摘要
研究背景与总体思路
近年来,防治缺血再灌注损伤( Ischemia reperfusion injury, IRI)一直是组织和器官保护研究的主要方向之一。血管内皮细胞(Vascμlar endothelial cells,VEC)缺血再灌注损伤可引起内皮屏障功能障碍、血栓形成和组织水肿。研究表明,在心脏IRI过程中,内皮细胞的结构和功能异常发生时间早于心肌细胞,是心脏缺血再灌注损伤早期发生、发展的病理生理基础,其功能恢复也晚于心肌细胞。因此,保护再灌注损伤内皮细胞具有重要意义。
在内皮细胞保护研究中,不少学者重视激活细胞内源性保护机制,关注焦点之一即是热休克蛋白(Heat shock proteins,HSPs)。HSPs是细胞在生理状态下必需,应激(热休克、缺血缺氧等)状态下表达增多的一类保护性蛋白质。热休克蛋白22(Heat shock protein, HSP22)是新近发现一种小分子热休克蛋白,广泛分布于哺乳动物的各种组织中。研究显示HSP22在缺血再灌注损伤心肌细胞中具有抗凋亡,抗氧化应激和分子伴侣等生物学功能,对再灌注损伤心肌的保护作用等同于缺血预适应。但HSP22是否对缺血再灌注损伤内皮细胞具有同样的保护作用?其可能信号通路情况如何?尚有待于进一步研究。
本研究,通过以建立内皮细胞缺氧/复氧模型模拟缺血/再灌注损伤,首先观缺氧/复氧损伤内皮细胞中HSP22基因表达水平的变化;其次,构建人HSP22过表达重组质粒,转染内皮细胞,通过G418筛选,获得稳定表达HSP22蛋白的内皮细胞株。在此基础上,观察HSP22对缺氧/复氧损伤内皮细胞的保护作用,并探讨其与NF-кB信号通路的关系。
第1部分缺氧复氧干预对内皮细胞中HSP22基因的影响
目的:
探讨热休克蛋白22(HSP22)在缺氧/复氧损伤内皮细胞中的表达及其规律。
方法:
培养人脐静脉内皮细胞株,分为正常培养组和缺氧/复氧组,缺氧环境在密闭的缺氧盒中经一次性缺氧催化袋(它能吸收O2,产生CO2)产生,复氧置于5%CO2、37℃培养箱内。实验分为两个部分:1.观察正常及缺氧24小时后复氧0、3、6、12h,倒置显微镜下观察各组内皮细胞形态,流式细胞仪及DNA电泳检测内皮细胞凋亡。2.应用RT-PCR及Western blot法检测HSP22的表达,明确缺氧/复氧损伤对HSP22基因的表达是否具有影响。
结果:
1.流式细胞仪及DNA电泳检测凋亡,缺氧24 h(复氧0h)仅少量内皮细胞发生凋亡,但复氧后3、6、12h内皮细胞凋亡逐渐增加, 12h凋亡率达55.03%; 2.RT-PCR及Western blot法,HSP22在正常内皮细胞中无表达,缺氧24 h复氧0h(缺氧24 h)出现表达,复氧3 h与复氧0h比表达进一步表升高(P﹤0.05),复氧6 h达峰值(P﹤0.01),但12 h表达回落。
结论:
在正常内皮细胞中无HSP22蛋白表达,但缺氧/复氧应激可以诱导内皮细胞中HSP22的表达,HSP22应激表达可能对缺氧/复氧损伤的内皮细胞具有保护作用。
第2部分:稳定表达人热休克蛋白22基因内皮细胞株的构建
目的:
构建pEGFP-N1/HSP22真核表达质粒并观察在内皮细胞中的表达。
方法:
RT-PCR从人MCF-7细胞中扩增HSP22基因,将HSP22基因连接到真核表达载体pEGFP-N1中,并转化DH5α,获得阳性克隆进行双酶切和测序鉴定。用脂质体法将重组质粒转染人脐静脉血管内皮细胞。G418筛选,获得G418抗性单克隆细胞,用荧光显微镜、逆转录-聚合酶链反应法(RT-PCR)及Western blot检测HSP22在人脐静脉内皮细胞中的表达。
结果:
琼脂糖凝胶电泳检测RT-PCR扩增产物, HSP22基因片段长约613 bp,与预期分子量相符,测序结果证实克隆完全正确;其阳性克隆进行酶切鉴定与热休克蛋白22理论值相符。稳定转染单克隆细胞中,荧光显微镜观察到每个细胞均有绿色荧光蛋白表达,RT-PCR和Western blot检测到HSP22mRNA和蛋白表达。
结论:
成功获得稳定表达人热休克蛋白22基因的内皮细胞株。
第3部分HSP22对缺氧/复氧致内皮细胞凋亡的保护作用的研究
目的:
探讨HSP22对缺氧/复氧致内皮细胞凋亡的保护作用
方法:
实验分为稳定表达pEGFP-N1空质粒对照组和稳定表达HSP22的pEGFP-N1/HSP22质粒组,缺氧培养24h后,分别复氧0、3、6、12h。应用流式细胞仪碘化丙啶(PI)染色法、原位缺口末端标记(TUNEL)法检测各组内皮细胞的凋亡,Western blot检测各组中Caspase-3、Bcl- 2和Bax蛋白的表达。
结果:
流式细胞仪和TUNEL法检测显示:内皮细胞缺氧24h后不同时间复氧, HSP22过表达组较pEGFP-N1对照组内皮细胞凋亡率显著下降。Western blot结果显示(1)随复氧时间延长,两组中Caspase-3水平升高,高峰见于复氧12h,但pEGFP-N1/HSP22复氧各时间点Caspase-3水平均低于空质粒对照组;(2)缺氧复氧后pEGFP-N1/HSP22组Bcl-2水平显著高于pEGFP-N1组(p<0.05),但两组中Bax蛋白表达无变化。
结论:
过表达HSP22可对缺氧复氧损伤内皮细胞产生显著保护作用,机制与HSP22上调内皮细胞中Bcl- 2水平有关。
第4部分HSP22对缺氧/复氧损伤内皮细胞中NF-кB转导通路的影响
目的:
探讨HSP22对缺氧/复氧损伤内皮细胞中NF-кB转导通路的影响及其机制。
方法:
实验分三组:未转染组,转染pEGFP-N1空质粒细胞组和pEGFP-N1/HSP22重组质粒细胞组,观察缺氧24小时复氧12小时内皮细胞,电泳迁移率(EMSA)检测NF-кB的表达;应用核浆分离技术和Western blot分析法检测NF-кBp65亚基在各组细胞核内的蛋白表达水平;应用Western blot分析法检测各组细胞NF-кB信号途径中,кB抑制因子-α(IкB-α)的降解以及IкB激酶IKKa的表达;免疫共沉淀检测HSP22在NF-кB激活通路里IкB-α和IKKa蛋白的关系。以HSP22抗体免疫共沉淀和Western blot法检测各组细胞中HSP22与IкB-α和IKKa蛋白的结合水平。
结果:
EMSA分析法显示,pEGFP-N1/HSP22组核内NF-кB活性水平显著低于对照组和pEGFP-N1组(P﹤0.05),应用核浆分离技术和Western blot分析各组NF-кB p65水平与EMSA结果一致。Western blot分析法检测各组细胞IкB-α、IKK-α,可见pEGFP-N1/HSP22组IкB-α表达水平显著升高,而IKK-α表达水平低于Control组和pEGFP-N1组(P<0.05)。应用免疫共沉淀—Western blot法检测各组细胞HSP22与IкB-α和IKKa蛋白结合水平,可见HSP22与IKK-α抗体共免疫沉淀,在pEGFP-N1/HSP22组出现特异性条带,提示HSP22抑制NF-кB激活可能是通过它与IKK-α相互作用。
结论:
在缺氧/复氧损伤内皮细胞中,HSP22可能通过影响IKK-α水平,升高细胞内IкB-α水平,抑制NF-кB的表达而对缺氧/复氧损伤内皮细胞发挥保护作用。
Background and Research Strategy
Recently, how to protect organs and tissues against ischemia/reperfusion injury (IRI) is one of main directions of research. Vascμlar endothelial cells injury induced by ischemic/reperfusion can lead to endothelial barrier dysfunction, thrombosis and edema. Further study indicated that apoptosis was first seen in the endothelial cells in the stages of reperfusion. Several studies demonstrate that the dysfunction and incompetence of VEC precedes myocyte cells apoptosis and restoration of its function is later than myocyte cells in ischemic/reperfusion. Therefore, it is of great significance to protect endothelial cells against ischemic/reperfusion injury.
More and more investigators emphasize on activation of endogenous protective mechanisms in endothelial cells, in which heat shock proteins (HSPs) are mostly focused. HSPs are a kind of self-preservation protein which are induced by various environmental stress, such as heat shock, ischemia and hypoxia. HSP22 is a relatively new member of the mammalian sHSPs family, which is expressed in many tissues. Recent studies have shown that HSP22 coμld protect ischemia/reperfusion injuried myocardial cells against apoptosis and oxidative stress. Furthermore, myocardial cells endogenous cytoprotection triggered by HSP22 is equivalent to ischemia preconditioning. Coμld this effect be extended to endothelial cells ischemia/reperfusion injury? How is the signaling pathway in endothelial cells? To determine the cytoprotective function of HSP22 in endothelial cells, we need further study.
In this study, using the model of cells hypoxia/reoxygenation to mimic ischemia/reperfusion injury, we intended to investigate the role of HSP22 in endothelial cells injury induced by hypoxia/reoxygenation. Firstly, we confirmed the expression of HSP22 in injuryed HUVECs induced by hypoxia/reoxygenation. Secondly, we cloned HSP22 gene and established endothelial cells Line, which can express HSP22 stably. And we determined the protective effects of HSP22 on hypoxia/reoxygenation injury and explored the effect of HSP22 on NF-кB signal pathway in vascμlar endothelial cells.
PartⅠEffects of HSP22 on Hypoxia /Reoxygenation Induced Endothelial Cell Injury
Objective:
To study the expression of HSP22 in injuryed HUVECs induced by hypoxia/ reoxygenation.
Methods:
Human umbilical vein endothelial cells (HUVECs) were divided into normal group and hypoxia/reoxygenation groups(0、3、6、12 hours). Hypoxia /reoxygenation model was achieved by using an anaerobic jar equipped with an AnaeroPack agent(disposable O2-absorbing and CO2-generating) and a 95% air 5%C02 incubator at 37℃. Whole experiment contained two parts: 1.Cells injury induced by hypoxia/reoxygenation was observed at different time periods (0h,3h,6h,12h),the cells morphology in each group were observed under inverted microscope,and cells apoptosis were measured by Flow Cytometer and DNA electrophoresis. 2. HSP22 expression was detected by RT-PCR and Western blot.
Resμlts:
1. In 24h-hypoxia group, the percentage of endothelial cell apoptosis was rather low (~2.06%) , while the apoptotic rate increased significantly at different time (3、 6、12 hours) in reoxygenation groups, especially in 12h-reoxygenation group ( 55.03% ).
2. RT-PCR and Western blot showed there was no HSP22 expression in the normal cμlture group. At 24h-hypoxia, the HSP22 expression occurred, increased at 3h-reoxygenation(P﹤0.05), peaked at 6h, and went down at 12h-reoxygenation(P﹤0.01).
Conclusions:
We approved that there was no HSP22 expression in the normal cμlture HUVECs, but hypoxia/reoxygenation stress coμld stimμlate HSP22 expression for the first time. The increase of HSP22 might protect endothelial cells against hypoxia /reoxygenation injury.
PartⅡEstablishment and Identification of Endothelial cell Line with Stable Expression of HSP22
Objective:
To clone HSP22 gene and establish human endothelial cells with stable expression of HSP22 gene.
Methods:
Fμll length of HSP22 cDNA was obtained from MCF-7 by RT-PCR and inserted into plasmid pEGFP-N1,and then transformed into DH5α. Positive clones were identified by double enzymes digestion and sequencing. The recombinant plasmid were transfected into HUVECs by Lipofectamine 2000, then HUVECs with stable HSP22 gene transfection were established by selection with G418. HSP22 expression was identified by fluorescence microscope, RT-PCR and Western blot.
Resμlts:
Agarose gel electrophoresis showed that HSP22 DNA segment was 613 bp, which was consistent with the expectation. Positive clones were identified by enzyme digestion and sequencing. In the stable cell line, fluorescence microscope confirmed green fluorescent protein expression, RT-PCR and Western blot proved HSP22 mRNA and protein expression.
Conclusions:
The HUVECs with stable HSP22 expression gene have been established successfμlly.
Part III HSP22 Protected Endothelial Cells Induced by Hypoxia-Reoxegenation Injury against Apoptosis
Objective:
To elucidate HSP22 protective effect on endothelial cells induced by hypoxia/ reoxegenation injury against apoptosis.
Methods:
This study is based on human umbilical vein endothelial cells with stable expression of pEGFP-N1 and pEGFP-N1/HSP22.The cells were subjected to reoxygenation 0h, 3h, 6h, and 12 h following 24h hypoxia. The apoptosis of cells were detected by the methods of Flow Cytometry and DeadEndTM Colorimetric TUNEL System assay. At the same time, the expression of caspase-3, Bcl- 2 and Bax protein were assayed by Western bot.
Resμlts:
There was lower percentage of apoptosis in pEGFP-N1/HSP22 group compared to pEGFP-N1 groups at reoxygenation 0h, 3h, 6h, and 12 h following 24h hypoxia. Apoptosis index calcμlation by DeadEnd TM Colorimetric TUNEL System assay also supported the resμlts. We found caspase-3 levels increased gradually after reoxygenation in both groups, while the expression of caspase-3 in pEGFP-N1/HSP22 group is lower than pEGFP-N1 group significantly (P<0.01). Our resμlts also indicated that expression of Bcl-2 in both groups gradually decreased after reoxygenation. However, Bcl-2 level in pEGFP-N1/HSP22 group was significantly higher than in pEGFP-N1/HSP22 group (p<0.05). Bax level did not change in both groups (p>0.05).
Conclusion:
In this study, we were first to approve HSP22 has a protective effect on endothelial cells injury and apoptosis induced by hypoxia/reoxygenation. The mechanisms might be correlated with improvement of Bcl-2 expression and inhibition of caspase-3 activation.
Part IV Effect of HSP22 on NF-кB Signal Pathway in Endothelial Cells Induced by Hypoxia/Reoxegenation Injury
Objective:
To explore the effect of HSP22 on NF-кB signal pathway in endothelial cells injury induced by hypoxia/reoxygenation and the molecμlar mechanisms.
Methods:
All the cells divided into normal group, pEGFP-N1 group and pEGFP- N1/HSP22 group. After subjected to 12h reoxygenation following 24h hypoxia, nuclear NF-кB expression was deteced by Electrophoretic mobilety shift assay , the level of NF-кB/P65 and the key signaling molecμles in NF-кB pathway such as IкB-α(the inhibitor factor of the NF-кB),and IKKa (IкB-αkinase) were assayed by Western blot. The association of IкB-αand IKKa with HSP22 were determined by Co-immunoprecipitation followed by Western blot.
Resμlts:
After the cells were subjected to 12h reoxygenation following 24h hypoxia, EMSA assay showed the activation of NF-кB was significantly lower in pEGFP-N1/HSP22 group than control and pEGFP-N1 group. NF-кB/p65 expression by Western blot was consistent with EMSA resμlts. The IкB-αlevel was higher in pEGFP-N1/HSP22 than other groups (P﹤0.05) , and there was no significant difference between the levels in pEGFP-N1 and control group. But IKK-αlevel in pEGFP-N1/HSP22 group decreased significantly comparing to pEGFP-N1 group(P﹤0.05). To further confirm this idea, co-immunoprecipitation indicated that HSP22 inhibited NF-кB pathway activation,it might be correlate that HSP22 influence the IKK-αdegradation instead of IкB-α.
Conclusions:
HSP22 coμld protect endothelial cells induced by hypoxia/reoxygenation injury against NF-кB activation. The mechanisms might be correclation with influencing IKKαdegradation, then enhancing IкB-αlevel in endothelial cells.
引文
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