负性共刺激分子B7-H3和B7-H4在HPV持续感染及宫颈癌发生发展中的机制研究

Uterine cervical cancer, as one of the most common gynecologic malignant tumor in China, is not only related to persistent high risk HPV infection, but also related to immune factors. B7-H3 and B7-H4, recently found as B7 family molecules, could inhibite proliferation and activation of T cells, are overexpressed in kinds of tumors. Our previous researches found that B7-H3 and B7-H4 were overexpressed in cervical cancer and their overexpression was related to clinical stages. Combined witn our previous researches, this project will detect the expression of B7-H3 and B7-H4 in HPV infected tissues, precancerous tissues and cervical cancer. Also, we will construct murine model of cervical cancer, regulate expression of B7-H3 and B7-H4 by gene transfection and gene knockout or antibody block. Then, we will observe changes of tumor formation as well as T cells function in murine model to explore the role of B7-H3 and B7-H4 in cervical tumor immunity and possible mechanisms.This project will elucidate the role of B7-H3 and B7-H4 in persistent HPV infection and cervical cancer development, providing new targets and theoretical evidence for cercical cancer immunotherapy.

宫颈癌是我国最常见的妇科恶性肿瘤之一，其发生的主要原因是高危型HPV持续感染，但免疫因素在HPV介导的致癌过程中也发挥重要作用。负性共刺激分子B7-H3和B7-H4是新近发现的B7家族成员，可抑制T细胞的增殖和活化，在多种肿瘤中高表达。前期研究发现，B7-H3和B7-H4在宫颈癌中表达增高，并与临床分期有关。本项目拟在前期研究的基础上，明确B7-H3和B7-H4在HPV感染组织、宫颈癌前病变（高级别CIN）组织、宫颈癌组织及细胞系中的表达；同时建立宫颈癌小鼠模型，采用基因转染、基因敲除、抗体阻断等方法调控B7-H3和B7-H4表达，观察小鼠成瘤情况和T细胞功能的变化，研究B7-H3和B7-H4在宫颈肿瘤免疫中的作用并探讨相关机制。通过本项目，明确B7-H3和B7-H4在HPV持续感染及宫颈癌发生发展中的作用及机制，为宫颈癌免疫治疗提供新的靶点和理论依据。

宫颈癌的免疫治疗成为继手术、放疗、化疗又一种全新的治疗模式。B7-H3(B7 homolog 3)和B7-H4(B7 homolog 4)是新近发现的B7家族成员，可表达在肿瘤细胞和抗原提呈细胞表面。本研究检测到B7-H3/B7-H4在高级别宫颈上皮内病变和宫颈癌中的表达明显高于正常宫颈组织。B7-H3与B7-H4的表达呈正相关关系，B7-H3高表达的患者比B7-H3低表达的患者预后差，B7-H3与B7-H4的高表达与宫颈癌深层间质浸润相关。同时，IL-10表达在宫颈癌组织＞高级别宫颈上皮内病变＞正常宫颈。B7-H3与IL-10、TGF-β1的表达呈正相关关系，B7-H4与IL-10的表达也呈正相关关系。IL-10高表达，宫颈癌分化程度越差，TGF-β1高表达，淋巴结转移可能性越大。敲除B7-H3/B7-H4后可抑制宫颈癌细胞的增殖、迁移、侵袭。干扰B7-H3/B7-H4能影响HPV致癌相关通路E7/Rb通路的表达水平及下游周期和凋亡相关分子。宫颈癌组织中CD8+T淋巴细胞与宫颈癌的期别相关，CD68+的单核巨噬细胞与宫颈癌的间质浸润有关。构建过表达B7-H3/B7-H4宫颈癌细胞-单核细胞U-937共培养体系，发现过表达B7-H3/B7-H4组中的单核细胞增殖速度加快，凋亡减少。过表达B7-H3/B7-H4组共培养体系中的上清液及单核细胞U-937中IL-10及TGF-β1的含量增加。过表达B7-H3和B7-H4能导致单核细胞中JAK2-STAT3通路的活化。利用宫颈癌小鼠细胞系U14通过C57BL/6小鼠建立宫颈癌小鼠皮下瘤模型，小鼠荷瘤后，给予腹腔注射单克隆抗体阻断剂B7-H3、B7-H4作为实验组，非免疫性IgG作为对照组。和对照小鼠相比，单克隆抗体处理后小鼠肿瘤的增长速率减缓。流式细胞术检测到腹腔注射单克隆抗体后肿瘤浸润淋巴细胞中CD8+T淋巴细胞及NK细胞中的IFN-γ和TNF-α高于对照组。.综上所述，本研究发现B7-H3/B7-H4可作为宫颈癌肿瘤标记物，明确了B7-H3/B7-H4可作为致癌分子影响E7/Rb通路，促进宫颈癌的发生与发展。初步探讨了B7-H3/B7-H4在宫颈癌患者中的负性免疫调节机制，发现B7-H3/B7-H4过表达能诱导单核细胞JAK2-STAT3通路的活化，并引起IL-10及TGF-β1的合成及分泌增加，形成有利于宫颈癌发展