【作者】 房孝生；

【导师】 王欣；

【作者基本信息】 山东大学 ， 肿瘤学， 2009， 博士

【摘要】 淋巴瘤是血液科常见疾病,占每年新发肿瘤病例的5%,肿瘤死亡病例的3%。近30年来其发病率增高了2倍,导致这种情况的原因尚不明确。在所有的恶性淋巴瘤中非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL)约占80%。多数NHL治疗效果较霍奇金淋巴瘤(Hodgkin’s lymphoma,HL)差。联合化疗及美罗华等靶向治疗虽然可以使NHL生存期明显延长,但仍有部分患者复发、耐药并最终死亡。因此迫切需要对NHL发病机理进行更深入研究,以期发现更有效的治疗手段和措施。热休克蛋白(Heat shock proteins,HSPs)广泛存在于多种细胞内,在热刺激、缺氧、创伤、氧化应激、药物等各种应激诱导下表达增加。通过参与细胞内蛋白质的折叠、伸展、转运和跨膜等过程HSPs发挥其“分子伴侣”功能,使细胞应对应激的能力增强。HSPs还具有抗凋亡的作用,它可以抑制凋亡通路中的多种蛋白,从而在凋亡通路的不同阶段阻断凋亡的发生。此外HSPs与肿瘤免疫也有密切的相关,研究表明HSPs能加强肿瘤的免疫原性,参与呈递肿瘤抗原,激发肿瘤免疫反应。由于在多种肿瘤细胞及组织中有高水平表达,并且与肿瘤发生、发展、生物学行为及预后有密切的关系,HSPs可以作为判断病情和预测预后的指标。部分研究还将HSPs作为肿瘤治疗的靶点。在体外实验中应用反义寡核苷酸或siRNA特异性抑制HSPs的表达,可以诱导肿瘤细胞凋亡和增强化疗敏感性。HSP90的抑制剂17-AAG在一期临床实验中显示了良好的疗效,目前已经进入二期临床实验。上述以HSPs为靶点的治疗为我们更好的治疗恶性疾病提供了新思路。在多数肿瘤中HSPs高表达是分期晚、治疗效果不佳和预后差的标志,但是HSPs在NHL中的相关表达通路及与疾病的生物学行为之间的关系目前仍不明确。本研究应用分子生物学技术探讨HSPs与淋巴瘤临床表现、恶性程度及治疗效果之间的相关性,并通过体外实验分析NHL Raji细胞中HSP70作用的相关通路和机理,希望对淋巴瘤的生物学特征有更深入的了解,为疾病的诊断和治疗服务。论文一PI3K/AKT通路在热处理诱导Raji细胞表达HSP70中的作用目的:热疗和化学治疗以及放射治疗联合可以用来治疗多种肿瘤。但是热疗的应用价值受到许多因素的影响,其中之一就是热休克反应。热休克反应是细胞内高度保守的分子应激反应,在这一过程中热休克蛋白(Heat Shock Proteins,HSPs)表达增加。而HSPs可以保护细胞免于一系列化学、物理和生物应激原的损伤。热休克蛋白70(Heat Shock Protein 70,HSP70)是热休克蛋白家族最主要的成员之一。研究表明HSP70对凋亡有抑制作用,HSP70表达增加可以减少化疗药物及射线诱导的肿瘤细胞凋亡。因此热处理诱导的HSP70表达增高,可能会对抗肿瘤治疗产生不良影响。HSP70的表达主要受热休克因子1(Heat shock factor 1,HSFl)的调控。最新研究显示PI3K可以影响HSFl的活性,进而影响HSP70的表达。抑制PI3K/AKT通路后,细胞内HSP70表达降低。因此PI3K/AKT通路可能成为一个新的抑制HSP70表达的靶点。本研究利用LY294002(一种PI3K抑制剂)抑制PI3K/AKT通路,以探讨该通路在热处理诱导Raji细胞表达HSP70过程中的作用,并检测热处理诱导的HSP70表达对Raji细胞凋亡和化疗敏感性的影响。方法:根据细胞处理的不同方式,实验分为3组:1)对照组:Raji细胞不经过热处理,直接加用化疗药物作用;2)热处理组:Raji细胞首先经42℃热处理,随后在37℃培养4h,然后加入化疗药物作用;3)LY294002预处理后加热处理组:细胞首先经LY294002预处理,随后按照2组的方法处理。用Western blotting分析不同处理后HSP70,AKT和P-AKT的表达。用AnnexinV-PI双染来检测细胞凋亡率的变化。用WST-8法检测ADM和DDP对Raji细胞的毒性。结果:1 PI3K抑制剂抑制热处理诱导的HSPTO表达42℃热处理后2小时即有HSP70的表达增加。热处理后8小时,Raji细胞内HSP70表达达到最高峰。在加热处理后24小时,HSP70表达仍然高于对照组。热处理后4小时HSP70表达明显增加,同时伴有AKT活化(P＜0.05)。而如果在热处理前用LY294002预处理细胞,则HSP70表达及AKT活化明显减弱(P＜0.05)。2热处理使Raji细胞抵抗DDP诱导的细胞凋亡42℃热处理后24小时,Raji细胞凋亡率从6.44±0.64%上升到6.95±0.57%,差异无统计学意义(P＞0.05)。用5ug/ml和10ug/ml DDP单独处理Raji细胞24小时,分别诱导了14.1%±0.5%和16.9%±1.14%的凋亡率。而如果在热处理后应用DDP,则凋亡率下降到9.73%±0.28%和10.50%±0.85%(P＜0.01)。3 PI3K抑制剂可以抵消热处理对Raji细胞的保护作用热处理前分别应用10umol和20umol的LY294002来抑制HSP70的表达,观察细胞凋亡率的改变。与未用LY294002相比,应用LY294002后5ug/ml DDP诱导的凋亡率有了明显的升高。10umol和20umolLY294002分别将凋亡率从9.73%±0.28%提高到15.33%±0.45%和19.5%±0.75%,差异有统计学意义(P＜0.01)。4 PI3K抑制剂增强了热处理后Raji细胞对ADM和DDP的敏感性在经42℃加热预处理后,Raji细胞对ADM的敏感性明显下降,IC50从5.2ug/ml上升到17.0ug/ml。而如果在热处理前应用LY294002处理细胞,则可以显著增强ADM的敏感性,并且呈计量依赖性。10umol、20umol和40umolLY294002分别使ADM的IC50下降至10.6 ug/ml、8.8ug/ml和3.6ug/ml。应用DDP进行实验,趋势与上述结果一致。结论:1.本研究首次证实在热诱导的Raji细胞内存在PI3K/AKT/HSP70表达通路。PI3K抑制剂LY294002可以抑制热处理诱导的HSP70表达。本研究为抑制Raji细胞中热诱导的HSP70表达提供了新思路。2.适当温度的热处理对Raji细胞有保护作用。当热处理诱导的HSP70表达被抑制后,该保护作用减弱甚至消失。因此热处理对Raji细胞的保护作用可能是通过诱导HSP70表达来实现的。论文二热休克蛋白70、90α在非霍奇金淋巴瘤中的表达及临床意义目的:HSP70和HSP90是热休克蛋白家族中两个最重要同时也是研究最深入的成员。多种肿瘤组织中有HSP70和HSP90的表达,并且与肿瘤的分期和预后相关。但是在不同肿瘤中HSP70和HSP90所体现的意义不尽相同,部分研究的结果甚至是相互矛盾的。这可能与肿瘤的特异性有关。有研究表明在淋巴瘤细胞中有HSP70和HSP90表达,但未能明确两者表达与淋巴瘤临床表现和治疗效果的关系。我们通过免疫组织化学技术检测两种热休克蛋白HSP70和HSP90α在NHL、坏死性淋巴结炎和淋巴结反应性增生组织中的表达并探讨其意义。方法:选取资料完整的NHL病例30例;年龄6～84岁,平均年龄43.6岁。所有标本均经两位病理学专家重新阅片证实。按Ann Arbor分期法对NHL进行分期。参照美国国立癌症研究所工作分型(1982年)进行病理分型。10例坏死性淋巴结炎和12例淋巴结反应性增生组织作为对照。用免疫组化法检测HSP70和HSP90α在三种组织中的表达。HSP70及HSP90α均以细胞核或胞浆黄染为阳性。计算阳性细胞百分数:≤5%为(-),6～25%为(+),26～50%为(++),＞50%为(+++);(+++)为高表达。结果:HSP70、90α均表达于NHL细胞胞浆和胞核。在NHL、坏死性淋巴结炎和淋巴结反应性增生组织中两种热休克蛋白均有高水平的表达,但差异无统计学意义(P＞0.05)。HSP70和HSP90α阳性表达与NHL恶性程度,分期,有无B症状,是否累计结外及治疗效果均无相关性(P＞0.05)。HSP70高表达(阳性率大于50%)与NHL恶性程度,分期及治疗效果密切相关(P＜0.05),与是否有B症状、是否累及结外无关(P＞0.05)。HSP90α高表达与上述指标无相关性(P＞0.05)。结论:1.本研究首次应用免疫组化技术检测了HSP70在NHL中表达情况,并证实在NHL组织中HSP70的高表达率与NHL恶性程度、分期及治疗效果相关。因此HSP70可以作为判断NHL病情和治疗效果的指标。2.首次明确了国内NHL患者亦有高水平HSP90α表达,但是HSP90α表达与NHL恶性程度、分期、B症状、节外浸润及治疗效果无相关性。3.HSP70及HSP90α在NHL中均有高水平表达,为在NHL中开展针对性靶向治疗提供了理论依据。更多还原

【Abstract】 Lymphoma is one of the very common diseases in hematology department and accounts for 5%of new cancer cases and 3%of cancer deaths.Its morbidity has increase approximately twofold over the last 30 years,the causes are still not clear. non-Hodgkin’s lymphoma(NHL) accounts for 80%of all lymphomas and has a more aggressive course than Hodgkin’s lymphoma(HL).Combined chemotherapy and immunotherapy like Rituximab had prolonged its survival,but few patients still suffered chemotherapy resistance and relapsed.So it is necessary to explore the pathogenesis of NHL and discovery more effective therapeutic methods.Heat shock proteins(HSPs) are widely expressed in many kinds of cells and elevated under stress conditions such as:heat shock,hypoxia,trauma,oxidative reaction and some kinds of drugs.In cells,HSPs assist the refolding,extension, transport and transmembrane of proteins,the function of HSPs has been named "molecular chaperon".HSPs also can inhibit apoptosis at different stages of apoptosis pathway.In addition,it closely relates to tumor immunity.Studies had indicated that HSPs could increase the tumor immunogenicity,present tumor antigen and contribute tumor immunity reaction.It had been proved that HSPs was abundantly expressed in many tumor cells and was correlated to the progress,biologic behavior and prognosis of tumors.Clinical researches had found HSPs to be a useful marker for the estimation of disease and predication of prognosis.HSPs had also been conducted as a therapy target.Some experiments had tried antisense oligonucleotides and siRNA to inhibit HSPs,and induced apoptosis and chemotherapy sensitivity.The exact relationship between HSPs and lymphoma is still not clear.Our research explored the correlation between HSPs and clinical manifestation of NHL by molecularbiology technology and studied the possible pathways of HSPs activation in Burkitt’s lymphoma Raji cell in vitro.All these works may be useful for the understanding of lymphoma and afford us new method for diagnosis and therapy.PartⅠPI3K/AKT Pathway is Needed for Hyperthermia Induced HSP70 Expression in Raji CellPurpose:Hyperthermia has been applied as an adjuvant therapy together with chemotherapy and radiotherapy.However,the full potential of hyperthermia is hindered by few considerations,one of this is heat shock response.Heat shock response is a highly conserved molecular stress response that is involved in the induction of heat shock proteins(HSPs).Hsp70 is one of the most well known and best-studied members of HSPs familiy.It protects cells against a variety of chemical, physical and biological stressors.So the efficiency of anticancer therapy may be diminished by hyperthermia induced HSP70 expression.The activation of HSF1 plays a key role in the induction of HSP70.Recent studies had shown that HSF1 could be activated via PI3K/AKT pathway.After PI3K/AKT pathway was inhibited,the expression of HSP70 also decreased.Therefore PI3K/AKT pathway might be a new target for the inhibiting of HSP70.This study examined the potential relationships between HSP70 expression and PI3K/AKT pathway in Raji cells,and the protection function of hyperthermia pretreatment.Method:According to the different sequences of treatments,the experiment had been divided into 3 groups:1) Raji cells were only treated with chemotherapy agents;2) Raji cells were first treated with hyperthermia and followed by chemotherapy agents; 3) Raji cells were pre-incubated with LY294002,then hyperthermia and chemotherapy agents were followed.The expressions of HSP70,AKT and P-AKT were detected by Western blotting.The apoptosis indexes were analyzed by Annexin V-PI double staining.The cytotoxicity of ADM and DDP were examined by WST-8. Results:1 LY294002 inhibits hyperthermia induced HSP70 expressionHyperthermia rapidly increased HSP70 expression within 2 hours.And HSP70 expression reached a maximum 8 hours after hyperthermia.24 hours later,the expression of HSP70 was still higher than untreated control cells.AKT was also activated after hyperthermia treatment.In the presence of 10umol or 20umol LY294002 before hyperthermia,HSP70 expression and the activation of AKT were drastically attenuated compared with controls(P＜0.05).2 Preheated Raji cells acquire tolerance to DDP induced apoptosis24 hours after hyperthermia,the apoptotic Raji cells increased from 6.44±0.64%to 6.95±0.57%,with no significant difference(P＞0.05).The ratio of apoptotic preheated Raji cells was 9.73±0.28%and 10.50±0.85%after treated with 5ug/ml or 10ug/ml DDP,which was significantly lower than non-preheated cells(14.1±0.5% and 16.9±1.14%)(P＜0.01).3 PI3K inhibitor increases the apoptosis index in preheated Raji cellsLY294002 was used to inhibited HSP70 expression before hyperthermia.After incubated with 10umol LY294002,5ug/ml DDP induced apoptosis index increased from 9.73±0.28%to 15.33±0.45%(P＜0.01) in preheated Raji cells.And 20umol LY294002 elevated the apoptosis index from 9.73±0.28%to 19.5±0.75%(P＜0.01).4 Effects of LY294002 and hyperthermia on cytotoxicity of ADM and DDPRaji cells were more resistant to ADM treatment after hyperthermia treatment,IC50 increased from 5.2ug/ml to 17.0ug/ml.If treated with LY294002 before hyperthermia, Raji cells’ sensitivity increased in a dose depended manner.10 umol,20 umol and 40umol LY294002 decreased IC50 to 10.6 ug/ml,8.8ug/ml and 3.6ug/ml.When the cells were treated with DDP,LY294002 also inhibited the protection of hyperthermia.Conclusion:1.Our study primarily demonstrated the existence of PI3K/AKT/HSP70 pathway in Raji cell.The result might afford a new strategy for the inhibition of HSP70 expression in Raji cell. 2.Hyperthermia treatment at 42℃could protect Raji cell against DDP induced apoptosis.The protection of hyperthermia might partly due to the expression of HSP70.PartⅡExpressions of HSP70、90αin non-Hodgkin’s lymphoma and its clinical significancePurpose:HSP70 and HSP90 are two of the most important and best studied members of heat shock protein family.Stduies have found HSP70 and HSP90 expressed in many kinds of tumors and their expressions were correlated with stages and prognosis. But HSP70 and HSP90 expressions indicated different significances in different tumors,some of the results even contradicted completely.The possible reason may due to the specificity of tumors.HSP70 and HSP90 were highly expressed in NHL, but their clinical significances were not clearly indicated.Our research detected the expressions of HSP70 and HSP90αin NHL by immunohistochemistry and explored their clinical significances.Methods:NHL tissues were collected from 30 NHL patients with an average age of 43.6 years(6～84years).All the pathology samples were reconfirmed by two pathologists.Staging was according to Ann Arbor classification and pathology typing was refered to American National Cancer Institute Work Group classification(1982). 10 necrotic lymphnoditis and 12 reactive hyperplastic lymphnode were used as controls.The expressions of HSP70 and HSP90αwere detected by immunohistochemistry.If the nucleus or cytoplasma were stained yellow,the cell was considered positive.The percentage of positive cells were calculated as fellows:≤5% (-),6～25%(+),26～50%(++),＞50%(+++);(+++) was also considered as high expression.Results:HSP70 and HSP 90αexpressions located in nucleus and cytoplasma.They were highly expressed in NHL,necrotic lymphnoditis and reactive hyperplastic lymphnode.There were no significant differences in the expressions of HSP 70 and HSP90αbetween NHL,necrotic lymphnoditis and reactive hyperplastic lymphnode (P＞0.05).The positive expression of HSP70 and HSP90αdid not correlate with the degree of malignancy,staging,therapy reaction,B syndrome and extra-lymphonode infestation(P＜0.05).High expression of HSP70(postive cell＞50%) was correlate with the degree of malignancy,staging and therapy reaction(P＜0.05),but was not relate to B syndrome and extra-lymphonode infestation(P＞0.05).None of such relationship was found between the high expression of HSP90αand clinical manifestation(P＞0.05).Conclusion:1.Our study measured HSP70 expression in NHL using immunohistochemistry for the first time and indicated high expression of HSP70 was correlated with the degree of malignancy,stage and therapy reaction.So HSP70 may be a useful indicator for the evaluation of malignancy,stage and therapy reaction of NHL.2.We also primarily demonstrated HSP90αwas highly expressed in china NHL patients,though its expression was not related with clinical manifestation and therapy reaction.3.The high expressions of HSP70 and HSP90αmight indicate them to be good therapy targets for NHL.更多还原