【作者】 崔雪莹；

【导师】 王建祥； 马小彤； 廖晓龙；

【作者基本信息】 北京协和医学院 ， 内科学， 2010， 博士

【摘要】 1980年Craig和McCarthy首次在黑腹果蝇基因组中分离了HSP22基因,2000年Smith等人从HELA细胞和黑色素瘤细胞基因文库中发现了人HSP22基因。它由196个氨基酸组成,分子量为22 kD,具有小分子热休克蛋白(sHSP)家族保守的中央“晶体蛋白结构域(a-crystallin) "。HSP22的mRNA在肌肉、胎盘、心脏和脑组织中高度表达,目前研究显示HSP22具有分子伴侣,自身激酶等生物活性,参与调节细胞的增殖、凋亡,其异常表达或突变与心肌肥大、阿尔茨海默症及远侧遗传性运动神经病等疾病密切相关。随着研究的不断深入,HSP22与肿瘤的关系越来越受到人们的关注,近几年来发现HSP22高表达于雌激素受体阳性的乳腺癌细胞并能被17β-雌二醇(17β-Estradiol, E2)诱导上调进而促进乳腺癌的发展；然而HSP22在有些肿瘤组织或细胞系中低表达,例如黑色素瘤、前列腺癌及肉瘤,而且通过去甲基化试剂5-aza-2’-deoxycytidine(地西他滨,Aza-C,达珂TM)处理,或转导HSP22能诱导人黑色素瘤细胞系SK-MEL-2和人前列腺癌细胞系PC-3凋亡。可见HSP22在肿瘤中的作用是多样的。为了研究HSP22在血液系统肿瘤中的表达和功能,我们首先采用RT-PCR方法检测HSP22在造血系统恶性肿瘤细胞系中的表达情况。在被检测的代表不同来源的13个造血系统恶性肿瘤细胞系、20例不同类型的白血病患者骨髓单个核细胞及10例健康志愿者骨髓单个核细胞中均未发现HSP22的表达。由于热休克蛋白家族是应激蛋白,当细胞受到高温、物理或化学刺激的时候其表达会显著上升。于是我们进一步用热休克、E2、阿糖胞苷(cytarabine, Ara-c)、柔红霉素(Daunorubicin, DRN)及Aza-C等处理白血病细胞系及白血病患者骨髓单个核细胞,发现只有地西他滨能诱导HSP22表达,且呈剂量依赖。于是我们推测在HSP22基因的启动子区存在甲基化。为了证实此推测,我们用甲基化特异的PCR方法检测了5个白血病细胞系及5例健康供者和15例不同类型的白血病患者骨髓单个核细胞中HSP22基因的启动子区甲基化状态,发现检测的所有样本呈现完全甲基化状态,而且,地西他滨处理过的白血病细胞系甲基化状态被部分逆转,呈现部分去甲基化状态。地西他滨是一种去甲基化药物,具有强烈的诱导细胞分化能力,目前临床用于治疗骨髓增生异常综合征(MDS)、慢性粒细胞白血病(CML)和急性髓细胞白血病(AML),并且旨在将其应用于更多肿瘤治疗中的临床实验正在不断开展。甲磺酸伊马替尼(伊马替尼,imatinib mesylate, gleevec)是BCR/ABL+白血病最有效的治疗药物,近年来有文献证明地西他滨与伊马替尼对BCR/ABL+的细胞具有协同的抗白血病功能,增强白血病细胞对伊马替尼的敏感性。我们的研究结果提示,接受地西他滨治疗会使患者白血病细胞表达HSP22,而且多数热休克蛋白家族成员都与药物的敏感性相关,那么HSP22的表达会对白血病细胞产生什么作用,是否参与介导了地西他滨增强白血病细胞对伊马替尼的敏感性呢?为了进一步探究HSP22对白血病细胞的影响,我们构建了HSP22慢病毒表达载体,感染K562和Namalwa细胞,荧光显微镜及流式细胞仪观察并测试瞬时感染效率,RT-PCR和Western Blot方法鉴定HSP22的表达。并通过生长曲线、细胞周期、集落形成实验来评价肿瘤细胞增殖、生长特性、集落形成能力,AnnexinV/7-AAD方法检测肿瘤细胞凋亡情况。结果发现,与转导空载体的对照细胞相比,HSP22能抑制K562细胞体内增殖和Namalwa细胞体外增殖,并抑制二者集落形成,但对于二者的细胞周期和凋亡没有明显影响。为了进一步探究HSP22是否可使白血病细胞对伊马替尼的的敏感性增强,是否参与介导了地西他滨与伊马替尼的协同作用,我们用伊马替尼处理转导HSP22和空载体的K562 (BCR/ABL+)细胞,并用AnnexinV/7-AAD方法检测细胞凋亡情况,发现HSP22能增强伊马替尼诱导的K562细胞凋亡,此现象具有剂量和时间依赖性。为了进一步明确HSP22增加肿瘤细胞对伊马替尼敏感性的机制,我们采用real-time PCR方法检测了转导HSP22和空载体的K562细胞mdr1和BCR/ABL基因的表达,结果显示转导HSP22使K562细胞中这两种基因的表达水平显著降低,提示HSP22增加肿瘤细胞对伊马替尼敏感性的作用可能是通过降低P-糖蛋白(P-glycoprotein, P-gp)转运体的功能从而减少伊马替尼的泵出以及减少BCR/ABL融合蛋白的表达从而降低其酪氨酸激酶的活性来实现的。由于地西他滨能够诱导多种基因表达,为了进一步确定HSP22是否在介导地西他滨与伊马替尼的协同抗白血病功能中起到特异的作用,我们在地西他滨诱导野生型K562细胞表达HSP22的基础上使用HSP22基因特异的siRNA干扰其表达,再使用伊马替尼处理,发现干扰HSP22的表达能降低伊马替尼诱导的K562细胞凋亡。提示HSP22可能在介导地西他滨与伊马替尼的协同作用中发挥特异的作用。综上所述,我们的工作首次证明,在血液系统中,启动子区甲基化是HSP22不表达的原因,去甲基化试剂地西他滨能够诱导HSP22的表达。异位表达HSP22能抑制白血病细胞的增殖和集落形成,并能增强BCR/ABL+的K562细胞系对伊马替尼的敏感性。而且此功能可能是通过降低P-糖蛋白(P-glycoprotein)转运体的功能和减少BCR/ABL融合蛋白的表达来实现的。RNA干扰实验显示降低HSP22的表达能减少伊马替尼诱导的K562细胞凋亡,提示HSP22可能在介导地西他滨与伊马替尼的协同作用中发挥特异的作用。更多还原

【Abstract】 Hsp22 gene was isolated by Craig and McCarthy from the Drosophila melanogaster genome in 1980. Then Smith et al. detected the HSP22 gene from the HeLa and melanoma cDNA libraries in 2000. Hsp22 (also known as HSPB8, H11, and E2IG1) was categorized as a member of the superfamily of sHSPs based on the presence of the conserved a-crystallin domain in this protein. It is composed of 196 amino acids with a calculated molecular mass of 21.6 kDa. Analysis of abundance of hHsp22 mRNA shows the highest expression of human Hsp22 was found in skeletal and smooth muscles, placenta, heart, and brain. Recent studies have demonstrated HSP22 displays chaperone activity and autokinase activity and was involved in regulation of cell proliferation, apoptosis. Abnormal expression of HSP22 correlates with development of cardiac hypertrophy, Alzheimer’s disease and different neuromuscular diseases. Hsp22 is also involved in tumors and its expression is cell-type-specific and can be altered in human tumors. Recently, it was found that overload of HSP22 triggers melanoma cell apoptosis but contribute to breast carcinogenesis and progression. The mRNA and protein levels of Hsp22 were reduced in various tumor tissues and cell lines (i.e., prostate cancer, sarcoma) relative to their normal counterparts and the percentage of apoptotic cells increased with time after Hsp22 induction.In these cells, expression was restored by the demethylating agent 5-aza-2-deoxycytidine (decitabine, Aza-C, DacogenTM) suggesting that Hsp22 expression was inhibited by aberrant DNA methylation. To find its role in leukemia, we examined the expression of HSP22 by RT-PCR both in 13 human hematopoietic malignant cell lines and bone marrow samples of 20 patients and 10 healthy volunteers, however no expression was found. Then we tried cytarabine (Ara-c), Daunorubicin (DRN),17B-estradiol (E2) and decitabine to induce HSP22 expression, the results showed only 5-aza-2’-deoxycytidine worked and it was does-dependent. Then we tested the genomic DNA in 5 human hematopoietic malignant cell lines and bone marrow samples of 15 patients and 5 healthy volunteers and found DNA methylation in the promoter region of HSP22 gene, using the MSP method. Lentivector expression system-mediated delivery of HSP22 into K562 and Namalwa cells can inhibit proliferation of Namalwa in vitro and K562 in vivo. HSP22 transduction can also inhibit colony forming of K562 and Namalwa cells. However there was no significant difference in cell cycle and apoptosis.Aza-C is an effective antileukemic agent because of its reactivation of several types of tumor (growth) suppressor genes and tumor-associated antigen genes. The selective BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate is now frontline therapy for chronic myeloid leukemia (CML). Recently published data indicate that Decitabine and imatinib mesylate have a synergistic antitumor activity, and decitabine may be a potential candidate to overcome imatinib resistance in patients with CML. In this study, our results suggest decitabine treated leukemic cells may express HSP22. To detect the fuction of HSP22 in leukemia and to find if HSP22 is involved in the synergistic antitumor activity of decitabine and imatinib mesylate, we treated the transducted K562 (BCR/ABL+) cells with imatinib mesylate and found that HSP22 can enhance imatinib mesylate-induced apoptosis. To figure out the mechanism of enhanced apoptosis of K562 cells by HSP22, we detected the expression of mdrl and BCR/ABL gene by real-time PCR. Our results show that HSP22 can significantly reduce the expression level of mdrl and BCR/ABL gene, suggesting that HSP22 may enhance imatinib mesylate-induced apoptosis through down-regulation P-glycoprotein and Bcr/Abl levels.To investigate the involvement of HSP22 in the synergistic antitumor activity of decitabine and imatinib mesylate, wild type K562 cells were treated with decitabine to induce HSP22 expression, then cells were transfected with HSP22-specific siRNA and Non targeting siRNA, respectively. Compared to Non targeting siRNA, Anti-HSP22 siRNA treatment reduce imatinib induced apoptosis of K562 cells. These findings further support the hypothesis that HSP22 could be involved in mediating the synergistic antitumor activity of decitabine and imatinib mesylate.In conclusion, our work firstly demonstrated that the promoter methlation is the reason for HSP22 silence in hematopoietic system. The effective demethlating agent decitabine can induce HSP22 expression. Ectopic HSP22 expression inhibits the proliferation and colony forming of leukemic cells. HSP22 can enhance imatinib mesylate induced apoptosis through down-regulation P-glycoprotein and Bcr/Abl levels in K562 cells. HSP22 knockdown can reduce imatinib induced apoptosis of K562 cells, suggesting that HSP22 could be involved in mediating the synergistic antitumor activity of decitabine and imatinib mesylate.更多还原