本文選題：Hsp70 + BRL-3A��； 參考：《黑龍江八一農(nóng)墾大學(xué)》2012年碩士論文

【摘要】：動(dòng)物在冷應(yīng)激過程中，肝臟因發(fā)生氧化應(yīng)激而受損。Hsp70是一種能夠提高機(jī)體應(yīng)激耐受的蛋白，通過腺病毒載體感染可以將外源的hsp70引入細(xì)胞中并表達(dá)�；诖�，本試驗(yàn)采用WST-1、原位染色、實(shí)時(shí)定量反轉(zhuǎn)錄PCR和western blotting等方法，針對(duì)腺病毒介導(dǎo)的hsp70對(duì)大鼠肝細(xì)胞氧化應(yīng)激的保護(hù)作用及機(jī)制展開研究：（一）利用不同濃度H_2O_2作用BRL-3A細(xì)胞，根據(jù)細(xì)胞存活率篩選最佳的H_2O_2作用濃度和時(shí)間。然后以此濃度處理細(xì)胞，檢測(cè)蛋白質(zhì)羰基含量，SOD、GSH-Px和CAT酶活力，并與大鼠冷應(yīng)激試驗(yàn)中肝臟氧化損傷指標(biāo)相對(duì)比，探討建立動(dòng)物冷應(yīng)激過程中肝臟氧化應(yīng)激損傷的細(xì)胞模型的可行性。（二）以帶有標(biāo)簽基因的腺病毒感染BRL-3A細(xì)胞，根據(jù)感染效率初篩病毒感染的最適濃度和作用時(shí)間。然后用帶有目的基因hsp70的腺病毒以初篩出的最適濃度和時(shí)間感染BRL-3A細(xì)胞，，并檢測(cè)hsp70的mRNA豐度，最終確定能使hsp70的mRNA豐度最高的感染濃度和作用時(shí)間。之后在蛋白水平上驗(yàn)證BRL-3A細(xì)胞Hsp70過表達(dá)模型是否建立成功。（三）將感染帶有hsp70腺病毒的Hsp70過表達(dá)細(xì)胞、感染空載體腺病毒的細(xì)胞和未感染病毒的細(xì)胞每一組都分為應(yīng)激組和無應(yīng)激組，給予相應(yīng)的處理后，檢測(cè)各組細(xì)胞的增殖情況，SOD、CAT和GSH-Px酶活力，LDH漏出率，GSH、MDA和蛋白質(zhì)羰基含量，hsp70mRNA豐度及Hsp70表達(dá)量。以此探究重組腺病毒介導(dǎo)的hsp70感染細(xì)胞的方法對(duì)大鼠肝細(xì)胞氧化應(yīng)激的保護(hù)作用，并希望能為Hsp70在動(dòng)物抗冷應(yīng)激相關(guān)領(lǐng)域的研究奠定基礎(chǔ)。 研究結(jié)果如下：（一）與對(duì)照組相比，500μmol/L H_2O_2作用于BRL-3A細(xì)胞3h，可導(dǎo)致細(xì)胞存活率下降（P＜0.01），蛋白質(zhì)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。結(jié)果表明，500μmol/L H_2O_2作用于BRL-3A細(xì)胞可導(dǎo)致氧化應(yīng)激的發(fā)生，其相關(guān)指標(biāo)與大鼠冷暴露后肝臟受損指標(biāo)改變趨勢(shì)相符。（二）通過篩選，濃度為1×107PFU/mL的病毒Ad-CMV-hsp70按MOI=20感染BRL-3A細(xì)胞48h，比其他濃度和時(shí)間組的細(xì)胞形態(tài)保持良好，感染效率高，hsp70的mRNA豐度高（P＜0.01）。檢測(cè)該組細(xì)胞和病毒Ad-CMV-Null處理的細(xì)胞以及無病毒處理的對(duì)照組細(xì)胞蛋白表達(dá)量，結(jié)果顯示Ad-CMV-hsp70組細(xì)胞Hsp70表達(dá)量最高，與其他兩組相比均差異極顯著（P＜0.01）。（三）與無病毒/應(yīng)激組細(xì)胞相比，Ad-CMV-hsp70/應(yīng)激組的增殖率高（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），蛋白質(zhì)羰基含量高（P＜0.01），hsp70mRNA豐度及Hsp70蛋白表達(dá)量均高（P＜0.01）。 結(jié)論：（一）500μmol/L H_2O_2作用于BRL-3A細(xì)胞3h可導(dǎo)致氧化應(yīng)激的發(fā)生，并基本模擬了大鼠冷應(yīng)激過程中肝臟所受的氧化應(yīng)激損傷程度。（二）以濃度為1×107PFU/mL的病毒Ad-CMV-hsp70按MOI=20感染BRL-3A細(xì)胞48h，能夠建立過表達(dá)Hsp70的肝細(xì)胞模型。（三）腺病毒介導(dǎo)的hsp70對(duì)大鼠肝細(xì)胞氧化應(yīng)激有一定的保護(hù)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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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