【作者】 王玲；

【导师】 王欣；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 在发达国家,非霍奇金淋巴瘤(non-Hodgkin lymphoma, NHL)是常见的血液恶性肿瘤之一,约占恶性肿瘤的4%；而弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma, DLBCL)是其最常见的一种亚型,约占40%。同样,在发展中国家,DLBCL也是最常见的肿瘤之一。DLBCL是一组在细胞遗传学、免疫表型和临床特征方面均存在很大异质性的肿瘤。虽然利妥昔单抗和联合化疗能提高DLBCL患者的疗效,但仍有部分患者耐药或复发。因此,寻求新的治疗方案迫在眉睫。中药在我国用于治疗恶性肿瘤历史悠久,并且取得了一定的临床疗效。传统中药砷剂包括二硫化二砷(arsenic disulfide,As2S2)、四硫化四砷(tetra-arsenic tetra-sulfide,As4S4)和三氧化二砷(arsenic trioxide, As2O3)。1996年,上海血液病研究所首次阐明了As203可诱导急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)细胞分化和凋亡,从而引起了国内外学者对砷剂治疗血液恶性肿瘤疾病的关注。二硫化二砷是雄黄的主要成分之一,无论是在中医还是在西医的治疗均历史悠久。基础研究证实,雄黄同样可诱导ATRA敏感的细胞株NB4和ATRA耐药的细胞株MR2发生凋亡和分化。进一步研究发现雄黄诱导HL-60、NB4和K562细胞凋亡的机制是通过抑制端粒酶活性；减少BCL-2和PNAS-2的表达；并且与MAPK通路相关连。同时,雄黄诱导HL-60细胞的分化不仅与丝氨酸、苏氨酸蛋白磷酸酶1型和2A型的活性的增强,而且通过线粒体膜电位的增强来实现的。而且,砷剂诱导非霍奇金淋巴瘤细胞凋亡机制多样。在人类Burkitt淋巴瘤细胞株Raji和人类T细胞淋巴瘤细胞株Jurkat中的研究中发现,三氧化二砷通过阻滞细胞周期、减少呼吸功能和线粒体膜电位,下调凋亡相关基因MCL-1进而活化caspase-3的途径来抑制增长和诱导凋亡的。进一步研究发现,Jurkat细胞和Raji细胞对砷剂诱导凋亡的敏感性存在差异,因为Jurkat细胞表达高水平的谷胱甘肽S-转移酶P1-1(glutathione S-transferase P1-1, GSTP-1)。同样,人T细胞淋巴瘤细胞株CEM研究中发现,雄黄诱导凋亡的机制是阻滞细胞停滞于G2/M期、下调Bcl-2和增加AP02.7蛋白的表达。最新研究证实三氧化二砷通过减少细胞周期蛋白D1的表达和上调凋亡相关基因的机制来抑制人滤泡淋巴瘤细胞(mantal cell lymphoma, MCL)的生长和诱导其凋亡。为了增强治疗效果并减少副作用,常采用联合化疗方案治疗肿瘤。复方黄黛片就是采用雄黄作为君药,而青黛和太子参作为臣药而组成的中药复方制剂,并且也证实其治疗APL效果显著。进一步的研究发现雄黄和青黛的有效活性成分是硫化砷(As2S2或AS4S4)和靛玉红。基础研究证实雄黄对人T细胞淋巴瘤细胞株(CEM-T)和B细胞淋巴瘤细胞株(Raji)有抑制增长和诱导凋亡作用。而且靛玉红对正正常淋巴细胞和恶性淋巴细胞,如T细胞淋巴瘤细胞株(Jurkat, CEM-T)和B细胞淋巴瘤细胞株(IM9, Reh6)均有抑制增长和诱导凋亡作用。有关AS2S2和靛玉红单药或联合对DLBCL细胞株的增殖和凋亡的影响未见提及。本研究以人DLBCL细胞株LY1和LY8作为研究对象,研究As2S2和靛玉红单药或联合对DLBCL细胞增殖和凋亡的影响,并初步探讨了其凋亡机制,为临床发掘新的联合治疗方案提供有力的理论支持。第一部分二硫化二砷诱导弥漫大B细胞淋巴瘤细胞凋亡及机制探讨目的：DLBCL是NHL中最常见的一种亚型,约占40%。DLBCL是一组在细胞遗传学、免疫表型和临床特征方面均存在很大异质性的肿瘤。虽然CD20单克隆抗体利妥昔单抗和联合化疗能提高DLBCL患者的疗效,但仍有部分患者耐药或复发。因此,寻求新的治疗方案成为研究的重点。目前含砷中药的抗肿瘤特点成为研究的热点。本研究选择了人DLBCL细胞株LY1和LY8,与As2S2在体外共培养,进行细胞增殖和凋亡分析,并探讨了其凋亡机制。材料和方法：1. DLBCL细胞株LY1和LY8与As,2S2共培养2.CCK-8测定细胞增殖3.流式细胞术测定细胞凋亡4.提取RNA,进行实时定量PCR5.蛋白提取和蛋白印迹分析6.统计学分析结果：1.CCK-8结果显示：随着As2S2作用时间的延长,DLBCL细胞活性逐渐减弱；同时药物浓度越高,细胞活性越低。其差异均有统计学意义。2.流式结果显示：As2S2诱导细胞凋亡效应呈现时间和剂量依赖性,其差异均有统计学意义。3.实时荧光定量结果显示：与对照组比较,10μMAs2S2与DLBCL细胞共孵育48h,Caspase-3mRNA的表达上升,Bcl-2mRNA的表达下降,Bax mRNA的表达上升,Bax/Bcl-2的比例升高(P<0.05)。4. Western blot结果显示：与对照组比较,10μM As2S2与DLBCL细胞共孵育48h, Procaspase-3蛋白的表达下降,Bcl-2表达下降,Bax表达上升,Bax/Bcl-2的比例升高(P<0.05),并且21-KDa Bax断裂成为18-KDa Bax。结论：1.As2S2以浓度和时间依赖的方式抑制DLBCL细胞生长和诱导凋亡。2.As2S2可能是通过线粒体凋亡途径来诱导DLBCL细胞凋亡,As2S2诱导凋亡过程中伴随着Bax断裂。第二部分靛玉红增强二硫化二砷诱导弥漫大B细胞淋巴瘤细胞凋亡作用及机制探讨目的：为了增强治疗效果并减少副作用,常采用联合化疗治疗肿瘤。复方黄黛片是采用硫化砷作为君药,靛玉红作为臣药的一种中药复方制剂。靛玉红及其衍生物早期用于治疗慢性粒细胞白血病的疗效得到肯定。随着靛玉红及其衍生物抗肿瘤活性的发掘,近年来,靛玉红也成为了肿瘤研究的热点。本实验研究了靛玉红单药或联合As2S2对DLBCL细胞株LY1和LY8的作用,探讨了靛玉红对AS2S2的增强诱导凋亡的机制,有可能为临床发掘新的联合治疗方案提供有力的理论支持。材料和方法：1.靛玉红单药或联合AS2S2与DLBCL细胞株LY1和LY8共培养2.CCK-8测定细胞增殖3.流式细胞术测定细胞凋亡4.提取RNA,进行实时定量PCR5.蛋白提取和蛋白印迹分析6.统计学分析结果：1.CCK-8结果显示：靛玉红单药与DLBCL细胞共培养,随着作用时间的延长和药物浓度的增加,DLBCL细胞活性无变化。2.随着靛玉红浓度的增加,DLBCL细胞与靛玉红共培养48h,细胞凋亡率无变化。3.与As2S2组比较,靛玉红联合As2S2组DLBCL细胞活性明显下降；凋亡率明显增高,差异有统计学意义。4.实时荧光定量结果显示：与10μMAs2S2组比较,20μM靛玉红联合10μMAs2S2组caspase-3、Bcl-2和Bax的表达在mRNA水平无差异(P>0.05)。5. Western blot结果显示：与10μMAs2S2组比较,20u M靛玉红联合10μMAs2S2组procaspase-3的表达下降,Bax/BCL-2的表达升高,凋亡过程中伴随着Bax断裂。结论：1.靛玉红单药对DLBCL细胞株无抑制增殖和诱导凋亡作用2.靛玉红可增强As2S2对DLBCL细胞株的促凋亡作用,因此,As2S2是作为君药而靛玉红作为臣药发挥抗肿瘤活性的。3.靛玉红增强As2S2对DLBCL细胞株的促凋亡作用可能是通过线粒体凋亡途径；并伴随着Bax断裂。更多还原

【Abstract】 NHL is the most common hematological neoplasm in the United States, and accounts for4%of all malignant tumors diagnosed each year. DLBCL is the most prevalent subtype of NHL worldwide and represents up to40%of all NHL cases among adults in the western world, and frequent in developing countries. DLBCL is the heterogeneous disease with variable cytogenetics and immunophenotype, as well as clinical features. Although the initial standard therapy with rituximab in combination with CHOP (R-CHOP) chemotherapy has improved outcomes for patients with DLBCL, there are still some patients who are refractory to initial treatment or relapse after standard therapy. Therefore, the discovery of novel and alternative therapeutic approaches is urgent.In ancient China, traditional Chinese medicine (TCM) has been used to treat malignant tumor and has been proven to be effective for a long time. And arsenic included As2S2, As4S4and As2O3. In1996, As2O3was been firstly used to induce APL differentiation and apoptosis by the institutes of Shanghai hematonosis. Consequently, arsenic drugs have become a’hot topic’and have attracted increased attention in regards to malignant hematological neoplasm.Realgar has been found to induce both apoptosis and differentiation simultaneously in ATRA-sensitive NB4and ATRA-resistant MR2PML-RARa+APL cell lines. Previous studies demonstrated that realgar induced apoptosis of HL-60, NB4and K562cell lines, which was associated with CD95/CD95L and MAPK pathway, inhibition of telomerase activity and decreased expression of BCL-2and PNAS-2. In addition, realgar induced the differentiation of HL-60cell line via not only the enhancement of the activity of serine/threonine protein phosphatase typel (PP1) and type2A (PP2A) but also oxidative stress and stress-related mitochondrial transmembrane potential (MTP).In addition, previous studies have demonstrated that arsenic induced apoptosis among in NHL cells by means of different mechanisms. They revealed that As2O3inhibited proliferation and induced apoptosis in the human Burkitt lymphoma cell line Raji and in the human T lymphoma cell line Jurkat through cell cycle arrest, decrease in respiratory function and MTP, downregulating the expression of MCL-1and subsequently activating caspase-3. However Jurkat cells were less sensitive to As2O3-induced apoptosis than Raji cells, as Jurkat cells express high levels of glutathione S-transferase P1-1(GSTP1-1). At the same time, realgar induced apoptosis of human T lymphocyte leukemia cell line CEM apoptosis through cell cycle arrest in the G2/M phase, a decrease in the expression of Bcl-2and an increase in APO2.7protein expression. A recent study demonstrated that As2O3inhibited the growth of mantle cell lymphoma (MCL) and inducesd apoptosis through a decrease in cyclinD1expression and increase in the expressin of apoptosis-related molecules.To enhance therapeutic efficacy and reduce adverse effects, it is common to combine multiple drugs to deal with diseases. Realgar-Indigo naturalis formula (RIF) was been constituted with realgar as a sovereign drug, whereas Indigo naturalis and Salvia miltiorrhiza as minister drugs. And further research demonstrated that the components of RIF were realgar and indigo with arsenic sulfide and indirubin as major ingredients. Realgar was known to be able to induce apoptosis in human T cell lymphoma cell (CEM) and human B cell lymphoma (Raji). Furthermore, indirubin was proven to induce apoptosis in human T cell lymphoma cell (Jurkat, CEM) and human B cell lymphoma (IM9, Reh6).But there was no study on whether indirubin and As2S2alone or in combination had effects in DLBCL cells. For the first time, our present manuscript focuses on indirubin and As2S2alone or in combination on the proliferation and apoptosis of DLBCL cell lines and its mechanism in an attempt to seek a more effective combination therapy scheme for DLBCL. Part Ⅰ Arsenic Disulfide Induces Apoptosis of Human Diffuse Large B-Cell Lymphoma Cells and The MechanismObjective:DLBCL is the most prevalent subtype of NHL worldwide and represents up to40%of all NHL cases among adults in the western world, and frequent in developing countries. DLBCL is the heterogeneous disease with variable cytogenetics and immunophenotype, as well as clinical features. Although the initial standard therapy with rituximab in combination with CHOP (R-CHOP) chemotherapy has improved outcomes for patients with DLBCL, there are still some patients who are refractory to initial treatment or relapse after standard therapy. Therefore, the discovery of novel and alternative therapeutic approaches is urgent. Currently, arsenic drugs have become a’hot topic’and have attracted increased attention in regards to malignant hematological neoplasm and other solid tumors. To investigate the effect of As2S2on the proliferation and apoptosis of DLBCL cell lines LY1and LY8in an attempt to find potential mechanism.Materials and Methods:1. Co culture with DLBCL cells and As2S22. CCK-8method to detect cell proliferation3. Flow cytometry to evaluate cell apoptosis4. RNA extraction and RT-PCR5. Protein extraction and western blot analysis6. Statistical analysisResults:1. We found that the DLBCL cells viability significantly decreased by As2S2at24h,48h and72h. Along with increasing As2S2concentration, the DLBCL cells viability was notably reduced compared with the control group, and was statistically significant (P<0.05).2. The apoptotic rates of DLBCL cells were significantly increased at24h,48h and72h with increasing As2S2concentration, and were statistically significant (P<0.05). 3. The quantitative real-time PCR results showed that the expression levels of Bax/Bcl-2ratio and caspase-3mRNA were up-regulated in As2S2-treated DLBCL cells.4. Western blot revealed that As2S2could down-regulate the expression of procaspase-3and up-regulate the ratio of Bax/Bcl-2. Our study also showed that21-KDa Bax was proteolytically cleaved into the more apoptotic18-KDa Bax in DLBCL cells exposed to As2S2at a concentration of10μM.Conclusions:1. As2S2could inhibit proliferation and induce apoptosis of LY1and LY8cells in a concentration-and time-dependent manner.2. The effect was partly due to the induction of mitochondria-dependent apoptosis involving Bax cleavage.Part Ⅱ Enhancing Effects of Indirubin on Arsenic Disulfide-Induced Apoptosis of Human Diffuse Large B-Cell Lymphoma Cells and The MechanismObjective:To enhance therapeutic efficacy and reduce adverse effects, it is common to combine multiple drugs to deal with diseases. RIF was been constituted with realgar as a sovereign drug, whereas Indigo naturalis as a minister drug. Indirubin and its derivatives have been made great progress on the treatment of chronic myeloid leukemia (CML). Extensive research has been performed to study the mechanism of their anti-tumor activity. To investigate the enhancing effect of indirubin on arsenic disulfide (As2S2) on the proliferation and apoptosis of DLBCL cells LY1and LY8in an attempt to find a better combination therapy scheme.Materials and Methods:1. DLBCL cells cultured with indirubin and As2S2alone or in combination2. CCK-8method to detect cell proliferation3. Flow cytometry to evaluate cell apoptosis4. RNA extraction and RT-PCR5. Protein extraction and western blot analysis 6. Statistical analysisResults:1. We found that the DLBCL cells viability had no significant change at24h,48h and72h with increasing indirubin concentration.2. Meanwhile the apoptotic rates of DLBCL cells weren’t significantly increased at48h with increasing indirubin concentration.3. Along with the combination of indirubin and As2S2, the viability and apoptotic rate of DLBCL cells were both notably changed compared with the As2S2-treated group, and was statistically significant (P<0.05).4. The quantitative real-time PCR results showed that indirubin had no enhancing effect on the expression levels of Bax/Bcl-2ratio and caspase-3mRNA.5. Western blot revealed that indirubin had enhancing effect on the expression levels of Bax/Bcl-2ratio and caspase-3protein. Our study also showed that21-KDa Bax was proteolytically cleaved into the more apoptotic18-KDa Bax in DLBCL cells exposed to the combination of indirubin and As2S2.Conclusions:1. Indirubin alone couldn’t inhibit proliferation and induce apoptosis of LY1and LY8cells.2. The combination of indirubin and As2S2yielded enhancing effects. Therefore our data demonstrated that As2S2served as the principal component, whereas indirubin served as the adjuvant ingredient on the anti-tumor activity.3. The enhancing effect was partly due to the induction of mitochondria-dependent apoptosis involving Bax cleavage.更多还原