本文選題：IRE1分子 + 人正常肝細(xì)胞　； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：內(nèi)質(zhì)網(wǎng)（ER）保持蛋白質(zhì)內(nèi)環(huán)境相對穩(wěn)定的這一功能的失衡，是由多種因素引起的。這其中就包含了缺氧和氧化應(yīng)激。這一功能的失衡就會導(dǎo)致蛋白質(zhì)折疊能力和蛋白質(zhì)負(fù)載能力之間的失衡。這些因素共同引發(fā)了內(nèi)質(zhì)網(wǎng)應(yīng)激（ER stress）以及未折疊蛋白反應(yīng)（UPR）。未折疊蛋白反應(yīng)（UPR）最初是作為細(xì)胞的一種適應(yīng)性反應(yīng)，但是在內(nèi)質(zhì)網(wǎng)應(yīng)激長期存在的情況下，也能誘導(dǎo)細(xì)胞凋亡。衣霉素(TM)是一種常用的體外內(nèi)質(zhì)網(wǎng)應(yīng)激的誘導(dǎo)試劑，在一定的濃度和作用時間下，能夠誘導(dǎo)細(xì)胞產(chǎn)生內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的反應(yīng)而對抗缺氧對細(xì)胞的損傷作用。通過篩選未折疊蛋白反應(yīng)的三條分子通路，我們重點研究IRE1通路中的下游蛋白RACK1分子，在內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的情況下探討其蛋白和RNA水平表達(dá)是否上調(diào)。再沉默其表達(dá)，通過Western-blot和RT-PCR檢測以及對細(xì)胞凋亡率的檢測和統(tǒng)計學(xué)分析，證明RACK1在內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的條件下，通過IRE1通路，作為一種保護(hù)性蛋白存在于肝細(xì)胞當(dāng)中。 實驗1．將正常人肝臟細(xì)胞HL-7702分成5組，分別為A，B，C，D，E組；A組為正常對照組，其余四組正常培養(yǎng)48小時后換液（分別含衣霉素濃度為0.05，0.10,0.20,0.40μg/ml的培養(yǎng)基）培養(yǎng)48小時后換正常培養(yǎng)基，即刻轉(zhuǎn)入缺氧培養(yǎng)箱分別培養(yǎng)2，4，8，16小時后再統(tǒng)一轉(zhuǎn)入正常培養(yǎng)箱培養(yǎng)6小時后觀察細(xì)胞形態(tài)并收集細(xì)胞測凋亡值。 結(jié)論： 根據(jù)普通光學(xué)顯微鏡和電子顯微鏡觀察結(jié)果和流式細(xì)胞檢測結(jié)果顯示D、E兩組細(xì)胞全部死亡；B組細(xì)胞與正常對照無差異；C組細(xì)胞凋亡率為20%。將衣霉素濃度0.1μg/ml，缺氧培養(yǎng)4小時再復(fù)氧培養(yǎng)6小時作為正常肝細(xì)胞缺氧模型的標(biāo)準(zhǔn)時限。 實驗2.根據(jù)實驗1的模型，將細(xì)胞分為4組：正常對照組（control組）；內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激組（ERS組）；缺氧損傷組（H/R組）；內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激+缺氧損傷組（ERS+H/R組）。收集各組細(xì)胞提取蛋白質(zhì)，通過Western-blot檢測內(nèi)質(zhì)網(wǎng)應(yīng)激特異性分子伴侶GRP78和UPR三條通路的蛋白ATF6，PERK以及RACK1的表達(dá)。 結(jié)論： 在內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的條件下，GRP78表達(dá)均上調(diào)，，證明衣霉素成功誘導(dǎo)了肝細(xì)胞內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的表達(dá)。UPR三條通路的蛋白分子ATF6，PERK以及RACK1的表達(dá)在內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的情況下有著不同程度的提高。均比正常對照和單純?nèi)毖鯎p傷組的表達(dá)要高。有統(tǒng)計學(xué)意義。通過GRP78這一標(biāo)志性蛋白的提升我們肯定衣霉素對肝細(xì)胞內(nèi)質(zhì)網(wǎng)預(yù)應(yīng)激的成功誘導(dǎo)，UPR三條通路的蛋白的表達(dá)也不同程度提高，本課題重點研究IRE1下游分子RACK1對肝細(xì)胞缺氧的保護(hù)機制。 實驗3．根據(jù)實驗1的模型，再將培養(yǎng)的人肝細(xì)胞分為5組：A組：正常對照；B組：內(nèi)質(zhì)網(wǎng)應(yīng)激+缺氧復(fù)氧損傷組；C組：單純內(nèi)質(zhì)網(wǎng)應(yīng)激組；D組：內(nèi)質(zhì)網(wǎng)應(yīng)激+空載轉(zhuǎn)染組；E組：內(nèi)質(zhì)網(wǎng)應(yīng)激+siRNA轉(zhuǎn)染組。收集各組細(xì)胞，以流式細(xì)胞儀檢測細(xì)胞凋亡，Western-bloting及RT-PCR檢測內(nèi)質(zhì)網(wǎng)應(yīng)激特異蛋白RACK1表達(dá)水平，并通過透射電鏡觀察各組細(xì)胞超微結(jié)構(gòu)改變。 結(jié)論： E組細(xì)胞凋亡率明顯升高；B C D組中RACK1的RNA和蛋白質(zhì)均高表達(dá)而E組細(xì)胞RNA和蛋白質(zhì)表達(dá)均降低。內(nèi)質(zhì)網(wǎng)應(yīng)激預(yù)處理對肝細(xì)胞缺氧損傷具有明顯的保護(hù)作用，沉默RACK1在細(xì)胞中的表達(dá)將大大提高肝細(xì)胞缺氧損傷后的凋亡值，內(nèi)質(zhì)網(wǎng)應(yīng)激特異性蛋白RACK1可能在肝細(xì)胞缺氧損傷中作為一種關(guān)鍵性的保護(hù)蛋白出現(xiàn)。
[Abstract]:The imbalance in the relative stability of the endoplasmic reticulum (ER) is caused by a variety of factors, including hypoxia and oxidative stress. The imbalance of the function leads to the imbalance between protein folding ability and protein load capacity. These factors jointly trigger the endoplasmic reticulum stress (ER stress) and Unfolded protein reaction (UPR). Unfolded protein reaction (UPR) is initially an adaptive response to cells, but it can also induce apoptosis in the presence of endoplasmic reticulum stress. TM is a common inducer to induce endoplasmic reticulum stress in vitro and can induce cell production at a certain concentration and time of action. By screening three molecular pathways of unfolded protein reaction, we focused on the downstream protein RACK1 molecules in the IRE1 pathway by screening the three molecular pathways of the unfolded protein reaction. We studied the up-regulated expression of the protein and the level of the protein and the level of the protein and the level of the RACK1 in the endoplasmic reticulum prestress. Then the expression was reticted and Western-blot was reticted. The detection of RT-PCR and the detection and statistical analysis of the rate of apoptosis show that RACK1 exists in the liver cells as a protective protein under the prestress of endoplasmic reticulum through the IRE1 pathway.
In experiment 1., normal human liver cell HL-7702 was divided into 5 groups, which were A, B, C, D, E group, and the A group was the normal control group. The other four groups were cultured for 48 hours after normal culture. After 48 hours, the culture medium was changed to the normal medium for 48 hours, and then transferred to the hypoxia incubator and then cultured for 2,4,8,16 hours respectively. The cells were then transferred to normal incubator for 6 hours to observe cell morphology and collect apoptotic values.
Conclusion:
According to the results of common optical and electron microscope observation and flow cytometry, all the cells in D, E two were dead, and there was no difference between the group B and the normal control. The apoptosis rate of the group C was 20%., the concentration of ycomycin was 0.1 mu, and the oxygen culture of the hypoxia culture for 4 hours was used as the standard of the normal liver cell hypoxia model. Limit.
Experiment 2. divided the cells into 4 groups: normal control group (group control), endoplasmic reticulum prestress group (group ERS), hypoxia injury group (group H/R) and endoplasmic reticulum prestress + hypoxia injury group (group ERS+H/R). The protein was collected from each group, and GRP78 and UPR three in endoplasmic reticulum stress were detected by Western-blot, and three UPR were detected by Western-blot. The expression of protein ATF6, PERK and RACK1 of the pathway.
Conclusion:
Under the prestress condition of endoplasmic reticulum, the expression of GRP78 was up-regulated. It was proved that the expression of ATF6, PERK and RACK1 in the.UPR three pathway of hepatocyte endoplasmic reticulum prestress was improved in varying degrees in the condition of endoplasmic reticulum prestress, which were higher than those of normal control and simple hypoxia injury groups. It is statistically significant. Through the promotion of the GRP78 marker protein, we affirm the successful induction of prestress on the endoplasmic reticulum of liver cells and the expression of protein in the UPR three pathway in different degrees. This topic focuses on the protection mechanism of RACK1 on the hypoxia of hepatocytes in the lower IRE1 molecule RACK1.
Experiment 3. according to the model of Experiment 1, the cultured human hepatocytes were divided into 5 groups: A group: normal control; group B: endoplasmic reticulum stress + hypoxia reoxygenation injury group; group C: simple endoplasmic reticulum stress group; D group: endoplasmic reticulum stress + transfection group; E group: endoplasmic reticulum stress +siRNA transfection group. Collection of cells in each group by flow cytometry Apoptosis, Western-bloting and RT-PCR were used to detect the expression level of RACK1 in the endoplasmic reticulum stress specific protein, and the ultrastructural changes in each group were observed by transmission electron microscope.
Conclusion:
The apoptosis rate of E group was significantly higher, the RNA and protein of RACK1 in the group of B C D were highly expressed, while the expression of RNA and protein in the E group decreased. The endoplasmic reticulum stress preconditioning had obvious protective effect on the hypoxia injury of liver cells. The expression of silent RACK1 in the cells would greatly increase the apoptosis value of the liver cells after hypoxia injury and endoplasmic reticulum stress. Specific protein RACK1 may be a key protective protein in hypoxic injury of hepatocytes.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉慧婷;胡金龍;;活化的蛋白激酶C受體1(RACK1 Receptor for Activated C Kinase1)在腫瘤相關(guān)方面的研究進(jìn)展[J];中國醫(yī)學(xué)工程;2012年05期

相關(guān)博士學(xué)位論文 前1條

1 高盈;RACK1對肺腺癌細(xì)胞侵襲轉(zhuǎn)移e笥跋旒傲俅慘庖錥D];中南大學(xué);2012年

相關(guān)碩士學(xué)位論文 前5條

1 雷玉平;冠狀動脈粥樣硬化患者外周血RACK1表達(dá)變化及與冠脈狹窄程度的關(guān)系[D];青島大學(xué);2012年

2 梁瑞;A549細(xì)胞中Rack1的敲低抑制細(xì)胞增殖促進(jìn)上皮—間充質(zhì)轉(zhuǎn)換[D];蘭州大學(xué);2012年

3 朱薇;大腸癌中RACK1的表達(dá)研究及意義[D];南華大學(xué);2013年

4 李秀萍;活化的蛋白激酶C受體1與Ki67在甲狀腺乳頭狀癌的表達(dá)及臨床意義[D];大連醫(yī)科大學(xué);2013年

5 張大林;支架蛋白RACK1對炎性細(xì)胞因子表達(dá)的影響[D];中南大學(xué);2013年

本文編號：1844382