从TLR2/NF-κB 通路调控炎症反应失衡切入研究“补气托毒”法对ABC-弥漫大B淋巴瘤的效应机制

The activation of TLR2/NF-κB signaling pathway resulted in releasing large amounts of inflammatory factors and inflammatory disequilibrium which highly increased the invasiveness of activate B-cell like diffuse large B cell lymphoma ( ABC-DLBCL) and made it progress ..Unfortunately, there are no drugs that could regulate such inflammation and then inhibit the progression of ABC-DLBCL.For the first time, our previous studies showed that the decoction of Reinforcing Qi and Expelling Toxins had anti-cancer effect by inhibiting the expression of NF-κB and reducing the level of inflammatory cytokines, such as IL-6,IL-12 etc. On these basis, we intend to study the effect of decoction of Reinforcing Qi and Expelling Toxins on TLR2/NF-κB signaling pathway in ABC-DLBCL with various experiment methods, including the immuno-precipitation(IP), RT-PCR, Western blot, FCM and ELISA.Furthermore, RNA interference will be used to silence TLR2 gene, confirming the target of the principle of “Reinforcing Qi and Expelling Toxins”. These studies are supposed to elucidate the regulation mechanism of Reinforcing Qi and Expelling Toxins on inflammatory disequilibrium and verify the key target of Reinforcing Qi and Expelling Toxins on ABC-DLBCL is the TLR2/NF-κB signal pathway. It is important to explore a new treatment for ABC-DLBCL with Chinese medicine .It also has great significance for implementing the creative treatment of ABC-DLBCL and improving its therapeutic effect.

TLR2/NF-κB通路在激活B细胞型弥漫大B细胞淋巴瘤(ABC-DLBCL)中的活化，导致大量炎症因子的释放，介导的炎症反应极大的增加了ABC-DLBCL侵袭性并促进其进展。但目前尚未发现有效的抗炎药物可以调控其炎症反应，发挥抗肿瘤作用。我们首次发现补气托毒方可以通过抑制炎症因子NF-κB的表达，降低炎症因子IL-6，IL-12等浓度，调控炎症失衡发挥抗肿瘤作用。在此基础上，本研究将用IP、RT-PCR、Western blot 、FCM 、ELISA等技术，对补气托毒方调控ABC-DLBCL中TLR2/NF-κB信号通路及炎症因子的水平进行研究，并用RNAi沉默TLR2基因，验证其作用靶点。从而阐明补气托毒方调控炎症反应发挥抗淋巴瘤作用的机制，确立TLR2/ NF-κB是补气托毒方治疗淋巴瘤的关键靶点。以期探讨出新的ABC-DLBCL中医药治疗策略，实现其治法的创新并提高其治疗疗效 。

本课题主要基于TLR2/NF-κB的炎症通路探讨“补气托毒”代表方透脓散干预弥漫大B淋巴瘤的疗效及其可能机制。（1）实验前期运用两种裸鼠模型，针对4种淋巴瘤细胞株，进行皮下成瘤实验，接种时无论是否加Matrixgel胶，均告失败，普通裸鼠无一只能成瘤，而NCG裸鼠6只中只有2只成瘤，且需要1个月的时间，与文献报道有较大的差距，提示淋巴瘤的皮下成瘤实验仍极具挑战。（2）细胞实验方面，本课题组使用大鼠含药血清和中药复方过滤原液干预多种淋巴瘤细胞，发现10%的含药血清能有效抑制细胞增殖，而直接中药干预效果不明显，只有在8 mg/ml的浓度时能抑制肿瘤细胞生长。进一步从细胞凋亡层面探讨其可能的机制，检测后未发现中药有诱导细胞凋亡的功效，（3）从炎症方面继续探索：①发现动物体内实验中，中药灌胃能有效减低LPS诱导的炎症因子TNF-α的升高，而IL-6、IFN-γ和IL12-P70的变化没有统计学意义。而对未用LPS诱导的小鼠的TNF-α不能降低。②中药干预后的淋巴瘤细胞培养基上清中的细胞因子未有统计学意义。③中药体外干预小鼠巨噬细胞系RAW264.7细胞，能降低其TNF-α的水平。④使用流式检测小鼠外周血和脾脏中的淋巴细胞，发现透脓散减低了其外周血中的巨噬细胞含量，由于巨噬细胞是TNF-α的重要来源，故与前期实验结果相吻合。（4）但western检测细胞表面的TLR2分子和NF-κB的活性成分p-p65，未发现细胞在给药后有明显改变，与预期有一定的差距。之后本课题组对于中药干预后的淋巴瘤细胞进行了转录组测序，时效和量效关系上显示中药能增加TIMP2表达水平，提示透脓散对肿瘤的抑制作用可能存在其他信号通路，具体有待进一步的实验验证。