中药天花粉蛋白促进小鼠肝癌T细胞免疫治疗的机理与应用研究

The mechanism of T lymphocyte immunotherapy of tumors involves specific recognition of the surface antigen on tumor cells by cytotoxic T lymphocytes (CTL), and the release of toxic substances, e.g. Granzyme B. Granzyme B can enter and kill tumor cells via its binding to the mannose 6-phosphate receptor (M6PR) on the cell membrane. It was first disclosed, in our previous investigation, that a low dose of the Chinese medicine, trichosanthin (TCS) could specifically bind to the Golgi-localized, Gamma-ear-containing, Arf-binding (GGA) protein in hepatoma cells. This competitively inhibited the binding between GGA and M6PR. Thus, the expression of M6PR on the cancer cell membrane was enhanced, which promoted the entry of granzyme B and inhibited the growth of hepatoma cells. In this project, we will use a variety of in vitro and in vivo methods, including hepatoma cell culture, gene transfection, immunofluorescence observation, mouse model of hepatoma, the united therapy of TCS and granzyme B, preparation of tutor vaccine, and the united therapy of TCS and CTL immumotherapy, to study the mechanism of up-regulation of M6PR by TCS, as well as the mechanism and application of TCS in promoting the chemotheraypy and CTL immunotherapy of hepatoma. This project will not only shed light on the immunotherapy of hepatoma, enrich and innovate the scientific connotation of the theory of traditional Chinese medicine, but will also be instrumental in the further development of the modern clinical application of the traditional Chinese medicine.

肿瘤的T细胞免疫治疗原理为具细胞毒性的T细胞CTL可特异性识别肿瘤细胞表面抗原，释放颗粒酶B等毒性物质，后者通过与肿瘤细胞膜表面的甘露糖-6-磷酸受体M6PR结合而进入和杀死肿瘤细胞。本课题组在前期研究中，首次发现小剂量的中药天花粉蛋白TCS可特异性地结合肝癌细胞高尔基体上的GGA蛋白，竞争性地抑制了GGA与M6PR的结合，增加了肝癌细胞膜表面M6PR的表达，从而促进颗粒酶B 进入并抑制肝癌细胞生长。本项目将运用肝癌细胞培养、基因转染、免疫荧光观察、肝癌小鼠建模、TCS与颗粒酶B联合药物治疗、肿瘤疫苗制备、TCS和细胞免疫联合治疗等动物体外和体内实验方法，探讨TCS上调肝癌细胞膜M6PR表达及TCS在肝癌药物和T细胞免疫治疗中协同作用的机理和应用前景。本项目不仅为肝癌免疫治疗带来了新思路，也丰富和创新了中医“扶正固本”理论的科学内涵，进一步开发了传统中药在现代临床治疗中的应用前景。

传统中药天花粉蛋白（Trichosanthin，TCS）属一型核糖体灭活蛋白,具有多种生物学及药理学活性。项目前期研究结果表明TCS不仅能够诱导肿瘤细胞凋亡，而且能够提升甘露糖-6-磷酸受体（M6PR）在肿瘤细胞膜上的表达，而 M6PR是颗粒酶B进入肿瘤细胞的主要受体，促进了颗粒酶B进入并杀伤肿瘤细胞。进一步研究发现TCS可能通过flotilin-1途径促进了M6PR的细胞膜转运。本项目在前期工作基础上，首先确证了TCS提高肿瘤细胞Flotilin-1的表达，进而促进了CI-MPR向细胞膜的转运，从而促进了外源颗粒酶B向细胞内的转运与杀伤作用的分子机理。在TCS和颗粒酶B的联合用药实验中，我们发现舌癌细胞比肝癌细胞对TCS更加灵敏，而且联合用药可通过诱导凋亡从而有效抑制舌癌生长。在肝癌荷瘤小鼠的免疫机理研究中发现，TCS能够通过增强小鼠机体T细胞免疫，提升CD4+T细胞和CD8+T细胞向肿瘤组织的趋化富集，并增加肿瘤组织中颗粒酶B的水平，促进小鼠肝癌细胞的凋亡，进而达到抑制肝癌的效果。为克服TCS的毒副作用，优化TCS抗肿瘤治疗效果，我们进行了现代化的纳米材料搭载重组修饰 TCS干预肝癌细胞实验。结果表明，重组TCS现代化纳米材料搭载增加TCS对肿瘤细胞的毒性作用，增强了TCS抗肿瘤的靶向性并减低了药物的副作用，在中药现代化方面进行了有意义的尝试。总体来说，本项目在TCS抑制肿瘤的分子机理、TCS和颗粒酶B联合用药、肝癌的免疫治疗及TCS中药现代化等方面的研究结果，为TCS进一步的临床转化研究奠定了良好的基础。

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