本文關(guān)鍵詞： 熱休克蛋白A12B 小鼠肺微血管內(nèi)皮細(xì)胞 CLP模型 內(nèi)毒素 急性肺損傷 HSPA12B mPMVEC 敲除 綠色熒光蛋白 熱休克蛋白A12B 肺微血管內(nèi)皮細(xì)胞 內(nèi)毒素 HSPA12B LPS損傷 磷酸化p38 p38MAPK信號(hào)通路　出處：《第二軍醫(yī)大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景及總體思路 膿毒癥時(shí)，肺臟是最易受損傷的重要臟器之一，急性肺損傷（ALI）出現(xiàn)早，發(fā)生率高，一直是膿毒癥研究的熱點(diǎn)之一。內(nèi)毒素（lipopolysaccharide，LPS）導(dǎo)致的肺微血管內(nèi)皮細(xì)胞（pulmonary micro-vascular endothelial cells，PMVECs)損傷，可引起肺血屏障功能障礙。膿毒癥時(shí)，內(nèi)毒素導(dǎo)致的急性肺損傷，其發(fā)生發(fā)展中關(guān)鍵細(xì)胞之一是肺微血管內(nèi)皮細(xì)胞。因此，保護(hù)膿毒癥損傷的PMVECs具有重要的臨床價(jià)值。 在膿毒癥相關(guān)性急性肺損傷研究中，不少學(xué)者重視細(xì)胞內(nèi)源性保護(hù)機(jī)制，關(guān)注熱點(diǎn)之一就是熱休克蛋白（Heat shock proteins，HSPs）。HSPs是細(xì)胞在應(yīng)激狀態(tài)下產(chǎn)生增多的一類保護(hù)性蛋白質(zhì)。而其中熱休克蛋白A12B（Heat shock proteinA12B,HSPA12B)是新近發(fā)現(xiàn)的一種熱休克蛋白，是HSP70家族中的遠(yuǎn)親，大量存在于動(dòng)脈粥樣硬化斑塊中，與血管生成保護(hù)相關(guān)。研究表明，HSPA12B在斑馬魚生長過程中和離體細(xì)胞血管形成中占有重要的作用。但膿毒癥時(shí)內(nèi)毒素對肺組織的PMVECs中HSPA12B表達(dá)變化產(chǎn)生何種影響？HSPA12B表達(dá)變化對PMVECs具有何種作用？其產(chǎn)生作用的可能機(jī)制怎樣？尚有待進(jìn)一步研究。 本研究通過在體的盲腸結(jié)扎穿孔（CLP）小鼠模型和LPS刺激小鼠肺微血管內(nèi)皮細(xì)胞的離體實(shí)驗(yàn)，檢測膿毒癥時(shí)HSPA12B在各時(shí)間點(diǎn)的表達(dá)水平；用小干擾RNAs（siRNAs）下調(diào)mPMVECs中HSPA12B的表達(dá)后，觀察mPMVECs遷移和凋亡的變化，以及檢測LPS刺激mPMVECs產(chǎn)生的炎癥因子，研究HSPA12B對LPS處理的PMVECs的作用，并探討其與MAPK信號(hào)通路中p38的關(guān)系。 第一部分膿毒癥時(shí)內(nèi)毒素對鼠肺組織中HSPA12B表達(dá)的影響 目的 探討HSPA12B在盲腸結(jié)扎穿孔(CLP)小鼠模型的肺組織中和LPS刺激的mPMVECs中的表達(dá)及其規(guī)律。 方法 1.構(gòu)建鼠CLP模型，分別在0h、3h、6h、9h、12h、18h、24h斷頭取肺。應(yīng)用RT-PCR法檢測肺組織中HSPA12B mRNA的水平，探討膿毒癥時(shí)肺組織中HSPA12B表達(dá)變化。 2.培養(yǎng)mPMVECs，倒置顯微鏡下觀察各時(shí)間點(diǎn)內(nèi)皮細(xì)胞形態(tài)，用終濃度為1μg/ml的LPS刺激mPMVECs，分別于0h、3h、6h、9h、12h、18h、24h收集細(xì)胞， 應(yīng)用RT-PCR法檢測HSPA12B mRNA的水平，探討LPS刺激的mPMVECs中HSPA12B表達(dá)變化。 結(jié)果 1. CLP模型3h、6h、9h、12h肺組織中HSPA12B表達(dá)逐步升高，，12h達(dá)峰值，后又逐漸下降。 2. LPS刺激mPMVECs，HSPA12B表達(dá)也呈現(xiàn)逐步升高，后又下降。 結(jié)論 膿毒癥時(shí)內(nèi)毒素對鼠肺組織和mPMVECs中HSPA12B表達(dá)具有一定影響，HSPA12B表達(dá)先升高后下降。 第二部分用siRNA干擾法下調(diào)原代mPMVECs上HSPA12B的表達(dá) 目的 用siRNA干擾法下調(diào)原代mPMVECs上HSPA12B的表達(dá)并驗(yàn)證之。 方法 分別將siRNA和空白對照的siRNA通過脂質(zhì)體Lippo2000TM轉(zhuǎn)入mPMVECs內(nèi)，實(shí)驗(yàn)分為兩部分：1.通過轉(zhuǎn)入帶熒光的siRNA觀察細(xì)胞的轉(zhuǎn)染效率；2.用RT-PCR及Western Blot法檢測HSPA12B的表達(dá)水平，驗(yàn)證siRNA的干擾效果。 結(jié)果 1.通過熒光顯微鏡觀察到mPMVECs均有綠色熒光蛋白表達(dá)，說明siRNA轉(zhuǎn)入到細(xì)胞內(nèi)。 2. RT-PCR和Western Blot檢測到mPMVECs中HSPA12B表達(dá)下調(diào)。 結(jié)論 成功構(gòu)建HSPA12B表達(dá)下調(diào)的mPMVECs。 第三部分HSPA12B表達(dá)下調(diào)后LPS對mPMVECs的遷移功能、炎癥反應(yīng)以及凋亡的影響 目的 探討HSPA12B對LPS刺激的mPMVECs遷移功能、炎癥反應(yīng)和凋亡情況的影響。 方法 根據(jù)以下情況將mPMVECs分為四組：①空白組、②LPS刺激組、③LPS+HSPA12B siRNA組、④LPS+空白siRNA組，分別置于37oC5%CO2潮濕孵箱內(nèi)培養(yǎng)，用LPS與細(xì)胞共培養(yǎng)24h，取出細(xì)胞后用RT-PCR檢測IL-6、IL-1β、IL-10和TNF-α mRNA表達(dá)情況,使用劃痕實(shí)驗(yàn)和transwell小室檢測細(xì)胞遷移情況，用流式細(xì)胞儀檢測細(xì)胞凋亡情況，用透射電子顯微鏡觀察細(xì)胞超微結(jié)構(gòu)變化。 結(jié)果 RT-PCR檢測結(jié)果表明：mPMVECs經(jīng)LPS刺激24h后，與平行對照組（LPS+空白siRNA組）相比，HSPA12B表達(dá)下調(diào)組IL-6和TNF-α mRNA表達(dá)水平顯著上升，而IL-10則表達(dá)下降，且均有統(tǒng)計(jì)學(xué)意義（P 0.05）。遷移實(shí)驗(yàn)表明：與平行對照組對比，HSPA12B表達(dá)下調(diào)組的mPMVECs的遷移功能明顯減弱；凋亡實(shí)驗(yàn)表明：與平行對照組相比，HSPA12B表達(dá)下調(diào)組LPS刺激后mPMVECs凋亡明顯增多；透射電子顯微鏡結(jié)果顯示：與平行對照組相比，HSPA12B表達(dá)下調(diào)組細(xì)胞超微結(jié)構(gòu)破壞加重。 結(jié)論 LPS刺激的HSPA12B表達(dá)下調(diào)的mPMVECs遷移功能減弱、炎癥反應(yīng)增強(qiáng)、凋亡明顯。說明HSPA12B可能對內(nèi)毒素刺激的小鼠肺微血管內(nèi)皮細(xì)胞具有一定的保護(hù)作用。 第四部分HSPA12B對LPS誘導(dǎo)的mPMVECs中MAPK信號(hào)通路中p38的作用 目的 研究HSPA12B對LPS誘導(dǎo)的mPMVECs中MAPK信號(hào)通路中p38的作用，初步探討HSPA12B保護(hù)鼠肺微血管內(nèi)皮細(xì)胞的可能機(jī)制。 方法 實(shí)驗(yàn)先分兩組：轉(zhuǎn)染空白siRNA組和轉(zhuǎn)染HSPA12B siRNA組，觀察LPS刺激24小時(shí)后，內(nèi)皮細(xì)胞上磷酸化p38（p-p38）、總p38（total p38）和β-actin蛋白表達(dá)水平；用Western blot法檢測p38表達(dá)變化情況。再將實(shí)驗(yàn)分成4組①LPS組、②LPS+siRNA組、③LPS+siRNA+p38MAPK抑制劑(SB203580)組和④LPS+siRNA+等劑量SB203580溶劑DMSO組，觀察mPMVECs遷移功能、凋亡和炎癥因子表達(dá)情況。最后LPS刺激mPMVECs24h后，用免疫熒光雙標(biāo)和免疫共沉淀方法觀察HSPA12B和p38MAPK蛋白間的相互作用。 結(jié)果 Western blot分析法檢測各組p-p38、total p38和β-actin，發(fā)現(xiàn)HSPA12B干擾組p-p38顯著增強(qiáng)，p-p38和total p38灰度值之比明顯增大，且有統(tǒng)計(jì)學(xué)意義（P 0.05）。SB203580可明顯逆轉(zhuǎn)HSPA12B表達(dá)下調(diào)所致的mPMVECs遷移功能抑制、凋亡增加和炎癥因子基因表達(dá)改變。免疫熒光雙標(biāo)和免疫共沉淀實(shí)驗(yàn)證實(shí)HSPA12B與p38MAPK信號(hào)通路中p38蛋白相互作用。 結(jié)論 HSPA12B表達(dá)下調(diào)的mPMVECs中p38磷酸化顯著增強(qiáng)，而LPS刺激的HSPA12B表達(dá)下調(diào)的mPMVECs遷移功能減弱、炎癥反應(yīng)增強(qiáng)、凋亡明顯，且SB203580可逆轉(zhuǎn)這些變化，免疫熒光雙標(biāo)和免疫共沉淀實(shí)驗(yàn)進(jìn)一步說明HSPA12B可能通過降低p38磷酸化水平來抑制LPS對內(nèi)皮細(xì)胞的損傷，從而對LPS誘導(dǎo)的內(nèi)皮細(xì)胞發(fā)揮保護(hù)作用。
[Abstract]:Research background and general idea
Sepsis, lung is one of the important organ most vulnerable to injury, acute lung injury (ALI) had high incidence of sepsis has been one of the hot research. Endotoxin (lipopolysaccharide, LPS) in pulmonary microvascular endothelial cells (pulmonary micro-vascular endothelial cells, PMVECs) can cause damage. Pulmonary blood barrier dysfunction. Sepsis, acute lung injury induced by LPS, one of the key cells in the development of the pulmonary microvascular endothelial cells. Therefore, the protection of septic injury PMVECs has important clinical value.
In the study of sepsis associated acute lung injury, many scholars pay attention to the endogenous protection mechanism is one of the hot heat shock protein (Heat shock proteins, HSPs.HSPs) is a kind of cell protective protein increased under stress. The heat shock protein A12B (Heat shock proteinA12B, HSPA12B) is a heat shock protein discovered recently, is a distant relative of the family HSP70, exist in the atherosclerotic plaque, and angiogenesis related protection. The results show that HSPA12B in zebrafish during growth and formation of somatic cells from blood vessels play an important role. But in sepsis, endotoxin impact of HSPA12B on the expression of lung tissue the expression of HSPA12B in the PMVECs? What is the effect on PMVECs? How could the mechanism of action remains to be further studied.?
This study through the perforations in the body of the cecal ligation (CLP) model and LPS mice stimulated mouse pulmonary microvascular endothelial cells in vitro, detection of sepsis HSPA12B expression level in each time point; using small interfering RNAs (siRNAs) expression of mPMVECs in HSPA12B, to observe the changes of migration and apoptosis of mPMVECs LPS, and the detection of mPMVECs stimulate the production of inflammatory factors, the effect of HSPA12B on LPS PMVECs, and to explore the relationship between MAPK and p38 signaling pathway.
The effect of endotoxin on the expression of HSPA12B in rat lung tissue in the first part of sepsis
objective
To investigate the expression and regularity of HSPA12B in the lung tissue of the cecum ligation perforation (CLP) mouse model and in the mPMVECs stimulated by LPS.
Method
1., we constructed rat CLP model, and took the lung at 0h, 3h, 6h, 9h, 12h, 18h and 24h respectively. The HSPA12B mRNA level in lung tissue was detected by RT-PCR method, and the expression of lung tissue in sepsis was also discussed.
2. mPMVECs was cultured. The morphology of endothelial cells at different time points was observed under inverted microscope. MPMVECs was stimulated by LPS with a final concentration of 1 g/ml, and cells were collected at 0h, 3h, 6h, 9h, 12h, 18h, and 24h, respectively.
The level of HSPA12B mRNA was detected by RT-PCR, and the changes of HSPA12B expression in mPMVECs stimulated by LPS were investigated.
Result
The expression of HSPA12B in the 1. CLP model 3h, 6h, 9h, 12h increased gradually, and the 12h reached its peak, and then decreased gradually.
2. LPS stimulated mPMVECs, and the expression of HSPA12B also increased gradually, and then decreased.
conclusion
In sepsis, endotoxin had a certain effect on the expression of HSPA12B in rat lung tissue and mPMVECs, and the expression of HSPA12B first increased and then decreased.
The second part uses siRNA interference to reduce the expression of HSPA12B on the original mPMVECs
objective
SiRNA interference method is used to reduce the expression of HSPA12B on the original mPMVECs and verify it.
Method
SiRNA siRNA and blank control respectively into mPMVECs by liposome Lippo2000TM, the experiment was divided into two parts: 1. by fluorescent siRNA to observe cell transfection efficiency; the expression level of 2. RT-PCR and Western Blot method for the detection of HSPA12B interference to verify the effect of siRNA.
Result
1. the expression of green fluorescent protein in mPMVECs was observed by fluorescence microscopy, indicating that siRNA was transferred into the cell.
2. RT-PCR and Western Blot detected the downregulation of HSPA12B expression in mPMVECs.
conclusion
Successful construction of a downregulated mPMVECs. in HSPA12B expression
The effect of LPS on the migration of mPMVECs, inflammatory reaction and apoptosis after the down regulation of HSPA12B expression in the third part
objective
To investigate the effect of HSPA12B on the function of mPMVECs migration, inflammatory response and apoptosis induced by LPS.
Method
According to the following mPMVECs were divided into four groups: blank group, LPS stimulation group, LPS+HSPA12B siRNA group, the LPS+ group were placed in the blank siRNA, 37oC5%CO2 wet incubator culture, 24h co cultured with LPS cells and remove cells detected by RT-PCR, IL-6, IL-1, IL-10 and TNF- alpha beta, mRNA expression use, scratch test and Transwell assay cell migration, cell apoptosis was detected by flow cytometry, cell ultrastructure changes were observed by transmission electron microscopy.
Result
The results of RT-PCR showed that mPMVECs stimulated by LPS 24h, and the parallel control group (LPS+ control group siRNA) compared to the expression of HSPA12B was significantly increased by group IL-6 and TNF- alpha mRNA expression, whereas IL-10 expression decreased, with statistical significance (P 0.05). The experimental results show that the migration and parallel control group, migration the function of HSPA12B expression of mPMVECs group obviously decreased; apoptosis compared with the parallel control group, HSPA12B expression group after LPS stimulation significantly increased mPMVECs apoptosis; transmission electron microscopy results showed that: compared with the parallel control group, HSPA12B expression group cell ultrastructure damage increase.
conclusion
LPS stimulated HSPA12B expression downregulated mPMVECs migration function weakened, inflammatory reaction increased, apoptotic obviously, indicating that HSPA12B may have a protective effect on endotoxin stimulated mouse lung microvascular endothelial cells.
The effect of fourth part HSPA12B on p38 in MAPK signaling pathway induced by LPS in mPMVECs
objective
The effect of HSPA12B on p38 in the MAPK signaling pathway induced by LPS in mPMVECs was studied, and the possible mechanism of HSPA12B protection of rat lung microvascular endothelial cells was preliminarily discussed.
Method
The first experiment was divided into two groups: blank group and siRNA transfection into HSPA12B siRNA group, LPS was observed after 24 h of stimulation, endothelial cells on the phosphorylation of p38 (p-p38), total p38 (total p38) and -actin protein expression level; using Western blot method to detect the expression of p38. The experiments were divided into 4 groups: LPS group, LPS+siRNA group, LPS+siRNA+p38MAPK inhibitor (SB203580) group and the LPS+siRNA+ dose of SB203580 solvent group DMSO, observe the mPMVECs migration function, apoptosis and expression of inflammatory factors. Finally, LPS after mPMVECs24h stimulation, the interaction between HSPA12B and p38MAPK protein were observed by immunofluorescence co precipitation and immunity.
Result
Western blot analysis method to detect the p-p38, total p38 and beta -actin, found the HSPA12B interference group p-p38 increased, p-p38 and total p38 significantly increased the ratio of the gray value, and there was statistical significance (P 0.05).SB203580 can significantly reverse the expression of HSPA12B mPMVECs induced down-regulation can inhibit the migration function, apoptosis and inflammatory cytokines gene expression changes. Immunofluorescence and immunoprecipitation experiments confirmed that p38 protein HSPA12B and p38MAPK signaling pathway in the interaction.
conclusion
The expression of HSPA12B p38 phosphorylation and down-regulation of mPMVECs in significantly enhanced LPS stimulated HSPA12B expression of mPMVECs reduced the migration function decreased, increased inflammation, apoptosis, and SB203580 could reverse these changes, experiments indicate that HSPA12B could decrease the phosphorylation level of p38 to the inhibition of LPS on endothelial cell injury by immunofluorescence and immunoprecipitation precipitation exerts a protective effect on LPS induced endothelial cells.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R459.7

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