TLR4及TLR9信号通路活化促进多发性骨髓瘤细胞生长的机制研究

Multiple myeloma (MM) arises from an accumulation of malignant plasma B cells in the bone marrow (BM), which accounts for about 10% of all hematological malignancies, however，the molecular mechanism of MM pathesenesis remains unclear.It was reported that plasma cells transformation, aberrant activation of signaling pathway and promotion of cytokines secretion might be responsible for the pathesenesis of MM. In our previous report we demonstrated that expression of Toll like receptor were higher in MM cells and TLR4 and TLR9 specific ligands could promote MM cells proliferation. Nevertheness, we hypothesized that activation of TLRs signaling pathway could promote MM cell growth.In this study, we want to confirm the expression of TLR4 ,TLR9 and other molecules downstream of TLR signaling pathway in MM patients and animal models, and specific interference of TLR4，TLR9 and the key adapt protein, MyD88, were further designed to verify the action of TLR signaling pathway on MM cell growth. In addition, sepecific inhibitor, microarray and cytokine analyse were employed to secreen the potential signaling molecules and search the dorminant cytokines. Take together, the expected results might enrich the molecular mechanism of MM pathesenesis and launch a solid basis of development of the new therapeutic targets.

多发性骨髓瘤（MM）是一种浆细胞恶性肿瘤，约占所有造血系统恶性肿瘤的10%，病原生物感染所 致的慢性抗原刺激导致浆细胞转化可能是发病机制之一。申请人在前期工作中发现病原生物的识别受体-TOLL样受体在MM细胞中呈高表达，TLR4及TLR9的配体能够促进MM细胞增殖。基于上述结果我们猜测TLR4/9信号通路激活是促进MM细胞生长的机制之一。本课题拟通过检测不同疾病状态TLR4/9的表达水平,特异性干扰TLR4、9及TLR通路中关键接头蛋白MyD88的表达，进一步证实TLR4及9通路激活可以促进MM细胞生长；在动物模型中寻找TLR4/9促进MM细胞生长和免疫逃逸机制；并进一步通过激活和抑制下游信号通路、基因芯片及细胞因子分析等手段，寻找其TLR通路下游可能起主导作用的信号通路、分子及细胞因子。本课题预期研究结果可丰富MM发病机制，为寻找新的治疗靶点提供理论和实验基础。

多发性骨髓瘤（MM）是一种浆细胞恶性肿瘤，约占所有造血系统恶性肿瘤的10%，其发病机制研究尚不充分。有报道表明细菌及病毒感染所致的慢性抗原刺激导致浆细胞转化，信号通路异常激活和细胞因子分泌增多可能是其发病机制。申请人在前期工作中发现病原生物的识别受体-TOLL样受体（TLR）在MM细胞中呈现高表达，TL4/TLR9配体能够促进MM细胞增殖。MyD88是TLR通路中最为关键的接头分子，因此我们猜测MyD88可能为TLR通路促进MM细胞生长的重要分子。有文献报道MyD88 L265P突变在多发性骨髓瘤患者中有10-20%发生率，但报道病例数较少，国内尚无类似的报道。本课题首先在传统的PCR测序基础上建立四引物毛细管电泳法，提高了MyD88 L265P的检测敏感性，收集了81例淋巴和浆细胞疾病患者进行检测，结果提示该突变多见于IgM型淋巴浆细胞疾病，以华氏巨球蛋白血症多见，伴此突变患者年龄较大，白细胞、血红蛋白、M蛋白计数较低，血小板计数较高。在体外筛选了7株MM细胞株，选取了表达不同MyD88水平的U226，LP1和L363细胞株作为研究对象，并构建了MyD88干扰和过表达质粒，结果提示MyD88是促进MM细胞生长的关键分子，干扰MyD88可以影响MM细胞增殖，同时构建了MyD88干扰的稳定细胞株，通过基因芯片及细胞因子分析等方法寻找发挥主要作用的细胞因子，揭示其下游的NF-kB通路可能发挥了重要作用。上述研究丰富了TLR通路激活促进MM细胞生长的机制，提示MyD88信号转导在多发性骨髓瘤的发生中有重要意义，为发展MyD88基因成为该类疾病的治疗靶点提供了实验基础。