从TGF-β/CCL9途径探讨保元解毒汤调控MDSCs改善肺转移前微环境的作用及机制研究

Pre-metastatic niche is the key step of tumor metastasis, and myeloid derived suppressor cells are the main bone marrow stromal cells in the pre-metastatic niche. CCL9 secreted by myeloid derived suppressor cells can promote directional migration of tumor cells, which can be regulated by TGF-β signaling pathway. Previous studies indicated that Baoyuan Jiedu Decoction significantly inhibited the migration of tumor cells to the lung and reduced the content of TGF-β and MDSCs. It is hypothesized that “Baoyuan Jiedu Decoction may regulate myeloid derived suppressor cells and the expression of CCL9 in the MDSC, improve the premetastatic niche of transfer target organs by interfering with the TGF-β/Smads signaling pathway”. To investigate the mechanism, we will copy spontaneous lung metastasis model of breast cancer and use of TGF-β RI inhibitor intervention, to observe the effect of Bao Yuan Jie Du Decoction on the expression of key molecules of MDSCs and TGF-β /CCL9 pathway in lung premetastatic niche. In vitro, we will establish tumor cells and MDSC co-culture system, and use of lentivirus transduction of MDSC by TGF- 1 siRNA, observe the effect of Baoyuan Jiedu Decoction on MDSCs and TGF-β/CCL9 signal pathway. It will provide a scientific illustration of molecular mechanism of Baoyuan Jiedu Decoction improving the pre-metastatic niche of lung , and provide effective traditional Chinese medicine prescriptions for the treatment and prevention of tumour metastasis.

转移前微环境是肿瘤转移的关键环节，MDSCs分泌的CCL9可以促进肿瘤细胞的定向迁移， CCL9的表达受TGF-β信号通路的调节。前期研究表明，保元解毒汤能够显著抑制肿瘤细胞向肺的迁移，降低血清TGF-β的含量和MDSCs的比例。据此提出假说：“保元解毒汤可能通过干预 TGF-β信号通路调控MDSCs细胞CCL9的表达，改善肺转移前微环境，抑制CCL9与CCR1的结合，影响肿瘤细胞的定向迁移”。本项目拟体内模拟肿瘤转移前微环境，体外进行肿瘤细胞的条件培养及共培养，以小动物活体荧光成像系统监测转移情况，探讨保元解毒汤对肺转移前微环境中MDSCs含量及TGF-β/CCL9途径中关键分子表达的影响。并以慢病毒转染技术及相关抑制剂进行干预，反证关键环节的调控作用，探讨保元解毒汤“扶正祛邪”改善肺转移前微环境的分子机制，为肿瘤转移的防治提供有效方药及科学依据。

本项目在体复制了自发性乳腺癌肺转移前模型，体外构建了条件培养、肿瘤细胞与MDSC共培养体系，分别通过小动物活体荧光成像动态监测肺转移情况、划痕和Transwell实验检测肿瘤细胞转移能力，研究保元解毒汤改善肺转移前微环境抑制肺转移的作用；通过流式细胞仪检测MDSC含量和亚群分布，ELISA检测TGF-β、CCL9等细胞因子含量，qRT-PCR、免疫荧光和Western Blot检测MDSC中TGF-βRⅠ、TGF-βRⅡ、Smad2、Smad3、Smad2/3、p-Smad2/3、Smad4和肿瘤细胞CCR1蛋白与mRNA的表达，并采用TGF-β信号通路阻断剂进行反证。分析各因素之间的关系，探讨保元解毒汤“扶正祛邪”改善肺转移前微环境的分子机制，为肿瘤转移的防治提供有效方药及科学依据。结果显示：1.保元解毒汤能够延长4T1荷瘤小鼠的生存时间。.2.自发性乳腺癌肺转移模型中MDSC的募集增多，保元解毒汤通过调控MDSC的数量降低其功能，从而肺转移前微环境的形成。.3.自发性乳腺癌肺转移模型中TGF-β信号通路能诱导MDSCs活化并产生CCL9，使肿瘤细胞微环境改变，通过和其受体CCR1的结合，可促进转移的肿瘤细胞的附着和生存；保元解毒汤通过干预MDSC细胞TGF-β信号通路中关键分子的表达，抑制其分泌CCL9，影响肿瘤细胞的定向迁移，从而改善肺转移前微环境。