胶质细胞瘤中HSP60和Caspase-3蛋白的表达及与病理分级关系的研究
摘要
一、研究背景：
胶质瘤是一种常见的颅内恶性肿瘤,约占颅内肿瘤的40%-50%,对其治疗至今尚没有明显进展,且发病原因仍不太清楚,目前越来越多的认为与某些蛋白的表达有关系。在胶质瘤的不同细胞类型,不同分化阶段,不同分级,都有不同的蛋白的表达发生改变,研究并检测这些蛋白标记物,同时结合对肿瘤细胞增殖活性的检测,对于疾病的诊断,治疗,和了解肿瘤的发生机制具有临床和理论意义。
热休克蛋白(Heat shock proteins,HSPs)是生物体受到物理、生物、化学、精神等刺激时所发生应激反应而合成的一种高度保守的蛋白质。这是因为温度升高后增强了这一区域的基因转录,从而相应生成的蛋白质被命名为热休克蛋白质(HSPs)。HSPs主要参与一些重要的细胞生理活动,比如蛋白质转位、装配和折叠,除此之外,各种刺激因素诱导HSPs合成后,可使细胞结构稳定,维持细胞正常生理功能,提高机体相应适应能力(如耐热、耐寒、抗感染、抗毒素等)。
热休克蛋白根据同源程度和分子量从小到大可分为：小分子HSP、HSP10、HSP40、HSP60、HSP70、HSP90、HSP110及泛素等几个家族。其中HSP60和HSP70具有高度的物种保守性,系热休克蛋白的两个重要的家族。HSP70已被深入研究,现已被证实HSP70在肿瘤表达异常,能与癌基因,抑癌基因产物结合,并参与肿瘤细胞的细胞周期调控、增殖、调往、分化、多药耐药、肿瘤免疫以及肿瘤细胞的发生发展密切相关。而对HSP60的研究相对较少,尤其是在胶质瘤方面的研究更加少见。
半胱氨酸天冬氨酸蛋白酶(Cysteinyl aspartate apecfic protease, Caspase)家族是一大类凋亡调节基因,是哺乳动物细胞凋亡的启动者和执行者。其中Caspase-3是Caspase级联“瀑布“下游最关键的凋亡执行蛋白酶,在各种因素启动的凋亡程序中起到最后枢纽作用,在其他下游的Caspase成员相比,可能是凋亡时间的真正执行者。在正常情况下,Caspase-3以无活动的酶原形式存在于哺乳动物的多种组织和细胞内,其在免疫细胞、脑和胚胎起源的细胞中有高水平表达。细胞凋亡机制调控失衡,导致细胞死亡减少,细胞异常增殖,相关肿瘤发生。既往研究显示,Caspase-3在脑肿瘤细胞凋亡过程扮演重要角色。并且对脑肿瘤的放疗、化疗过程中,Caspase-3诱导的细胞凋亡机制研究提示,脑肿瘤的发生不仅与神经细胞的异常增强和分化有关,也与细胞凋亡的异常有关。
本文用免疫组化方法检测64例胶质瘤手术标本中HSP60、Caspase-3的表达,分析两者与肿瘤增殖、凋亡和细胞分化的关系。
二、研究对象和方法
1.研究对象：收集来自中南大学湘雅附属第二医院病理科2005年3月到2009年12月间诊断为胶质细胞瘤的手术存档标本共64例。按2000年WHO制定的神经系统肿瘤分类和分级标准分为：Ⅰ级10例,Ⅱ级18例,Ⅲ级19例,Ⅳ级17例。其中男性41名,女性23名,年龄7～65岁,平均34.6岁。
2.免疫组化染色免疫组化染色用SP法。一抗分别为鼠抗人Caspase3单克隆抗体和鼠抗人HSP60单克隆抗体。
3.结果判定：采用双盲法对每张切片在高倍镜(x400)下计数5个高倍视野。以染色阳性细胞比例分别计为：阳性细胞蕊5%为阴性(一)；阳性细胞数6%-25%弱阳性(+)；阳性细胞数26%-50%为阳性(++)；阳性细胞数51～100%为强阳性(+++)。
三、结果
1.HSP60与Caspase 3蛋白免疫组织化学染色阳性结果为棕黄色颗粒,HSP 60阳性颗粒主要定位于肿瘤细胞核和细胞浆内,Caspase3阳性颗粒主要定位于肿瘤细胞浆内。
2.随着胶质瘤病理级别的增高,HSP60的表达强度增加,Ⅰ-Ⅳ级胶质瘤的HSP60阳性率分别为20%、72.2%、89.4%、100%(P<0.01)。
3.随着胶质瘤病理级别的增高,Caspase-3的表达强度减弱,Ⅰ-Ⅳ级胶质瘤的Caspase3阳性率分别为90%、77.7%、36.8%、23.5%(P<0.01)。
4.胶质瘤中,Caspase 3与HSP60的蛋白表达间存在负相关关系(P<0.01)。
四、结论
HSP60与Caspase 3蛋白的表达结果提示,两者与胶质瘤的生物学行为和分化程度有关,对不同级别胶质细胞瘤中HSP60和Caspase3蛋白的表达进行联合检测,不仅有助于判断胶质细胞瘤恶性程度,并且有助于为肿瘤的发病机制提供理论依据。
Backgrounds
Glioma is a frequent malignant tumor of intracalvarium. Account 40%-50% in tumor of intracalvarium,up to now, the treatment is not obviously advancement and its morbility reason is not understanded, at present more and more people consider follow with some protein express. There is different protein express follow with different cell types,cell differentiation and class. To study and resea- rch these protein maker and combine with the cell proliferation examination sho- uld be help for the diagnosis and treatment of tumor and understanding the me- chanism of the carcinogenesis of glioma.
Heat shock proteins are kinds of highly conservative proteins which are synth- esized by the irritation of physics chemmistry and psyche reasons in creatures. This is because when the tempreture rises, the genetic transcription of this area is strengthen. So, the carresponding proteins are named Heat shock proteins. HSPs mainly attend some important cellophysiology activity, for example:protein transposition, assemblage,and aliasing, otherwise, after the sythesis of HSPs which are induced by many kind of irritations, the HSPs can stabilize the struct- ure of cell, sustain normal physiological function of cell, and to inhence the ada- ptive capacity of body (for example:thermostabilization; heat-resistant, cold- resistant, anti-infection, anti-toxin).
The HSPs can be divided into tiny HSP, HSP10, HSP40, HSP60, HSP70, HSP 90, HSP 110, and HSP60, etc, according to the degree of homology and that of molecular weight. The HSP60 and HSP70 are highly conservative in evolution, and very important in HSP family.
HSP70 has already been studied vastly. It may express abnormally in tissues of tumor, by combining with the production of oncogene and the anti-oncogene, It may take part in controlling the cellular cycle of the tumor cell. It may also affect the multiplication, the apoptosis, the differentiation, the mulri-drugs resistance, the tumor immunology, and the development of the tumor cells. But, we still kn- ow less now about the HSP60, especially on its role in Glioma.
Cysteinyl aspartate specfic protease (Caspase) family is a large set of apoptotic regulated genes for the initiation and execution of mammalian programmed cell death. Apoptosis may be thought of as a signaling cascade, which Caspase-3 is a key protease. In normal status, Caspase-3 as inactivity zymogen exists in some tissues and cells of mammalian, and overexpression in immunocyte, brain,and embry cells. The irregulation of cell apoptosis result in the cell death reducing and uncontrolled proliferate that associated the development and progression of tumor. It is reported that Caspase-3 plays an important role in the cell apoptosis in brain tumor, which treatment by radiotherapy and chemotherapy, show that the tumor development not only related the nervous cells abnormal proliferate, but also related the cells abnormal apoptosis.
The present study use immunohistochemistry to investigate the clinical signif- icance of HSP60 and Caspase-3 expression and analysis the relationship between the expression and the proliferation, apoptosis, and differentiation of cells in Gli-oma.
Msterials and Methods
Patients and specimens
We consecutively collected tumors from 64 patients (mean age, 34.6; range,4-67 years; male:female,41:23)who had undergone surgery for glioma at the Xiangya second hospital of the PLA from March 2005 to December 2009. The histopathological diagnosis was made according to the WHO classification:10 cases were WHOⅠgrade; 18cases were WHOⅡgrade; 19 cases were WHOⅢgrade; and 17 cases were WHOⅣgrade.
Immunohistochemistry
Immunohistochemical staining of HSP60 and Caspase 3 monoclonal antibody was done using the SP Staining System.
Evaluationofimmunostaining
All of the immunostained sections were reviewed by double blinded studies. For expression of HSP60 and Caspase 3 protein, five areas (X400) were selected at random and scored in cases with multiple areas of high intensity that occurred during evaluation of immunostain-ing.The degree of immunohistochemical stai- ning was recorded that considered the proportion of tumor cells with an unequiv- ocal positive reaction (Area: -,≤5% staining;+positive staining in 6%～25% of tumor cells;++, positive staining in 26% to 50% of tumor cells;+++, positives taining in> 50% of tumor cells).
Results
1. Immunohistochemistry were done to evaluate HSP60 and Caspase-3 protein expression, which show brown, respectively. The HSP60 Protein expression had a cytoplasmic and nuclear staining pattem, and the Caspase 3 had a cyt- oplasmic staining in tumor cells.
2. The HSP60 expressed intensity of tumor cells were increase with the hist- ological grade up in Gliomas, and the percentage of stained tumor cells from histological gradeⅠtoⅣwas 20%,72.2%,89.4% and 100%(P<0.01), respectively.
3. The Caspase -3 expressed intensity of tumor cells were decrease with the hi- stological grade up in Gliomas,and the percentage of stained tumor cells from histological grade I to IV was 90%,77.7%,36.8% and 23.5%(p<0.01), respectively.
4. Caspase 3 expression was reverse correlated with HSP60 expression (P<0.01) in Gliornas
Conclusion
The Caspase-3 and HSP60 Protein expression are related to the biological be-havior and differentiation of tumor cell in Glioma.The irnrnunohistochemical ex-amination of Caspase3 and HSP 60 expression in Glioma, which have different histological grade, should be help for evaluated the tumor cell malignant degree and have significant to demonstrate the mech- anism of the carcinogenesis of Gli- oma.
胶质瘤是一种常见的颅内恶性肿瘤,约占颅内肿瘤的40%-50%,对其治疗至今尚没有明显进展,且发病原因仍不太清楚,目前越来越多的认为与某些蛋白的表达有关系。在胶质瘤的不同细胞类型,不同分化阶段,不同分级,都有不同的蛋白的表达发生改变,研究并检测这些蛋白标记物,同时结合对肿瘤细胞增殖活性的检测,对于疾病的诊断,治疗,和了解肿瘤的发生机制具有临床和理论意义。
热休克蛋白(Heat shock proteins,HSPs)是生物体受到物理、生物、化学、精神等刺激时所发生应激反应而合成的一种高度保守的蛋白质。这是因为温度升高后增强了这一区域的基因转录,从而相应生成的蛋白质被命名为热休克蛋白质(HSPs)。HSPs主要参与一些重要的细胞生理活动,比如蛋白质转位、装配和折叠,除此之外,各种刺激因素诱导HSPs合成后,可使细胞结构稳定,维持细胞正常生理功能,提高机体相应适应能力(如耐热、耐寒、抗感染、抗毒素等)。
热休克蛋白根据同源程度和分子量从小到大可分为：小分子HSP、HSP10、HSP40、HSP60、HSP70、HSP90、HSP110及泛素等几个家族。其中HSP60和HSP70具有高度的物种保守性,系热休克蛋白的两个重要的家族。HSP70已被深入研究,现已被证实HSP70在肿瘤表达异常,能与癌基因,抑癌基因产物结合,并参与肿瘤细胞的细胞周期调控、增殖、调往、分化、多药耐药、肿瘤免疫以及肿瘤细胞的发生发展密切相关。而对HSP60的研究相对较少,尤其是在胶质瘤方面的研究更加少见。
半胱氨酸天冬氨酸蛋白酶(Cysteinyl aspartate apecfic protease, Caspase)家族是一大类凋亡调节基因,是哺乳动物细胞凋亡的启动者和执行者。其中Caspase-3是Caspase级联“瀑布“下游最关键的凋亡执行蛋白酶,在各种因素启动的凋亡程序中起到最后枢纽作用,在其他下游的Caspase成员相比,可能是凋亡时间的真正执行者。在正常情况下,Caspase-3以无活动的酶原形式存在于哺乳动物的多种组织和细胞内,其在免疫细胞、脑和胚胎起源的细胞中有高水平表达。细胞凋亡机制调控失衡,导致细胞死亡减少,细胞异常增殖,相关肿瘤发生。既往研究显示,Caspase-3在脑肿瘤细胞凋亡过程扮演重要角色。并且对脑肿瘤的放疗、化疗过程中,Caspase-3诱导的细胞凋亡机制研究提示,脑肿瘤的发生不仅与神经细胞的异常增强和分化有关,也与细胞凋亡的异常有关。
本文用免疫组化方法检测64例胶质瘤手术标本中HSP60、Caspase-3的表达,分析两者与肿瘤增殖、凋亡和细胞分化的关系。
二、研究对象和方法
1.研究对象：收集来自中南大学湘雅附属第二医院病理科2005年3月到2009年12月间诊断为胶质细胞瘤的手术存档标本共64例。按2000年WHO制定的神经系统肿瘤分类和分级标准分为：Ⅰ级10例,Ⅱ级18例,Ⅲ级19例,Ⅳ级17例。其中男性41名,女性23名,年龄7～65岁,平均34.6岁。
2.免疫组化染色免疫组化染色用SP法。一抗分别为鼠抗人Caspase3单克隆抗体和鼠抗人HSP60单克隆抗体。
3.结果判定：采用双盲法对每张切片在高倍镜(x400)下计数5个高倍视野。以染色阳性细胞比例分别计为：阳性细胞蕊5%为阴性(一)；阳性细胞数6%-25%弱阳性(+)；阳性细胞数26%-50%为阳性(++)；阳性细胞数51～100%为强阳性(+++)。
三、结果
1.HSP60与Caspase 3蛋白免疫组织化学染色阳性结果为棕黄色颗粒,HSP 60阳性颗粒主要定位于肿瘤细胞核和细胞浆内,Caspase3阳性颗粒主要定位于肿瘤细胞浆内。
2.随着胶质瘤病理级别的增高,HSP60的表达强度增加,Ⅰ-Ⅳ级胶质瘤的HSP60阳性率分别为20%、72.2%、89.4%、100%(P<0.01)。
3.随着胶质瘤病理级别的增高,Caspase-3的表达强度减弱,Ⅰ-Ⅳ级胶质瘤的Caspase3阳性率分别为90%、77.7%、36.8%、23.5%(P<0.01)。
4.胶质瘤中,Caspase 3与HSP60的蛋白表达间存在负相关关系(P<0.01)。
四、结论
HSP60与Caspase 3蛋白的表达结果提示,两者与胶质瘤的生物学行为和分化程度有关,对不同级别胶质细胞瘤中HSP60和Caspase3蛋白的表达进行联合检测,不仅有助于判断胶质细胞瘤恶性程度,并且有助于为肿瘤的发病机制提供理论依据。
Backgrounds
Glioma is a frequent malignant tumor of intracalvarium. Account 40%-50% in tumor of intracalvarium,up to now, the treatment is not obviously advancement and its morbility reason is not understanded, at present more and more people consider follow with some protein express. There is different protein express follow with different cell types,cell differentiation and class. To study and resea- rch these protein maker and combine with the cell proliferation examination sho- uld be help for the diagnosis and treatment of tumor and understanding the me- chanism of the carcinogenesis of glioma.
Heat shock proteins are kinds of highly conservative proteins which are synth- esized by the irritation of physics chemmistry and psyche reasons in creatures. This is because when the tempreture rises, the genetic transcription of this area is strengthen. So, the carresponding proteins are named Heat shock proteins. HSPs mainly attend some important cellophysiology activity, for example:protein transposition, assemblage,and aliasing, otherwise, after the sythesis of HSPs which are induced by many kind of irritations, the HSPs can stabilize the struct- ure of cell, sustain normal physiological function of cell, and to inhence the ada- ptive capacity of body (for example:thermostabilization; heat-resistant, cold- resistant, anti-infection, anti-toxin).
The HSPs can be divided into tiny HSP, HSP10, HSP40, HSP60, HSP70, HSP 90, HSP 110, and HSP60, etc, according to the degree of homology and that of molecular weight. The HSP60 and HSP70 are highly conservative in evolution, and very important in HSP family.
HSP70 has already been studied vastly. It may express abnormally in tissues of tumor, by combining with the production of oncogene and the anti-oncogene, It may take part in controlling the cellular cycle of the tumor cell. It may also affect the multiplication, the apoptosis, the differentiation, the mulri-drugs resistance, the tumor immunology, and the development of the tumor cells. But, we still kn- ow less now about the HSP60, especially on its role in Glioma.
Cysteinyl aspartate specfic protease (Caspase) family is a large set of apoptotic regulated genes for the initiation and execution of mammalian programmed cell death. Apoptosis may be thought of as a signaling cascade, which Caspase-3 is a key protease. In normal status, Caspase-3 as inactivity zymogen exists in some tissues and cells of mammalian, and overexpression in immunocyte, brain,and embry cells. The irregulation of cell apoptosis result in the cell death reducing and uncontrolled proliferate that associated the development and progression of tumor. It is reported that Caspase-3 plays an important role in the cell apoptosis in brain tumor, which treatment by radiotherapy and chemotherapy, show that the tumor development not only related the nervous cells abnormal proliferate, but also related the cells abnormal apoptosis.
The present study use immunohistochemistry to investigate the clinical signif- icance of HSP60 and Caspase-3 expression and analysis the relationship between the expression and the proliferation, apoptosis, and differentiation of cells in Gli-oma.
Msterials and Methods
Patients and specimens
We consecutively collected tumors from 64 patients (mean age, 34.6; range,4-67 years; male:female,41:23)who had undergone surgery for glioma at the Xiangya second hospital of the PLA from March 2005 to December 2009. The histopathological diagnosis was made according to the WHO classification:10 cases were WHOⅠgrade; 18cases were WHOⅡgrade; 19 cases were WHOⅢgrade; and 17 cases were WHOⅣgrade.
Immunohistochemistry
Immunohistochemical staining of HSP60 and Caspase 3 monoclonal antibody was done using the SP Staining System.
Evaluationofimmunostaining
All of the immunostained sections were reviewed by double blinded studies. For expression of HSP60 and Caspase 3 protein, five areas (X400) were selected at random and scored in cases with multiple areas of high intensity that occurred during evaluation of immunostain-ing.The degree of immunohistochemical stai- ning was recorded that considered the proportion of tumor cells with an unequiv- ocal positive reaction (Area: -,≤5% staining;+positive staining in 6%～25% of tumor cells;++, positive staining in 26% to 50% of tumor cells;+++, positives taining in> 50% of tumor cells).
Results
1. Immunohistochemistry were done to evaluate HSP60 and Caspase-3 protein expression, which show brown, respectively. The HSP60 Protein expression had a cytoplasmic and nuclear staining pattem, and the Caspase 3 had a cyt- oplasmic staining in tumor cells.
2. The HSP60 expressed intensity of tumor cells were increase with the hist- ological grade up in Gliomas, and the percentage of stained tumor cells from histological gradeⅠtoⅣwas 20%,72.2%,89.4% and 100%(P<0.01), respectively.
3. The Caspase -3 expressed intensity of tumor cells were decrease with the hi- stological grade up in Gliomas,and the percentage of stained tumor cells from histological grade I to IV was 90%,77.7%,36.8% and 23.5%(p<0.01), respectively.
4. Caspase 3 expression was reverse correlated with HSP60 expression (P<0.01) in Gliornas
Conclusion
The Caspase-3 and HSP60 Protein expression are related to the biological be-havior and differentiation of tumor cell in Glioma.The irnrnunohistochemical ex-amination of Caspase3 and HSP 60 expression in Glioma, which have different histological grade, should be help for evaluated the tumor cell malignant degree and have significant to demonstrate the mech- anism of the carcinogenesis of Gli- oma.
引文
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[16]Van der Straten A,Rommel C,Dickson B,et al.The heat shock protein83 (HSP83) is required for Raf2 mediated signaling in Drosophila[J].EMBO J,1997, 16(8):1961-1969.
[17]Ja at tela M,Wissing D.Heat shock proteins protect cells from monocyte cyto toxicity:possible mechanismof self2 protection [J].J Exp Med,1993,177(1):231-236.
[18]Van LG, Van GM, Depuydt B, et al. The serine protease omi/Htr A2 is released from mitochondria during apoptosis. Omi interacts with Caspase-inhibitor XIAP and induces enhanced caspase activity[J]. Cell Death Differ,2002,9(1):20-26.
[19]Gupta S, Know Iton AA. HSP60, Bax, apoptosis and the heart. J CellMolMed,2005,9:51～58.
[20]Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes:role of the exosomalpathway. Am J Physiol Heart Circ Physio,l 2007,292:H3052～H3056.
[21]Cappello F, Czarnecka AM, Rocca GL, et al. Hsp60 and Hsp10 as antitumor molecular agents. Cancer Biol Ther,2007,6:487～489.
[22]Sivaslava PK. Heast shock proteins implieations of the stress respionse[J].Adv Caneer Bes.1993,62:153-159.
[23]JuliannG et al. Phannacol[J]. Ther 1998 80(2):183-201.
[24]Chan JY, ChengHL, Chou JL, eta.l Heatshock protein 60 or 70 activates nitric-oxide synthase (NOS) I-and inhibits NOS Ⅱ-associated signaling, and depresses themitochondrial apoptotic cascade during brain stem death. J Biol Chem,2007,282: 4585～4600.
[25]ChangAY, Chan JY, Chou JL, et al. Heatshock protein 60 in rostralventr-olateralmedulla reduces cardiovascular fatality during endotoxaemia in the rat. J Physio,1 2006,574(Pt2):547～564.
[26]VeereshwarayyaV, KumarP, RosenKM, eta.l Differential effects of mitocho-ndrial heat shock protein 60 and related molecular chaperones to prevent intrac-ellularβ-amyloid-induced inhibition of complex IV and limit apoptosis. J Biol-Chem, 2006,281:29468～29478.
[27]Kondan S, Barns BP, Morimura T, etal.Intedeukin-1B coverting, enzyme mediates cisplatin-indueed apoptosis in malignant glloma cells[J].Cancer Res,1995, 55:6166-6171.
[28]Srinivasula SM, Abmad M, et al.Generation of constitutively active recom-binant caspase-3 and 6 reammgement of their subunits[J] J Biol Chem,1998,273: 10107-10511.
[29]Thoraberry NA, lazebnik Y.Caspase[J].Science,1998,281(5381):1312-1316.
[30]Shariat SF, DesaiS, Song W, et al. Adenovirus-mediated transfer of inducible caspase:a novel "death swith" gene therapeutic appreach to prostate caneer[J].Cancer Res,2001,61:2562-2571.
[1].Kerr JFR, Wyllie AH, Currie AR, et al. Apoptosis:abasic boilogicl Phenomenon with wide ranging implication intuissue Kinet ics[J]. B r J caneer,1972, 26:239
[2].Han YC, LinTL,Pruett SB. Thymic atrophy caused byethanol in a mousemodel for binge drinking:involvement of endogenous glucocofticoids [J]. Toxico-Appl-Pharma2cal,1993,123:16.
[3].ThompsonCB. Apoptosis in the pathofenesis and treatment of disease [J].Seienee,1995,267:1456.
[4]JULIANN G K,GEORGE C T.Heat shock protein 70 kDa:Molecular biology,bioche mistry and physiology [J].Pharmacol Ther,1998,80(2):183-201.
[5]FEDER M E,HOFMANN G E.Heat shock proteins,molecular chaperones and the stress response[J].Annu Rev Physiol,1999,61:243-282.
[6]BECHMANN R P,MIZZEN L A,WELCH W Interaction of HSP70 newly synthesized proteins:Implications for protein folding and assembly events [J]. Science,1990,248:850-854.
[7]WANG X Y,MANJLILI M H,CHEN X,et al.Development of cancer vaccines using autologous and recombinant high molecular weight stress proteins [J]. Methods,2004,32(1):13-20.
[8]Wick GAtherosclerosis-an autoimmune disease due to an immune reaction against heat-shock protein 60.Herz,2000 Mar;25(2):87-90.
[9]Lorca O,Perez-Perez J,Carrascosa JL,et a.Effects of the inter-ring communi-cation in GroEL structural and functional asymmetry.J Biol Chem,1997 Dee 26;272(52):32925-32932.
[10]Gupta S, Know Iton AA. HSP60, Bax, apoptosis and the heart. J CellMolMed,2005,9:51～58.
[11]Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes:role of the exosomalpathway. Am J Physiol Heart Circ Physio,1 2007,292:H3052～H3056.
[12]Deocaris CC, Kaul SC, Wadhwa R. On the brotherhood of the mitochondrial chaperonesmortalin and heatshock protein 60. CellStress Chaperones,2006,11: 116～128.
[13]Chen W,Syldath U,Bellmann K,et al.Human 60-kDa heat-shock protein:a danger signal to the innate immune system. J Immunol.1999, Mar15; 162 (6):3212-3219.
[14]Habich C,Baumgart K,Kolb H,et al.The receptor for heat hock protein 60 on macrophages is saturable,specific,and distinct from receptors for other heat shock proteins. J Immunol.2002 Jan 15;168(2):569-576.
[15]Barazi HO,Zhou L,Templeton NS,et al.Identification of heat shockprotein 60 as a molecular mediator of alpha 3 beta 1 integrin activation.Cancer Res,2002, Marl:62(5):1541-1548.
[16]Wick G.Atherosclerosis-an autoimmune disease due to an immunereaction against heat-shock protein 60.Herz,2000,Mar;25(2):87-90.
[17]Kerr JFR,Wyllie AH,Currie AR.Apoptosis:a bassic biological phenoenon with wideranging implications in tissue kinetics.Br J Cancer 1972;26:239-257.
[18]Stewart BW.Mechanisms of apoptosis:integration of genetic, biomedical,and cellular indications.J Natl Cancer Inst 1994; 86(17):1286-1295.
[19]Cohen JJ.Apoptosis.Immunol-Today 1993,Mar;14(3):126-130.
[20]Cappello F, Czarnecka AM, Rocca GL, et al. Hsp60 and HsplO as antitumor molecular agents. Cancer Biol Ther,2007,6:487～489.
[21]Alard JE, DueymesM, Youinou P, eta.l Modulation of endothelial cell damages by anti-Hsp60 autoantibodies in systemic autoimmune diseases. Autoimmun Rev,2007,6:438-443.
[22]Barinaga M.Is apoptosis key in alzheimer's disease? Seienee.1998 281(5381):1303-1304.
[23]Lin XY. Choi MS.Porter AG. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopoly saccharide and interferon-gamma. J Biol Chem.2000 275(51):39920-39926.
[24]Howard Y, Chang HY, Yang X. Proteases for cell suicide:functions and regulation of caspases. Microbiology Mol Biol.2000 Dec:64(4):821-846.
[25]Douglas R, Green, Reed JC. Mitochondria and apoptosis. Seience.1998 Aug:28:281(5381):1309-1312.
[26]Gotlieb RA. Mitoehondria and apoptosis. Biol Signals Recept.2001. May-Aug:10(3-4):147-61.
[27]ZouH, Yang R, Hao J, et al.Regulation of the Apaf-11Caspase9 apoptosome by Caspase-3 and XIAP. J Biol Chem.2002 Dec:27
[28]Juliann Get al. Phannacol[J]. Ther,1998,80(2):183～201.
[29]Tschopp J, Martiron F, et al. Mechanisms of caspase activation Kelly M Boatright and Guy S Salvesen Current Opinion in Cell[J].Biology,2003,15:725-731.
[30]Chang DW,Xing Z,Capacio VL,Peter ME,Yang X.Inter-dimer processing mechanism of procaspase-8 activation[J].EMBO,2003,22:4132-4142.
[31]Green DR,Reed JC.Mitochondria and apoptosis[J].Science,1998,281(5381): 1309-1312.
[32]Saikumar P,Dong Z,Mikhailov V,et al.Apoptosis:definition, mechanism, and relevance to disease[J].Am J Med,1999,107(5):489-506.
[33]Budihardjo I,Oliver H,Lutter M,et al.Biochemical pathways of caspase activation during apoptosis[J].Annau Rev Cell Dev Biol,1999,15:269-290.
[2]Thomberry N A, Lazebnik Y. Caspases:enemies within[J]. Seienee,1998, 281 (5381):1312-1316.
[3]秦佳,杨金莹,伊淑莹,等.热激蛋白对细胞凋亡的调节作用[J].生命科学,19(2),159～163.
[4]Barinaga M.Is apoptosis key in alzheimer's disease? Seienee.1998 281(5381): 1303-1304.
[5]Lin XY. Choi MS.Porter AG. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopoly saccharide and interferon-gamma. J Biol Chem.2000 275(51):39920-39926.
[6]Howard Y, Chang HY, Yang X. Proteases for cell suicide:functions and regulation of caspases. Microbiology Mol Biol.2000 Dec:64(4):821-846.
[7]Douglas R, Green, Reed JC. Mitochondria and apoptosis. Seience.1998 Aug: 28:281(5381):1309-1312.
[8]Nicholson DW, ThotherryNA.Caspase:killerProtease[J].Themds Biehem Sci, 1997,22:299～306.
[9]Uetsuki T, Takeffiot K, Yashikawa K, et al. Activation of neuron by intracelar Accumulation of wild-type Alzhelmer's amyloid preeursor protein[J] J Neurosei, 1996, (76):6955～6954.
[10]Srinivasula SM, Femandes-Alnemri T, Zangrilli J, et al.The Ced-3/inte-deukinlbeta coverting enzyme-like homolog Mch 6 and the lamin-cleaving enzymeh Mch 2 alpha are substrates for the apoptotie mediator CPP32[J].J Biol Chem,1996,271 (43):27099-27106.
[11]Krajewsks M, Wallg H C, Kajewski S, et al. Lminunohistochemicalanalysia of in vivo patterns of expression of CPP32 caspase-3), a cell death protease [J]. CancerRes,1997,57(8):1605-1613.
[12]秦佳,杨金莹,伊淑莹,等.热激蛋白对细胞凋亡的调节作用[J].生命科学,19(2),159～163.
[13]Feng H,Zeng Y,White sell L,et al.Stresse dapoptotic tumor cells Express heat shock proteins and elicit tumor-specific immunity.Blood,2001 Jun 1;97(11): 3505-3512.
[14]Yano M,Naito Z,Tanaka S,et al.Expression and roles of heat shock proteins in human breast cancer.Jpn J Cancer Res,1996 Sep;87(9):908-915.
[15]施一江,赵枚,徐先发,等.主要人热休克蛋白在癌组织及癌旁正常组织中表达水平的比较研究[J].中华肿瘤杂志,1998,20(4)：277-279.
[16]Van der Straten A,Rommel C,Dickson B,et al.The heat shock protein83 (HSP83) is required for Raf2 mediated signaling in Drosophila[J].EMBO J,1997, 16(8):1961-1969.
[17]Ja at tela M,Wissing D.Heat shock proteins protect cells from monocyte cyto toxicity:possible mechanismof self2 protection [J].J Exp Med,1993,177(1):231-236.
[18]Van LG, Van GM, Depuydt B, et al. The serine protease omi/Htr A2 is released from mitochondria during apoptosis. Omi interacts with Caspase-inhibitor XIAP and induces enhanced caspase activity[J]. Cell Death Differ,2002,9(1):20-26.
[19]Gupta S, Know Iton AA. HSP60, Bax, apoptosis and the heart. J CellMolMed,2005,9:51～58.
[20]Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes:role of the exosomalpathway. Am J Physiol Heart Circ Physio,l 2007,292:H3052～H3056.
[21]Cappello F, Czarnecka AM, Rocca GL, et al. Hsp60 and Hsp10 as antitumor molecular agents. Cancer Biol Ther,2007,6:487～489.
[22]Sivaslava PK. Heast shock proteins implieations of the stress respionse[J].Adv Caneer Bes.1993,62:153-159.
[23]JuliannG et al. Phannacol[J]. Ther 1998 80(2):183-201.
[24]Chan JY, ChengHL, Chou JL, eta.l Heatshock protein 60 or 70 activates nitric-oxide synthase (NOS) I-and inhibits NOS Ⅱ-associated signaling, and depresses themitochondrial apoptotic cascade during brain stem death. J Biol Chem,2007,282: 4585～4600.
[25]ChangAY, Chan JY, Chou JL, et al. Heatshock protein 60 in rostralventr-olateralmedulla reduces cardiovascular fatality during endotoxaemia in the rat. J Physio,1 2006,574(Pt2):547～564.
[26]VeereshwarayyaV, KumarP, RosenKM, eta.l Differential effects of mitocho-ndrial heat shock protein 60 and related molecular chaperones to prevent intrac-ellularβ-amyloid-induced inhibition of complex IV and limit apoptosis. J Biol-Chem, 2006,281:29468～29478.
[27]Kondan S, Barns BP, Morimura T, etal.Intedeukin-1B coverting, enzyme mediates cisplatin-indueed apoptosis in malignant glloma cells[J].Cancer Res,1995, 55:6166-6171.
[28]Srinivasula SM, Abmad M, et al.Generation of constitutively active recom-binant caspase-3 and 6 reammgement of their subunits[J] J Biol Chem,1998,273: 10107-10511.
[29]Thoraberry NA, lazebnik Y.Caspase[J].Science,1998,281(5381):1312-1316.
[30]Shariat SF, DesaiS, Song W, et al. Adenovirus-mediated transfer of inducible caspase:a novel "death swith" gene therapeutic appreach to prostate caneer[J].Cancer Res,2001,61:2562-2571.
[1].Kerr JFR, Wyllie AH, Currie AR, et al. Apoptosis:abasic boilogicl Phenomenon with wide ranging implication intuissue Kinet ics[J]. B r J caneer,1972, 26:239
[2].Han YC, LinTL,Pruett SB. Thymic atrophy caused byethanol in a mousemodel for binge drinking:involvement of endogenous glucocofticoids [J]. Toxico-Appl-Pharma2cal,1993,123:16.
[3].ThompsonCB. Apoptosis in the pathofenesis and treatment of disease [J].Seienee,1995,267:1456.
[4]JULIANN G K,GEORGE C T.Heat shock protein 70 kDa:Molecular biology,bioche mistry and physiology [J].Pharmacol Ther,1998,80(2):183-201.
[5]FEDER M E,HOFMANN G E.Heat shock proteins,molecular chaperones and the stress response[J].Annu Rev Physiol,1999,61:243-282.
[6]BECHMANN R P,MIZZEN L A,WELCH W Interaction of HSP70 newly synthesized proteins:Implications for protein folding and assembly events [J]. Science,1990,248:850-854.
[7]WANG X Y,MANJLILI M H,CHEN X,et al.Development of cancer vaccines using autologous and recombinant high molecular weight stress proteins [J]. Methods,2004,32(1):13-20.
[8]Wick GAtherosclerosis-an autoimmune disease due to an immune reaction against heat-shock protein 60.Herz,2000 Mar;25(2):87-90.
[9]Lorca O,Perez-Perez J,Carrascosa JL,et a.Effects of the inter-ring communi-cation in GroEL structural and functional asymmetry.J Biol Chem,1997 Dee 26;272(52):32925-32932.
[10]Gupta S, Know Iton AA. HSP60, Bax, apoptosis and the heart. J CellMolMed,2005,9:51～58.
[11]Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes:role of the exosomalpathway. Am J Physiol Heart Circ Physio,1 2007,292:H3052～H3056.
[12]Deocaris CC, Kaul SC, Wadhwa R. On the brotherhood of the mitochondrial chaperonesmortalin and heatshock protein 60. CellStress Chaperones,2006,11: 116～128.
[13]Chen W,Syldath U,Bellmann K,et al.Human 60-kDa heat-shock protein:a danger signal to the innate immune system. J Immunol.1999, Mar15; 162 (6):3212-3219.
[14]Habich C,Baumgart K,Kolb H,et al.The receptor for heat hock protein 60 on macrophages is saturable,specific,and distinct from receptors for other heat shock proteins. J Immunol.2002 Jan 15;168(2):569-576.
[15]Barazi HO,Zhou L,Templeton NS,et al.Identification of heat shockprotein 60 as a molecular mediator of alpha 3 beta 1 integrin activation.Cancer Res,2002, Marl:62(5):1541-1548.
[16]Wick G.Atherosclerosis-an autoimmune disease due to an immunereaction against heat-shock protein 60.Herz,2000,Mar;25(2):87-90.
[17]Kerr JFR,Wyllie AH,Currie AR.Apoptosis:a bassic biological phenoenon with wideranging implications in tissue kinetics.Br J Cancer 1972;26:239-257.
[18]Stewart BW.Mechanisms of apoptosis:integration of genetic, biomedical,and cellular indications.J Natl Cancer Inst 1994; 86(17):1286-1295.
[19]Cohen JJ.Apoptosis.Immunol-Today 1993,Mar;14(3):126-130.
[20]Cappello F, Czarnecka AM, Rocca GL, et al. Hsp60 and HsplO as antitumor molecular agents. Cancer Biol Ther,2007,6:487～489.
[21]Alard JE, DueymesM, Youinou P, eta.l Modulation of endothelial cell damages by anti-Hsp60 autoantibodies in systemic autoimmune diseases. Autoimmun Rev,2007,6:438-443.
[22]Barinaga M.Is apoptosis key in alzheimer's disease? Seienee.1998 281(5381):1303-1304.
[23]Lin XY. Choi MS.Porter AG. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopoly saccharide and interferon-gamma. J Biol Chem.2000 275(51):39920-39926.
[24]Howard Y, Chang HY, Yang X. Proteases for cell suicide:functions and regulation of caspases. Microbiology Mol Biol.2000 Dec:64(4):821-846.
[25]Douglas R, Green, Reed JC. Mitochondria and apoptosis. Seience.1998 Aug:28:281(5381):1309-1312.
[26]Gotlieb RA. Mitoehondria and apoptosis. Biol Signals Recept.2001. May-Aug:10(3-4):147-61.
[27]ZouH, Yang R, Hao J, et al.Regulation of the Apaf-11Caspase9 apoptosome by Caspase-3 and XIAP. J Biol Chem.2002 Dec:27
[28]Juliann Get al. Phannacol[J]. Ther,1998,80(2):183～201.
[29]Tschopp J, Martiron F, et al. Mechanisms of caspase activation Kelly M Boatright and Guy S Salvesen Current Opinion in Cell[J].Biology,2003,15:725-731.
[30]Chang DW,Xing Z,Capacio VL,Peter ME,Yang X.Inter-dimer processing mechanism of procaspase-8 activation[J].EMBO,2003,22:4132-4142.
[31]Green DR,Reed JC.Mitochondria and apoptosis[J].Science,1998,281(5381): 1309-1312.
[32]Saikumar P,Dong Z,Mikhailov V,et al.Apoptosis:definition, mechanism, and relevance to disease[J].Am J Med,1999,107(5):489-506.
[33]Budihardjo I,Oliver H,Lutter M,et al.Biochemical pathways of caspase activation during apoptosis[J].Annau Rev Cell Dev Biol,1999,15:269-290.