热应激心肌细胞内Hsp110表达及其抗热应激性损伤机理研究
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摘要
过高或过低的养殖环境温度、生畜禽因贸易需要而长途运输等因素常导致畜禽发生应激,导致应激性损伤、肉品品质降低、甚至死亡等,给畜禽养殖业造成了严重的经济损失。因此,对应激及其应激损伤展开研究并提出合理的降低应激损伤对养殖业造成危害的措施尤为必要。热休克蛋白(Hsp)是广泛存在于从酵母菌至哺乳动物细胞内的一类具有保护作用的内源性蛋白质,在生理条件或应激条件下,能在有机体组织细胞内大量、稳定表达,是应激条件下组织细胞生存必不可缺的分子伴侣。Hspl10是热休克蛋白家族中的重要一员,能保护应激损伤的细胞或目的蛋白,而且高诱导表达的Hsp110能精确识别变性蛋白并使之保持在一个稳定、可溶的状态,使变性蛋白恢复至具有活性的状态。因此,对热应激过程中Hsp110的研究也是热休克蛋白家族研究中的一个重要方向,同时心肌细胞损伤也是导致应激动物猝死的可能原因之一。本文主要通过对体外培养大鼠原代心肌细胞培养条件进行优化,建立体外心肌细胞热应激模型,观察热应激过程中心肌细胞内Hspl10及其相应hsp110mRNA的表达与转录、Hspl1O变化规律与热应激心肌细胞损伤之间的相关性,探讨Hspl10在热应激过程中抗应激性损伤的保护作用与机制。
运用细胞培养技术、细胞爬片技术、免疫荧光技术、组织学技术及MTT试验等手段对原代培养的大鼠心肌细胞进行条件优化。结果表明,取2日龄SD乳大鼠心脏,依次经过胶原酶Ⅰ消化14h,吹打7～9次,细胞差速贴壁60min,加入0.1～0.3MBRDU,逐点铺细胞等过程,使免疫荧光试验细胞铺板总数为1.8-2.0×106个/mL, Wlestem blot试验细胞铺板总数为2.2-2.3×10个/mL,MTT试验细胞铺板总数为0.25～1×106个/mL时,体外培养的心肌细胞纯度可达到94%,满足试验要求。而逐点铺细胞等是获得高纯度体外培养心肌细胞的必要条件。
将大鼠原代心肌细胞在37℃二氧化碳细胞培养箱中进行72h的适应性培养后,迅速将心肌细胞置于预热的42℃二氧化碳培养箱中,分别进行0min、10min、20min、40min、60min、120min、240min、360min及480min的热应激处理,应用组织学、心肌特异性酶检测技术、免疫荧光检测技术、荧光定量PCR技术和Western blot检测技术对热应激处理心肌细胞出现的应激性损伤及保护性蛋白Hsp110变化规律进行相关性分析研究。热应激心肌细胞培养液中AST、LDH、CK的酶活性显著升高,表明心肌细胞结构的完整性发生了改变；组织学检测结果显示,热应激40-60min期间,心肌细胞体积急性肿胀,胞浆内出现大量红色变性颗粒,细胞核深染、浓缩；Westernblot检测结果显示,热应激处理20min后,Hspl10的表达量呈显著上升趋势,并且在热应激处理240min后,Hsp110的表达量比对照组提高约1.2倍；免疫组织化学结果显示,颗粒状Hsp110分布于心肌细胞的胞浆与细胞核内,42℃热应激240min后,核内Hsp110的分布密度显著增加；hsp110mRNA在热应激处理20min后也明显增加,并于240min时达到高峰,增加量约为对照组的10倍。研究结果表明,Hsp110在体外培养原代心肌细胞内的表达对热应激敏感,在基因和蛋白表达水平均呈现出表达量的明显增加,可能参与心肌细胞获得热耐受过程,在心肌细胞对抗或减轻热应激损伤中发挥重要作用。
将大鼠原代心肌细胞在37℃二氧化碳培养箱中进行72h的适应性培养,迅速将心肌细胞置于预热的42℃二氧化碳细胞培养箱中,分别进行0min、10min、20min、40min、60min、120min、240min、360min及480min的热应激处理。免疫荧光检测结果显示,阳性染色Hsp110颗粒主要位于胞浆内,胞核内也有少量分布,在热应激4h后,阳性染色Hspll0颗粒在细胞浆与细胞核内均大量增加。不同于Hspll0的是,应激组与对照组心肌细胞内的HSF-1仅分布在细胞核内；在42℃热应激120min后,应激心肌细胞内hsp110mRNA的转录量增加,同时可见HspllO表达量也显著增加,而且,同一时期内HSF-1的表达量也增加。结果表明,HspllO与HSF-1表达与分布具有时间依赖的方式,且对热敏感。热应激期间Hsp110表达增加以及HSF-1的表达先下降后升高的特点,表明二种蛋白在心肌细胞热损伤的保护中发挥着不同的作用。
用N-乙酰-L-半胱氨酸(NAC)对离体培养的大鼠原代心肌细胞进行预处理,然后进行热应激,对热应激心肌细胞内Hspll0表达与线粒体凋亡信号通路以及核转录因子(NF-κB)主要调节蛋白表达进行检测。结果表明,在热应激处理1h内,NAC显著抑制了热应激诱导的心肌细胞凋亡,而且随着热应激时间的延长,这种抑制作用呈现减弱的趋势；NAC对热应激心肌细胞核转录因子信号通路的调节蛋白NF-κB具有显著的抑制效果。
将体外37℃条件下适应性培养48h的心肌细胞利用Hsp siRNA与脂质体Lipofectamine2000进行24h转染,采用弃掉转染试剂复合物溶液和保留转染试剂复合物溶液二种方法进行Hspll0基因沉默,然后在37℃条件下继续培养至84h,分别在24h、36h、48h、60h、72h、84h收获细胞,对Hsp110siRNA引物沉默大鼠原代心肌细胞内Hsp110基因表达与转染方式、沉默时间及培养基中血清之间相互关系进行研究。结果表明,弃掉转染试剂复合物溶液后,阴性对照组及试验组心肌细胞内Hsp110的相对表达量在36-48h达峰值,然后呈现逐渐下降的趋势,36～72h内的沉默效率为40%左右,84h时的沉默效率为82%；保留转染试剂复合物溶液后,阴性对照组心肌细胞内Hsp110的相对表达量在72h达到峰值,然后逐渐下降,试验组心肌细胞内Hsp110的相对表达量在36h时达到峰值,然后呈逐渐下降趋势,在84h时最低,72～84h的沉默效率在86%～95%。这一结果说明,转染试剂以及siRNA对转染效率及沉默效率是有显著影响的,而且,我们合成的Hsp110siRNA引物在40-120nM时,转染试剂siRNA复合体对细胞持续转染72～84h,获得的对心肌细胞Hsp110表达的沉默效率最佳。
用Hsp110siRNA引物转染并沉默体外37℃培养48h的心肌细胞,37℃继续培养60h后,弃掉细胞培养液,改用含15%FBS的高糖DMEM培养基继续培养12h；然后于42℃二氧化碳细胞培养箱内进行4h的热应激处理,采用Western blot、ELISA、流式细胞术等检测技术,对体外培养心肌细胞内Hsp110相对表达量的变化、心肌细胞相关酶AST、CK、CK-MB、LDH的改变以及心肌细胞凋亡变化进行了比较研究。结果表明,Hsp110siRNA引物转染并沉默体外培养的心肌细胞后,经过4h的热应激处理后,试验组心肌细胞内Hsp110的相对表达量显著下降；心肌特异性酶AST、CK、 CK-MB在细胞培养液中的含量升高,但差异不显著；心肌细胞早、晚期凋亡减少,但差异不显著。这一结果说明,Hsp110在持续性热应激过程中的所呈现的变化与以往的报道有差异,其作用及其相应分子机制仍然需要进一步的研究验证。
Stress factors including long-distance transportation for livestock trade and higher temperature in living environment for poultry often cause the stressing damage, PSE (pale, soft, exudative) meat, and even to death in the effected animals. It is necessary to study the relation between stress and its damage to stressed animals and provide reasonable methods to reduce the stress influences to the farming industry. Heat shock proteins (Hsps), widespread in the organism from yeast to mammalian, are some endogenous proteins which have protective role. Furthermore, they can overexpress stably in the organism under physiological conditions or stress conditions, and act as the role of molecular chaperone. Hsp110is one of the most important of heat shock proteins family (HSPs) and can protect cells or targeted protein against stressed damages. The overexpression of Hsp110can indentify the denatured proteins and make them keep in a stable and soluble state, and then restore the activities of the denatured protein. In view of consideration that sudden death of stressed animal might be the damage of myocardial cells and heart failure, the protective role and mechanism of Hsp110against stressing damages during heat stress were studied through establishing the mode of myocardial cells stressed by heat in vitro, observing the expression of Hsp110and transcription of hsp110mRNA as well as the correlations between the variations of Hsp110expression and stressing damages of myocardial cells in vitro.
The optimizations of the culture conditions of in vitro myocardial cells were carried out by using cell culture techniques, cell growing on coverslips technology, immunofluorescence technique, histological techniques and MTT assay etc. The results showed that the purity of the myocardial cells incubated in vitro were up to94%and met the test requirements under the following conditions, the cells quantities of the immunofluorescence test were plated at a total of1.8-2.0×106cells/mL, the cells quantities of western blot test were plated at a total of2.2-2.3×106cells/mL, the cells quantities of the MTT test were plated at a total of0.25-1x106cells/mL. The results also showed that it was necessary to obtain optimal primary myocardial cells by selecting2-day-old SD neonatal rats, digesting heart tissue with collagenase I for14h, blowing gently heart tissue mass7-9times with the pipette, making the cells adherent and differential for60min, adding0.1-0.3M BRDU into the cells after cell adherence, seeding cells into the dishes with point by point.
After incubated at37℃for72h, myocardial cells were heat stressed in a incubator at42℃with a humidified atmosphere of5%CO2and95%air for0min,10min,20min,40min,60min,120min,240min,360min, and480min, respectively. The correlations between the cell damage and the variation of Hsp110expression were carried out by using the histological examination, special myocardial enzyme detections, the immunofluorescence technology, real time PCR technology as well as western blot technology. Significant increases of AST, LDH, and CK enzymatic activities in the suspension of myocardial cell were observed during the heat stress, suggesting that the integrity of the myocardial cells was altered. The results of HE staining showed that acute cell swelling characterized by a number of red granules in the cytoplasm deeply stained and concentrated nuclei were observed during heat stress from40min to120min. The results of the western blot test displayed that the overall levels of Hsp110expression increased significantly after20min and the Hsp110levels were approximately1.2-fold higher than that of the control after240min of heat stress. Immunocytochemical analysis revealed that the expressed Hsp110was constitutively localized in the cytoplasm with small amounts in the nuclei characterized by a granular pattern, nuclear Hsp110levels increased significantly after240min of heat stress in comparison with the control. The results of real time PCR proved that increasing levels of hsp110mRNA were observed after20min of heat stress, and the levels peaked with a10-fold increase after240min of heat stress. These results above indicate that the expression of Hsp110in primary myocardial cells in vitro is sensitive to hyperthermic stress by increasing both hsp110mRNA transcription and Hspl10expression levels, Hsp110is involved in the potential acquisition of thermotolerance after heat stress. Therefore, Hsp110might play a fundamental role in opposing and alleviating the stress damages caused by hyperthermic stress in primary myocardial cells.
After incubated at37℃for72h, myocardial cells were heat stressed in an incubator at42℃with a humidified atmosphere of5%CO2and95%air for0min,10min,20min, 40min,60min,120min,240min,360min, and480min, respectively. The results of the immuofluorescence test showed that Hsp110positive staining distributed with enrichment in the cytoplasm and small amount in the nuclei, however, Hsp110increased both in the cytoplasm and the nuclei of myocardial cells after4h of heat stress. In contrast to Hsp110, HSF-1staining was only observed in the nuclei of primary rat myocardial cells. Both of Hsp110and HSF-1increased in the heat-stressed myocardial cells, meanwhile the increasing of hsp110mRNA and HSF-1mRNA transcript were observed after120min of heat stress at42℃. The results showed that Hsp110and HSF-1were sensitive to heat stress in a time-dependent manner. The characteristics of Hsp110inductions during heat stress, nuclear HSF-1expression initial decrease and then significant increase between240and480min, suggesting that both Hsp110and HSF-1have different roles in protecting myocardial cells from heat stress damage.
Rat primary myocardial cells in vitro were pretreated for1h with N-acetyl-L-cysteine (NAC) and then heat-stressed. The proteins associated with mitochondrial apoptosis signaling pathway, nuclear transcription factor (NF-κB) signaling pathway as well as the expression levels of Hsp110in the heat stressed myocardial cells were studied. The results showed that NAC inhibited significantly myocardial cells apoptosis induced by heat stress within1h, and the role became weaker with the elongation of heat stress. However NAC inhibited significantly the expression levels of NF-κB belonging to NF-κB signaling pathway.
After incubated at37℃with lipofectamine2000for48h and Hsp110siRNA primer for24h, then continued to incubate the cells until84h with or without transfection mixture solution including Hsp110siRNA primer and lipofectamine2000at37℃, and then the transfected myocardial cells were harvested at24h,36h,48h,60h,72h,84h, respectively. The correlations among the expression Hsp110, the transfection methods, silencing time as well as with or without serum in the media were studied in the rat primary myocardial cells silenced by Hsp110siRNA primer in vitro. The results showed that the relative expression levels of Hsp110in the myocardial cells between the negative control and the test groups reached the peak level from36to48h and decreased gradually after discarding the transfection mixture solution. The silencing efficiency within36to72h was about40%as well as the silencing efficiency at84h was about82%; Hsp110expression in the myocardial cells of the negative control reached the peak level at72h and declined gradually, however, Hsp110expression of the test groups went up to the top level at36h and declined gradually with the transfection mixture solution and arrived the lowest levels at84h. The silencing efficiency of Hsp110in the test group from72to84h was about from86%to95%. The results indicate that the effect of transfection reagent and Hsp110siRNA on the transfection and silencing efficiency is significant. Furthermore, the optimal silencing efficiency of Hsp110could be obtained through silencing the cells from72to84h with the transfection solution containing40-120nM Hsp110siRNA primer.
The myocardial cells incubated at37℃for48h were transfected with Hsp110siRNA primer and continued to be incubated for60h at37℃. After changed the medium with DMEM medium containing15%FBS and continued to incubate the cells for12h at37℃, the myocardial cells were stressed in a incubator at42℃with a humidified atmosphere of5%CO2and95%air for4h. The comparative study of the correlations among the expression levels of Hsp110, the levels of the related enzyme including AST, CK, CK-MB, LDH in the suspension of myocardial cell, as well as the apoptosis of the myocardial cells were carried out by using Western blot test, E1ISA test, flow cytometry test. The results showed that the expression levels of Hsp110reduced significantly in the myocardial cells of the test groups. The levels of enzyme AST, CK, CK-MB which indicating myocardial cells damages rose without significant difference, as well as the early and late apoptosis rate reduced without significant difference, suggesting that the present results are different from the former reports, the protective role and mechanism of Hsp110during heat stress still need to be verified further.
运用细胞培养技术、细胞爬片技术、免疫荧光技术、组织学技术及MTT试验等手段对原代培养的大鼠心肌细胞进行条件优化。结果表明,取2日龄SD乳大鼠心脏,依次经过胶原酶Ⅰ消化14h,吹打7～9次,细胞差速贴壁60min,加入0.1～0.3MBRDU,逐点铺细胞等过程,使免疫荧光试验细胞铺板总数为1.8-2.0×106个/mL, Wlestem blot试验细胞铺板总数为2.2-2.3×10个/mL,MTT试验细胞铺板总数为0.25～1×106个/mL时,体外培养的心肌细胞纯度可达到94%,满足试验要求。而逐点铺细胞等是获得高纯度体外培养心肌细胞的必要条件。
将大鼠原代心肌细胞在37℃二氧化碳细胞培养箱中进行72h的适应性培养后,迅速将心肌细胞置于预热的42℃二氧化碳培养箱中,分别进行0min、10min、20min、40min、60min、120min、240min、360min及480min的热应激处理,应用组织学、心肌特异性酶检测技术、免疫荧光检测技术、荧光定量PCR技术和Western blot检测技术对热应激处理心肌细胞出现的应激性损伤及保护性蛋白Hsp110变化规律进行相关性分析研究。热应激心肌细胞培养液中AST、LDH、CK的酶活性显著升高,表明心肌细胞结构的完整性发生了改变；组织学检测结果显示,热应激40-60min期间,心肌细胞体积急性肿胀,胞浆内出现大量红色变性颗粒,细胞核深染、浓缩；Westernblot检测结果显示,热应激处理20min后,Hspl10的表达量呈显著上升趋势,并且在热应激处理240min后,Hsp110的表达量比对照组提高约1.2倍；免疫组织化学结果显示,颗粒状Hsp110分布于心肌细胞的胞浆与细胞核内,42℃热应激240min后,核内Hsp110的分布密度显著增加；hsp110mRNA在热应激处理20min后也明显增加,并于240min时达到高峰,增加量约为对照组的10倍。研究结果表明,Hsp110在体外培养原代心肌细胞内的表达对热应激敏感,在基因和蛋白表达水平均呈现出表达量的明显增加,可能参与心肌细胞获得热耐受过程,在心肌细胞对抗或减轻热应激损伤中发挥重要作用。
将大鼠原代心肌细胞在37℃二氧化碳培养箱中进行72h的适应性培养,迅速将心肌细胞置于预热的42℃二氧化碳细胞培养箱中,分别进行0min、10min、20min、40min、60min、120min、240min、360min及480min的热应激处理。免疫荧光检测结果显示,阳性染色Hsp110颗粒主要位于胞浆内,胞核内也有少量分布,在热应激4h后,阳性染色Hspll0颗粒在细胞浆与细胞核内均大量增加。不同于Hspll0的是,应激组与对照组心肌细胞内的HSF-1仅分布在细胞核内；在42℃热应激120min后,应激心肌细胞内hsp110mRNA的转录量增加,同时可见HspllO表达量也显著增加,而且,同一时期内HSF-1的表达量也增加。结果表明,HspllO与HSF-1表达与分布具有时间依赖的方式,且对热敏感。热应激期间Hsp110表达增加以及HSF-1的表达先下降后升高的特点,表明二种蛋白在心肌细胞热损伤的保护中发挥着不同的作用。
用N-乙酰-L-半胱氨酸(NAC)对离体培养的大鼠原代心肌细胞进行预处理,然后进行热应激,对热应激心肌细胞内Hspll0表达与线粒体凋亡信号通路以及核转录因子(NF-κB)主要调节蛋白表达进行检测。结果表明,在热应激处理1h内,NAC显著抑制了热应激诱导的心肌细胞凋亡,而且随着热应激时间的延长,这种抑制作用呈现减弱的趋势；NAC对热应激心肌细胞核转录因子信号通路的调节蛋白NF-κB具有显著的抑制效果。
将体外37℃条件下适应性培养48h的心肌细胞利用Hsp siRNA与脂质体Lipofectamine2000进行24h转染,采用弃掉转染试剂复合物溶液和保留转染试剂复合物溶液二种方法进行Hspll0基因沉默,然后在37℃条件下继续培养至84h,分别在24h、36h、48h、60h、72h、84h收获细胞,对Hsp110siRNA引物沉默大鼠原代心肌细胞内Hsp110基因表达与转染方式、沉默时间及培养基中血清之间相互关系进行研究。结果表明,弃掉转染试剂复合物溶液后,阴性对照组及试验组心肌细胞内Hsp110的相对表达量在36-48h达峰值,然后呈现逐渐下降的趋势,36～72h内的沉默效率为40%左右,84h时的沉默效率为82%；保留转染试剂复合物溶液后,阴性对照组心肌细胞内Hsp110的相对表达量在72h达到峰值,然后逐渐下降,试验组心肌细胞内Hsp110的相对表达量在36h时达到峰值,然后呈逐渐下降趋势,在84h时最低,72～84h的沉默效率在86%～95%。这一结果说明,转染试剂以及siRNA对转染效率及沉默效率是有显著影响的,而且,我们合成的Hsp110siRNA引物在40-120nM时,转染试剂siRNA复合体对细胞持续转染72～84h,获得的对心肌细胞Hsp110表达的沉默效率最佳。
用Hsp110siRNA引物转染并沉默体外37℃培养48h的心肌细胞,37℃继续培养60h后,弃掉细胞培养液,改用含15%FBS的高糖DMEM培养基继续培养12h；然后于42℃二氧化碳细胞培养箱内进行4h的热应激处理,采用Western blot、ELISA、流式细胞术等检测技术,对体外培养心肌细胞内Hsp110相对表达量的变化、心肌细胞相关酶AST、CK、CK-MB、LDH的改变以及心肌细胞凋亡变化进行了比较研究。结果表明,Hsp110siRNA引物转染并沉默体外培养的心肌细胞后,经过4h的热应激处理后,试验组心肌细胞内Hsp110的相对表达量显著下降；心肌特异性酶AST、CK、 CK-MB在细胞培养液中的含量升高,但差异不显著；心肌细胞早、晚期凋亡减少,但差异不显著。这一结果说明,Hsp110在持续性热应激过程中的所呈现的变化与以往的报道有差异,其作用及其相应分子机制仍然需要进一步的研究验证。
Stress factors including long-distance transportation for livestock trade and higher temperature in living environment for poultry often cause the stressing damage, PSE (pale, soft, exudative) meat, and even to death in the effected animals. It is necessary to study the relation between stress and its damage to stressed animals and provide reasonable methods to reduce the stress influences to the farming industry. Heat shock proteins (Hsps), widespread in the organism from yeast to mammalian, are some endogenous proteins which have protective role. Furthermore, they can overexpress stably in the organism under physiological conditions or stress conditions, and act as the role of molecular chaperone. Hsp110is one of the most important of heat shock proteins family (HSPs) and can protect cells or targeted protein against stressed damages. The overexpression of Hsp110can indentify the denatured proteins and make them keep in a stable and soluble state, and then restore the activities of the denatured protein. In view of consideration that sudden death of stressed animal might be the damage of myocardial cells and heart failure, the protective role and mechanism of Hsp110against stressing damages during heat stress were studied through establishing the mode of myocardial cells stressed by heat in vitro, observing the expression of Hsp110and transcription of hsp110mRNA as well as the correlations between the variations of Hsp110expression and stressing damages of myocardial cells in vitro.
The optimizations of the culture conditions of in vitro myocardial cells were carried out by using cell culture techniques, cell growing on coverslips technology, immunofluorescence technique, histological techniques and MTT assay etc. The results showed that the purity of the myocardial cells incubated in vitro were up to94%and met the test requirements under the following conditions, the cells quantities of the immunofluorescence test were plated at a total of1.8-2.0×106cells/mL, the cells quantities of western blot test were plated at a total of2.2-2.3×106cells/mL, the cells quantities of the MTT test were plated at a total of0.25-1x106cells/mL. The results also showed that it was necessary to obtain optimal primary myocardial cells by selecting2-day-old SD neonatal rats, digesting heart tissue with collagenase I for14h, blowing gently heart tissue mass7-9times with the pipette, making the cells adherent and differential for60min, adding0.1-0.3M BRDU into the cells after cell adherence, seeding cells into the dishes with point by point.
After incubated at37℃for72h, myocardial cells were heat stressed in a incubator at42℃with a humidified atmosphere of5%CO2and95%air for0min,10min,20min,40min,60min,120min,240min,360min, and480min, respectively. The correlations between the cell damage and the variation of Hsp110expression were carried out by using the histological examination, special myocardial enzyme detections, the immunofluorescence technology, real time PCR technology as well as western blot technology. Significant increases of AST, LDH, and CK enzymatic activities in the suspension of myocardial cell were observed during the heat stress, suggesting that the integrity of the myocardial cells was altered. The results of HE staining showed that acute cell swelling characterized by a number of red granules in the cytoplasm deeply stained and concentrated nuclei were observed during heat stress from40min to120min. The results of the western blot test displayed that the overall levels of Hsp110expression increased significantly after20min and the Hsp110levels were approximately1.2-fold higher than that of the control after240min of heat stress. Immunocytochemical analysis revealed that the expressed Hsp110was constitutively localized in the cytoplasm with small amounts in the nuclei characterized by a granular pattern, nuclear Hsp110levels increased significantly after240min of heat stress in comparison with the control. The results of real time PCR proved that increasing levels of hsp110mRNA were observed after20min of heat stress, and the levels peaked with a10-fold increase after240min of heat stress. These results above indicate that the expression of Hsp110in primary myocardial cells in vitro is sensitive to hyperthermic stress by increasing both hsp110mRNA transcription and Hspl10expression levels, Hsp110is involved in the potential acquisition of thermotolerance after heat stress. Therefore, Hsp110might play a fundamental role in opposing and alleviating the stress damages caused by hyperthermic stress in primary myocardial cells.
After incubated at37℃for72h, myocardial cells were heat stressed in an incubator at42℃with a humidified atmosphere of5%CO2and95%air for0min,10min,20min, 40min,60min,120min,240min,360min, and480min, respectively. The results of the immuofluorescence test showed that Hsp110positive staining distributed with enrichment in the cytoplasm and small amount in the nuclei, however, Hsp110increased both in the cytoplasm and the nuclei of myocardial cells after4h of heat stress. In contrast to Hsp110, HSF-1staining was only observed in the nuclei of primary rat myocardial cells. Both of Hsp110and HSF-1increased in the heat-stressed myocardial cells, meanwhile the increasing of hsp110mRNA and HSF-1mRNA transcript were observed after120min of heat stress at42℃. The results showed that Hsp110and HSF-1were sensitive to heat stress in a time-dependent manner. The characteristics of Hsp110inductions during heat stress, nuclear HSF-1expression initial decrease and then significant increase between240and480min, suggesting that both Hsp110and HSF-1have different roles in protecting myocardial cells from heat stress damage.
Rat primary myocardial cells in vitro were pretreated for1h with N-acetyl-L-cysteine (NAC) and then heat-stressed. The proteins associated with mitochondrial apoptosis signaling pathway, nuclear transcription factor (NF-κB) signaling pathway as well as the expression levels of Hsp110in the heat stressed myocardial cells were studied. The results showed that NAC inhibited significantly myocardial cells apoptosis induced by heat stress within1h, and the role became weaker with the elongation of heat stress. However NAC inhibited significantly the expression levels of NF-κB belonging to NF-κB signaling pathway.
After incubated at37℃with lipofectamine2000for48h and Hsp110siRNA primer for24h, then continued to incubate the cells until84h with or without transfection mixture solution including Hsp110siRNA primer and lipofectamine2000at37℃, and then the transfected myocardial cells were harvested at24h,36h,48h,60h,72h,84h, respectively. The correlations among the expression Hsp110, the transfection methods, silencing time as well as with or without serum in the media were studied in the rat primary myocardial cells silenced by Hsp110siRNA primer in vitro. The results showed that the relative expression levels of Hsp110in the myocardial cells between the negative control and the test groups reached the peak level from36to48h and decreased gradually after discarding the transfection mixture solution. The silencing efficiency within36to72h was about40%as well as the silencing efficiency at84h was about82%; Hsp110expression in the myocardial cells of the negative control reached the peak level at72h and declined gradually, however, Hsp110expression of the test groups went up to the top level at36h and declined gradually with the transfection mixture solution and arrived the lowest levels at84h. The silencing efficiency of Hsp110in the test group from72to84h was about from86%to95%. The results indicate that the effect of transfection reagent and Hsp110siRNA on the transfection and silencing efficiency is significant. Furthermore, the optimal silencing efficiency of Hsp110could be obtained through silencing the cells from72to84h with the transfection solution containing40-120nM Hsp110siRNA primer.
The myocardial cells incubated at37℃for48h were transfected with Hsp110siRNA primer and continued to be incubated for60h at37℃. After changed the medium with DMEM medium containing15%FBS and continued to incubate the cells for12h at37℃, the myocardial cells were stressed in a incubator at42℃with a humidified atmosphere of5%CO2and95%air for4h. The comparative study of the correlations among the expression levels of Hsp110, the levels of the related enzyme including AST, CK, CK-MB, LDH in the suspension of myocardial cell, as well as the apoptosis of the myocardial cells were carried out by using Western blot test, E1ISA test, flow cytometry test. The results showed that the expression levels of Hsp110reduced significantly in the myocardial cells of the test groups. The levels of enzyme AST, CK, CK-MB which indicating myocardial cells damages rose without significant difference, as well as the early and late apoptosis rate reduced without significant difference, suggesting that the present results are different from the former reports, the protective role and mechanism of Hsp110during heat stress still need to be verified further.
引文
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