CCN2以肝素依赖形式促单核细胞“归巢”增强肝癌恶性潜能的研究

The liver tumor microenvironment is a complex mixture of stromal cells and proteins contributing to the carcinogenic process seeming as the soil for tumor growth. Monocyte-macrophage is an important part of the liver tumor microenvironment. CCN2 is a secreted protein contains CT module, which is implicated in protein-protein interactions and mediated cell adhesion by binding to Heparin sulfate proteoglycans (HSPG) and integrin receptors simultaneously. And blocking CT module or damage HSPG could abolish the binding between CCN2 and target cells. In the previous studies, hepatocellular carcinoma (HCC) with the expression of OPN could promote macrophage recruitment. And further studies showed HCC with OPN over-expression significantly increased the secretion of CCN2. Thus , we hypothesized that HCC high expression of OPN could promote monocyte homing to the microenvironment by upregulating CCN2 in heparin dependent manner. In the further study, we will analyze the relationship among OPN, CCN2 and macrophage in HCC. And then, the mechanism of CCN2 in monocyte homing to the tumor microenvironment will be verified. And finally, heparin inhibiting HCC malignant potential will be explored through reducing peritumoral macrophages in vivo. During this study, the mechanism of CCN2 enhancing HCC malignant potential through promoting monocytes homing to tumor microenvironment in a heparin-dependent manner will be revealed, and which will provide a theoretical basis for the establishment of individualized therapy combined with heparin especially for HCC with high expression of CCN2.

肝癌微环境为肝癌生长提供土壤，单核-巨噬细胞系统是微环境的重要组成部分。结缔组织生长因子（CCN2）是一种分泌蛋白，其CT区能同时与细胞膜硫酸肝素糖蛋白（HSPG）的肝素链和整合素受体结合参与细胞粘附，而封闭CCN2的CT区或破坏HSPG则能够抑制CCN2与细胞结合。前期发现，肝癌表达骨桥蛋白（OPN）可促癌周M2巨噬细胞募集，进一步研究，高表达OPN肝癌CCN2分泌明显增加。我们推测高表达OPN肝癌通过上调CCN2以肝素依赖形式促单核细胞向微环境“归巢”从而增强自身恶性潜能。本课题首先分析OPN、CCN2、巨噬细胞与肝癌进展的关系，而后验证肝癌旁分泌CCN2在促单核细胞“归巢”中的作用及肝素的干预机制，最后在体探索肝素通过减少癌周巨噬细胞抑制肝癌恶性潜能。本课题将首次揭示肝癌旁分泌CCN2在单核细胞向微环境“归巢”的促癌机制，为针对高分泌CCN2的肝癌建立联合肝素的个性化治疗提供理论依据

原发性肝细胞性肝癌（简称肝癌）居恶性肿瘤发病率的第5位，死亡率居第三位，大多数患者就诊时已是晚期，失去了根治性治疗的机会。目前，索拉非尼作为晚期肝癌患者的首选姑息性治疗方案，然而该药物的有效率仍较低，且极容易出现耐药。因此以铂类为基础的局部和全身化疗仍是晚期肝癌患者的可选治疗方案，因此探究铂类化疗耐药的分子特征在制定肝癌个体化治疗方案中具有重要意义。前期研究中，本人构建了奥沙利铂耐药的细胞及动物模型，并通过全基因组芯片筛查发现CCN2和ID-1均表达上调。本课题中，我们验证奥沙利铂耐药肝癌中CCN2和ID-1表达明显升高，并发现两者具有明显相关性。采用人肝癌组织标本检测发现ID-1高表达患者愈后较差，而ID-1与CCN2同时高表达的肝癌患者生存期最短，复发率最高。进一步慢病毒过表达及干扰肝癌ID-1构建稳定细胞株并体内外验证其功能并发现，过表达ID-1肝癌转移及成瘤能力明显增强，奥沙利铂耐药程度亦增加，而干扰ID-1表达则能够明显降低肝癌的成瘤及转移能力。全基因组芯片检测提示过表达CCN2后ID-1和MAPK信号明显变化，而过表达ID-1同样能够部分上调CCN2表达，提示CCN2为ID-1的上游基因。进一步研究发现，CCN2能够明显激活MAPK信号通路、增强肝癌奥沙利铂耐药，而通过索拉非尼或U0126阻断CCN2/MAPK/ ID-1正反馈信号环则能够明显抑制肝癌恶性进展，降低肝癌奥沙利铂耐药。通过本课题研究，我们证实CCN2-MAPK-ID-1反馈环在肝癌恶性进展及耐药中发挥重要作用，而阻断该反馈环则能够明显抑制肝癌的恶性进展，在减轻奥沙利铂耐药中发挥重要作用。

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