Bv8诱导N2型肿瘤相关中性粒细胞在宫颈癌转移中的作用及机制研究

Metastasis and relapse are the primary cause of treatment failure of cervical cancer. The key point to improve curative effect and prognosis is to identify the exact mechanism of metastasis. We have observed that Bv8 was over expressed in cervical cancer and its expression was correlated with the clinical stages and distant metastasis. Furthermore, we found that the increased expression of MMP-9 in neutrophils treated by Bv8. Taken together, we suppose that Bv8 might promote the metastasis of cervical cancer by inducing tumor-associated neutrophils polarized toward to N2 type, making it the key objective in this project. We will firstly validate the independent prognostic role of Bv8 in a prospectively established large database of cervical cancer specimen in local area with complete follow-up information. Secondly, the Bv8 eukaryotic expression vector will be injected into nude mice, from which the separated PMN will be mixed with human cervical cancer cell lines and subcutaneously injected into the nude mice, so as to investigate the impacts of added PMNs on the tumor invasion and metastasis in vivo.Thirdly, the Bv8 protein will be used to study its ability to induce the tpye and function changes of human peripheral blood PMNs, and the Bv8 disposed PMNs will be co-cultivated with human cervical cancer cells to study their impact on the invasion and metastasis of cervical cancer cells in vitro. Lastly, the signaling pathway inhibitors will be applicated to study the underlying molecular mechanisms. This study is expected to verify Bv8 as an independent prognostic factor in cervical cancer metastasis, and help to establish a novel strategy against Bv8 to prevent metastasis of cervical cancer.

转移与复发是宫颈癌治疗失败的主要原因，明确其转移的机制是提高疗效和预后的关键。前期研究发现，Bv8在宫颈癌组织中高表达，其表达与临床分期及远处转移密切相关；且Bv8能诱导中性粒细胞MMP-9表达，据此推测Bv8可通过诱导N2型肿瘤相关中性粒细胞促进宫颈癌转移。本研究拟首先在大样本宫颈癌标本库中证实Bv8是影响患者转移的独立预后因素；第二，在裸鼠体内转染表达Bv8载体后分离其骨髓 PMN，将其与人宫颈癌细胞株混合接种，体内研究PMN对肿瘤成瘤能力及转移能力的影响；第三，分离获取正常人外周血PMN，用Bv8蛋白处理后体外检测其表型及功能改变，以及其与肿瘤细胞共培养后PMN对宫颈癌细胞侵袭与转移能力的影响；第四，应用信号通路抑制剂等在分子水平初步探讨在宫颈癌中Bv8诱导中性粒细胞促瘤潜能及促进肿瘤转移中的分子信号机制。研究成果有望为宫颈癌转移提供新的预测指标，为临床治疗宫颈癌转移提供理论依据。

目的：研究中性粒细胞（PMN）在宫颈癌发生、发展及预后的作用，为宫颈癌的防治提供理论依据。方法：1、采用IL-6诱导小鼠骨髓中性粒细胞（PMN）生成和释放基质金属蛋白酶-9（MMP-9），通过实时荧光定量聚合酶链式反应及Western blot检测法探讨其作用机制及激活途径；2、采用免疫组织化学法检测宫颈癌组织中CD66b阳性的PMN数量，探讨其在预测宫颈癌复发中的作用；3、采用粒细胞集落刺激因子（G-CSF）诱导小鼠骨髓中性粒细胞（PMN）生成Bv 8，通过体外及体内试验探讨其对肿瘤生长和肿瘤血管生成的作用。结果：研究结果证实，1、IL-6刺激后，PMN中MMP-9 mRNA水平明显升高，明胶酶谱检测的释放到胞外MMP-9活性明显高于对照组，IL-6刺激后PMN中STAT3磷酸化水平明显升高。在IL-6作用时加入特异性STAT3抑制剂Ⅷ后，MMP-9 mRNA水平、培养上清液中MMP-9活性以及PMN中磷酸化STAT3蛋白水平与IL-6刺激组相比均明显下降；2、PMN浸润数量>PMN平均值组的RFS无复发生存（recurrence free survival，RFS）期明显短于PMN浸润数量≤PMN平均值组，PMN浸润数量增加是宫颈癌患者RFS的独立危险因素；3、G-CSF刺激后， PMN中 Bv8 的 mRNA 水平明显升高， PMN中 ERK 磷酸化水平明显升高。在 G-CSF作用时加入特异性 ERK 抑制剂 PD98059后， Bv8的 mRNA水平、PMN中磷酸化ERK蛋白的水平与G-CSF刺激组相比均明显下降。NBM-PMN抑制肿瘤生长及血管生成，TBM- PMN促进肿瘤生长及血管生成，加入 G-CSF及抗 Bv8抗体后，TBM -PMN的促肿瘤生长及促血管生成作用减弱。结论：中性粒细胞（PMN）与宫颈癌的发生、发展及预后关系密切，在临床实践中，可根据检测宫颈癌组织中PMN数量预测宫颈癌患者的预后。