HSPA12B对内毒素刺激的小鼠肺微血管内皮细胞的作用及其机制

发布时间：2018-02-04 20:48

  本文关键词： 热休克蛋白A12B 小鼠肺微血管内皮细胞 CLP模型 内毒素 急性肺损伤 HSPA12B mPMVEC 敲除 绿色荧光蛋白 热休克蛋白A12B 肺微血管内皮细胞 内毒素 HSPA12B LPS损伤 磷酸化p38 p38MAPK信号通路　出处：《第二军医大学》2013年硕士论文　论文类型：学位论文

【摘要】：研究背景及总体思路 脓毒症时，肺脏是最易受损伤的重要脏器之一，急性肺损伤（ALI）出现早，发生率高，一直是脓毒症研究的热点之一。内毒素（lipopolysaccharide，LPS）导致的肺微血管内皮细胞（pulmonary micro-vascular endothelial cells，PMVECs)损伤，可引起肺血屏障功能障碍。脓毒症时，内毒素导致的急性肺损伤，其发生发展中关键细胞之一是肺微血管内皮细胞。因此，保护脓毒症损伤的PMVECs具有重要的临床价值。 在脓毒症相关性急性肺损伤研究中，不少学者重视细胞内源性保护机制，关注热点之一就是热休克蛋白（Heat shock proteins，HSPs）。HSPs是细胞在应激状态下产生增多的一类保护性蛋白质。而其中热休克蛋白A12B（Heat shock proteinA12B,HSPA12B)是新近发现的一种热休克蛋白，是HSP70家族中的远亲，大量存在于动脉粥样硬化斑块中，与血管生成保护相关。研究表明，HSPA12B在斑马鱼生长过程中和离体细胞血管形成中占有重要的作用。但脓毒症时内毒素对肺组织的PMVECs中HSPA12B表达变化产生何种影响？HSPA12B表达变化对PMVECs具有何种作用？其产生作用的可能机制怎样？尚有待进一步研究。 本研究通过在体的盲肠结扎穿孔（CLP）小鼠模型和LPS刺激小鼠肺微血管内皮细胞的离体实验，检测脓毒症时HSPA12B在各时间点的表达水平；用小干扰RNAs（siRNAs）下调mPMVECs中HSPA12B的表达后，观察mPMVECs迁移和凋亡的变化，以及检测LPS刺激mPMVECs产生的炎症因子，研究HSPA12B对LPS处理的PMVECs的作用，并探讨其与MAPK信号通路中p38的关系。 第一部分脓毒症时内毒素对鼠肺组织中HSPA12B表达的影响 目的 探讨HSPA12B在盲肠结扎穿孔(CLP)小鼠模型的肺组织中和LPS刺激的mPMVECs中的表达及其规律。 方法 1.构建鼠CLP模型，分别在0h、3h、6h、9h、12h、18h、24h断头取肺。应用RT-PCR法检测肺组织中HSPA12B mRNA的水平，探讨脓毒症时肺组织中HSPA12B表达变化。 2.培养mPMVECs，倒置显微镜下观察各时间点内皮细胞形态，用终浓度为1μg/ml的LPS刺激mPMVECs，分别于0h、3h、6h、9h、12h、18h、24h收集细胞， 应用RT-PCR法检测HSPA12B mRNA的水平，探讨LPS刺激的mPMVECs中HSPA12B表达变化。 结果 1. CLP模型3h、6h、9h、12h肺组织中HSPA12B表达逐步升高，，12h达峰值，后又逐渐下降。 2. LPS刺激mPMVECs，HSPA12B表达也呈现逐步升高，后又下降。 结论 脓毒症时内毒素对鼠肺组织和mPMVECs中HSPA12B表达具有一定影响，HSPA12B表达先升高后下降。 第二部分用siRNA干扰法下调原代mPMVECs上HSPA12B的表达 目的 用siRNA干扰法下调原代mPMVECs上HSPA12B的表达并验证之。 方法 分别将siRNA和空白对照的siRNA通过脂质体Lippo2000TM转入mPMVECs内，实验分为两部分：1.通过转入带荧光的siRNA观察细胞的转染效率；2.用RT-PCR及Western Blot法检测HSPA12B的表达水平，验证siRNA的干扰效果。 结果 1.通过荧光显微镜观察到mPMVECs均有绿色荧光蛋白表达，说明siRNA转入到细胞内。 2. RT-PCR和Western Blot检测到mPMVECs中HSPA12B表达下调。 结论 成功构建HSPA12B表达下调的mPMVECs。 第三部分HSPA12B表达下调后LPS对mPMVECs的迁移功能、炎症反应以及凋亡的影响 目的 探讨HSPA12B对LPS刺激的mPMVECs迁移功能、炎症反应和凋亡情况的影响。 方法 根据以下情况将mPMVECs分为四组：①空白组、②LPS刺激组、③LPS+HSPA12B siRNA组、④LPS+空白siRNA组，分别置于37oC5%CO2潮湿孵箱内培养，用LPS与细胞共培养24h，取出细胞后用RT-PCR检测IL-6、IL-1β、IL-10和TNF-α mRNA表达情况,使用划痕实验和transwell小室检测细胞迁移情况，用流式细胞仪检测细胞凋亡情况，用透射电子显微镜观察细胞超微结构变化。 结果 RT-PCR检测结果表明：mPMVECs经LPS刺激24h后，与平行对照组（LPS+空白siRNA组）相比，HSPA12B表达下调组IL-6和TNF-α mRNA表达水平显著上升，而IL-10则表达下降，且均有统计学意义（P 0.05）。迁移实验表明：与平行对照组对比，HSPA12B表达下调组的mPMVECs的迁移功能明显减弱；凋亡实验表明：与平行对照组相比，HSPA12B表达下调组LPS刺激后mPMVECs凋亡明显增多；透射电子显微镜结果显示：与平行对照组相比，HSPA12B表达下调组细胞超微结构破坏加重。 结论 LPS刺激的HSPA12B表达下调的mPMVECs迁移功能减弱、炎症反应增强、凋亡明显。说明HSPA12B可能对内毒素刺激的小鼠肺微血管内皮细胞具有一定的保护作用。 第四部分HSPA12B对LPS诱导的mPMVECs中MAPK信号通路中p38的作用 目的 研究HSPA12B对LPS诱导的mPMVECs中MAPK信号通路中p38的作用，初步探讨HSPA12B保护鼠肺微血管内皮细胞的可能机制。 方法 实验先分两组：转染空白siRNA组和转染HSPA12B siRNA组，观察LPS刺激24小时后，内皮细胞上磷酸化p38（p-p38）、总p38（total p38）和β-actin蛋白表达水平；用Western blot法检测p38表达变化情况。再将实验分成4组①LPS组、②LPS+siRNA组、③LPS+siRNA+p38MAPK抑制剂(SB203580)组和④LPS+siRNA+等剂量SB203580溶剂DMSO组，观察mPMVECs迁移功能、凋亡和炎症因子表达情况。最后LPS刺激mPMVECs24h后，用免疫荧光双标和免疫共沉淀方法观察HSPA12B和p38MAPK蛋白间的相互作用。 结果 Western blot分析法检测各组p-p38、total p38和β-actin，发现HSPA12B干扰组p-p38显著增强，p-p38和total p38灰度值之比明显增大，且有统计学意义（P 0.05）。SB203580可明显逆转HSPA12B表达下调所致的mPMVECs迁移功能抑制、凋亡增加和炎症因子基因表达改变。免疫荧光双标和免疫共沉淀实验证实HSPA12B与p38MAPK信号通路中p38蛋白相互作用。 结论 HSPA12B表达下调的mPMVECs中p38磷酸化显著增强，而LPS刺激的HSPA12B表达下调的mPMVECs迁移功能减弱、炎症反应增强、凋亡明显，且SB203580可逆转这些变化，免疫荧光双标和免疫共沉淀实验进一步说明HSPA12B可能通过降低p38磷酸化水平来抑制LPS对内皮细胞的损伤，从而对LPS诱导的内皮细胞发挥保护作用。
[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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7

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