东亚钳蝎氯毒素抑制MMP-2重塑抗肿瘤免疫效应的机制研究

Glioma, as a malignant tumor, is rarely curable. The prognosis for patients with high-grade gliomas is generally poor. The lack of response to treatment in glioma patients may be attributed to the immunosuppressed microenvironment that is characteristic of invasive glioma. Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme degrading the extracellular matrix and overexpressed by glioma. MMP-2-specific CD4+T lymphocytes display a suboptimal inflammatory Th2 profile. MMP-2-conditoned DCs prime Th2 responses against several tumor-associated antigens, suggesting that MMP-2 can create a Th2 skewing microenvironment in a bystander fashion. Previously, We showed that BmK CT could specifically interact with glioma cells and inhibit the invasion of those cells via MMP-2. This project will find out the key binding sites and modes of action between BmK CT and its recptor MMP-2 .The cytokine expression of glioma cells will examine after treatment with BmK CT- by elisa . Both flow cytometric and pathological analysis will reveal that immune reconstitution of serum, lymph tissue adjacent to the tumor and tumor tissue in BmK CT treatment glioma-bearing mice. This research results will explain the mechanism of anti-tumor immune reconstitution though BmK CT inhibting MMP-2.

神经胶质瘤是严重危害人类健康的恶性肿瘤之一，其在脑部的发生发展与免疫监管缺失有很大关系。胶质瘤免疫逃逸的主要影响因素是自身高表达的基质金属蛋白酶2（MMP-2）作为一个内生调节器促使CD4+T细胞转化成Th2细胞来逃脱机体对肿瘤细胞的免疫抑制。本课题前期研究发现东亚钳蝎氯毒素（BmK CT）蛋白可以特异的结合MMP-2蛋白并抑制其胞外活性和胞内表达，从而抑制胶质瘤的侵袭和迁移。本项目将进一步阐述BmK CT与MMP-2结合位点及作用方式；检测BmK CT作用胶质瘤细胞对细胞因子分泌的影响；利用荷瘤鼠模型，阐明BmK CT治疗前后血清、瘤旁淋巴组织、肿瘤区域免疫重塑情况；最终揭示BmK CT通过抑制MMP-2重塑机体抗肿瘤免疫效应的机制。研究结果为BmK CT治疗神级胶质瘤提供理论支持并为临床应用奠定基础。

本项目在分子和细胞水平上研究东亚钳蝎氯离子通道毒素蛋白抑制神经胶质瘤细胞活性的机制，取得的重要成果及关键数据包括以下五个方面。.1．.根据以色列蝎氯毒素和东亚钳蝎氯毒素同源性进行蛋白三维结构同源建模，结果显示东亚钳蝎氯毒素的33位半胱氨酸可能结合基质金属蛋白酶2活性中心的锌离子。我们利用功能缺失性突变证实了关键氨基酸的结合位点。.2．.东亚钳蝎氯毒素通过激活神经胶质瘤细胞的丝裂原活化蛋白激酶进而活化下游丝裂原活化蛋白激酶激活的蛋白激酶-2引起细胞周期阻滞从而抑制胶质瘤细胞增殖。.3．.东亚钳蝎氯毒素联合替莫唑胺能更好地抑制胶质瘤细胞增殖并促进细胞凋亡，主要机制是东亚钳蝎氯毒素通过抑制蛋白激酶B的磷酸化提高替莫唑胺对胶质瘤细胞的敏感性，表明了东亚钳蝎氯毒素和神经胶质瘤现有临床用药替莫唑胺起到了协同作用。.4．.东亚钳蝎氯毒素通过下调基质金属蛋白酶2的活性缓解了神经胶质瘤细胞上清对T淋巴细胞增殖抑制，并促使T淋巴细胞更多地转化为Th1细胞。.5．.东亚钳蝎氯毒素处理神经胶质瘤细胞前后的代谢组学结果表明其抑制了谷氨酰胺代谢从而降低了胶质瘤细胞活性。.以上结果阐明东亚钳蝎氯离子通道毒素蛋白抗神经胶质瘤的分子机制，为联合临床用药提供了理论支持。