【作者】 侯芸；

【导师】 巴一； 王华庆；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2014， 博士

【摘要】 研究背景恶性淋巴瘤是国人常见恶性肿瘤之一。弥漫大B细胞淋巴瘤(DLBCL)是恶性淋巴瘤中最常见的类型,化疗是治疗的主要手段。多数的DLBCL患者经标准化疗方案治疗后可以缓解,但是25～30%的DLBCL患者对化疗药物耐药,属于难治性DLBCL,尤其是活化B细胞(ABC)型DLBCL(ABC-DLBCL),是目前临床上治疗的难点。细胞分裂周期相关蛋白激酶7(CDC7)是一种丝氨酸/苏氨酸激酶,通过磷酸化激活其下游的微小染色体维持蛋白MCMs(minichromosome maintenance complex)发挥作用,尤其对MCM2的磷酸化作用最强。MCM2磷酸化后被激活,通过空间构象的改变来解除对解旋酶的抑制,从而促使DNA的复制,参与肿瘤细胞的增殖与细胞周期调控。有关CDC7、MCM2与DLBCL预后关系的研究不断有报道,但是尚无一致结论。CDC7在人类许多肿瘤细胞系中过表达,包括DLBCL。那么CDC7是否与ABC-DLBCL患者的耐药相关?干预CDC7蛋白的表达是否能诱导ABC-DLBCL细胞的凋亡?干预CDC7蛋白与一线治疗联合是否能提高ABC-DLBCL细胞的凋亡率?针对这些问题,目前尚无报道。这些问题的解决将有助于我们阐明难治性DLBCL患者的耐药机制,并改善这部分患者的治疗疗效。研究目的1、研究CDC7、MCM2是否与DLBCL患者的预后相关。2、研究CDC7、MCM2与传统预后指标的相关性,CDC7、MCM2能否成为DLBCL的特异性预后指标。3、干预CDC7蛋白表达对ABC-DLBCL凋亡的影响,明确CDC7蛋白介导ABC-DLBCL的耐药机制。4、干预CDC7蛋白表达与一线治疗联合诱导ABC-DLBCL凋亡是否有协同增效作用,提出靶向调控CDC7蛋白可能成为改善ABC-DLBCL患者治疗疗效的潜在方案。研究方法本研究将从临床试验和基础实验两方面进行:1、临床试验:选择天津医科大学肿瘤医院2008年1月至2010年1月收治的60例初治DLBCL患者,取骨髓、外周血及组织蜡块标本。患者一线均接受RCHOP方案化疗。回顾性分析其性别、年龄、ECOG评分、AnnArbor分期、IPI评分、骨髓是否累及、B症状有无、结外受累数目、血乳酸脱氢酶(LDH)数值、治疗模式、组织细胞来源等临床资料,按照实体瘤评价标准进行疗效评价,随访至2010年12月。应用免疫组化法测定组织蜡块中CDC7、MCM2蛋白的表达,并应用荧光定量PCR法测定患者外周血及骨髓中CDC7、MCM2mRNA的表达,阐明CDC7、MCM2与DLBCL患者预后的关系,明确CDC7、MCM2与传统预后指标的相关性。试图找到针对DLBCL患者的特异性预后指标。2、基础实验:体外培养 ABC、GCB-DLBCL 细胞系,Western-Blot、RT-PCR法检测细胞中CDC7蛋白及CDC7mRNA的表达,将CDC7shRNA转染至ABC-DLBCL细胞,干预CDC7蛋白的表达,检测其下游MCM2、pMCM2蛋白表达水平的变化,探讨CDC7与MCM2相互作用的关系;检测干预CDC7蛋白后诱导ABC-DLBCL细胞凋亡水平的变化,CDC7蛋白干预与美罗华联合诱导ABC-DLBCL的凋亡是否有协同增效作用,Western-Blot、RT-PCR法检测凋亡通路相关分子指标的变化,明确凋亡机制。试图阐明CDC7蛋白介导ABC-DLBCL的耐药机制,为研制针对CDC7的新型靶向药物提供理论依据。结果1、DLBCL患者的组织腊块、外周血及骨髓中CDC7、MCM2表达水平是患者的预后不良因素,CDC7、MCM2高表达组患者的2年生存率明显低于低表达组患者(P<0.05),差异具统计学意义。2、CDC7、MCM2表达水平与DLBCL患者的IPI评分、Ann-Arbor分期呈正相关(P<0.05),CDC7、MCM2与IPI评分、Ann-Arbor分期结合可以成为DLBCL患者的特异性预后指标。3、DLBCL患者外周血CDC7mRNA、MCM2mRNA水平与组织及骨髓中CDC7蛋白、MCM2蛋白、CDC7mRNA、MCM2mRNA水平呈显著正相关(P<0.05),可以用患者外周血代替组织、骨髓标本,检测其中CDC7、MCM2表达水平,分析其与DLBCL患者预后的关系,将为临床工作带来很大便利,适合于临床广泛应用。4、干预CDC7蛋白表达可通过下调抗凋亡蛋白Bcl-2的表达和上调促凋亡蛋白Bax的表达来诱导ABC-DLBCL细胞凋亡,与美罗华联合具有协同增效作用。结论1、CDC7、MCM2表达与DLBCL患者预后的关系,CDC7、MCM2与IPI评分、Ann-Arbor分期结合可以成为DLBCL患者的特异性预后指标,较传统预后指标更准确地评价DLBCL患者的预后,指导个体化治疗方案的制定,以提高DLBCL患者治疗的有效率及生存期。2、CDC7介导了 ABC-DLBCL患者对于一线治疗的耐药,靶向调控CDC7蛋白可以成为治疗ABC-DLBCL的新方法,与美罗华联合可延长ABC-DLBCL患者的生存期。更多还原

【Abstract】 BackgroundMalignant lymphoma is a common kind of cancer in China.Diffuse large B cell lymphoma(DLBCL)is the most common subtype in malignant lymphoma.Chemotherapy is the primary means to treat malignant lymphoma.Most patients with DLBCL treated with standard chemotherapy regimen can receive response,but 25～30%patients are resistant to chemotherapy.This kind of resistant lymphoma belongs to refractory lymphoma,such as ABC-DLBCL,which is still difficult to treat nowadays.CDC7 is a serine/threonine kinase that is essential for the initiation of eukaryotic DNA replication through phosphorylating Mem proteins.The studys about the relationship between CDC7&MCMs and malignant lymphoma were reported constantly,but the outcomes have not shown the consistent conclusions.CDC7 is high expressed in many kinds of human tumor cells including DLBCL.Whether the expression of CDC7 is related with drug resistance of ABC-DLBCL?Whether inhibiting CDC7 expression can improve the response rate of ABC-DLBCL patients?Whether inhibiting CDC7 expression with rituximab can prolong the overall survival of ABC-DLBCL?These questions need to be explored.Objective1.To study the relationship between CDC7.MCM2 and prognosis of patients withDLBCL.2.To study the relationship between CDC7.MCM2 and traditional prognosticfactors of patients with DLBCL.3.To confirm the drug resistance mechanism of ABC-DLBCL mediated by CDC7.4.To confirm the effect of CDC7 inhibition with rituximab in the apoptosis ofABC-DLBCL in order to explore new therapy in the treatment of ABC-DLBCL.Methods1.Clinical trial:Treatment-naive patients with DLBCL in Tianjin Medical University Cancer Hospital from January 2008 to January 2010 were enrolled into this study.We collected the peripheral blood、tissue、bone marrow of DLBCL patients.The expression level of CDC7&MCM2 were examined by immunohistochemistry and RT-PCR to clarify the relationship between CDC7&MCM2 and prognosis of DLBCL patients.2.Basic research:ABC.GCB-DLBCL cells were cultured in vitro.The expression level of CDC7 was examined by Western-blotting.CDC7 was down-regulated by shRNA,then co-cultured with rituximab,the effect of inducing apoptosis was detected to find the related mechanism of resistance in ABC-DLBCL.The down-stream factors were examind in order to find the mechanism of apoptosis mediated by CDC7 inhibition and rituximab.Results1.The expression level of CDC7&MCM2 was poor prognostic factor of DLBCL patients.2.The expression level of CDC7&MCM2 was positively correlated with IPI score and Ann Arbor stage,CDC7&MCM2 in addition with IPI score and Ann Arbor stage can be the particular prognostic factors for DLBCL patients.3.The expression level of CDC7&MCM2 in peripheral blood was positively correlated with tissue and bone marrow.We can examined peripheral blood of patients to replace other samples.4.Inhibition of CDC7 can induce apoptosis of ABC-DLBCL cells through down-regulating Bcl-2 meanwhile up-regulating bax.CDC7 inhibition with rituximab has synergistic effect in apoptosis of ABC-DLBCL cells.Conclusions1.The expression level of CDC7&MCM2 in addition with IPI score and Ann Arbor stage can be the particular prognostic factors for DLBCL patients.Based on the particular prognostic factors,we can find more individual regimen in order to prolong overall survival of DLBCL patients.2.CDC7 mediated the drug resistance of ABC-DLBCL.CDC7 is the new target for ABC-DLBCL patients.CDC7 inhibition with rituximab can prolong the overall survival of ABC-DLBCL patients.更多还原