the status is similar to a real cold-exposed-and-damaged rat liver.（B） By transfecting theBRL-3A cells with the Ad-CMV-hsp70virus at the concentration of1×10⁷PFU/mL, MOI=20, for48h, anHsp70-overexpressing liver cell model can be established.（C） Adenoviral transfection of Hsp70has someprotective effects for rat liver cells against oxidative stress.更多还原