脂质筏神经鞘磷脂在增强T淋巴细胞杀伤卵巢癌细胞中的作用及机制

Ovarian cancer is a common malignant tumor of female genital organs. Ovarian cancer is the most common cause of death from gynecological cancers,and seriously threaten the life of woman. Although the operation and chemotherapy showed some mitigation on treatment of advanced ovarian cancer,but the chemotherapy induced side effects,drug resistance and poor curative effect were big problem. recently, immunotherapy is in spotlight as new treatment method on ovarian cancer, including cell immunotherapy, cancer vaccines and monoclonal antibodies. During T cell activation, TCRs gathers at supramolecular activation cluster- interaction position of T cell and antigen-presenting cell.It has been suggested that sphingolipid- and cholesterol-rich microdomains termed lipid rafts, form platforms for the regulation and transduction of TCR signals. An actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has few reports. In previous study，we cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1，as well as clarified that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.This study will overexpress sphingomyelin synthase, on T cells and lead small lipid rafts coalesce into larger structures,to increase T cell activities;T cell recognizability and cytotoxicity against tumor cells.The study will provide new method and approach for clinical tumor immunotherapy.

卵巢癌是女性生殖器官常见的恶性肿瘤。卵巢癌致死率占各类妇科肿瘤的首位。虽然手术和化学治疗对晚期卵巢癌患者有一定的缓解，但化疗副作用大，容易产生耐药性，疗效差。近年来免疫学疗法引人关注，细胞免疫疗法、癌症疫苗和单克隆抗体是治疗卵巢癌的新免疫疗法。在T细胞活化中, T细胞受体集聚在T细胞与抗原提呈细胞接触处的超分子活化簇，这些由神经鞘磷脂(sphingomyelin; SM)和胆固醇富含的微结构域叫脂质筏，它构成了调控和传递TCR信号的平台。SM是脂质筏的主要构成成分，目前有关SM在细胞膜脂质筏中所起重要作用报道甚少。本项目在前期工作中阐明了细胞膜SM在T细胞信号传导过程中的重要作用。本研究利用高表达SM合成酶的方法，使得T淋巴细胞膜表面聚集更多的SM，引起小的脂质筏会凝聚成更大的结构，从而增强T细胞活化，提高T淋巴细胞对肿瘤的识别和杀伤能力。该研究为临床肿瘤免疫治疗提供一种新的方法和途径。

卵巢癌致死率占各类妇科肿瘤的首位。虽然手术和化学治疗对晚期卵巢癌患者有一定的缓解，但化疗副作用大，容易产生耐药性，疗效差。近年来免疫学疗法引人关注，细胞免疫疗法、癌症疫苗和单克隆抗体是治疗卵巢癌的新免疫疗法。在T细胞活化中，T细胞受体集聚在T细胞与抗原提呈细胞接触处的超分子活化簇，这些由神经鞘磷脂(sphingomyelin; SM)和胆固醇富含的微结构域叫脂质筏，它构成了调控和传递TCR信号的平台。本研究利用高表达SM合成酶的方法，使得T淋巴细胞膜表面聚集更多的SM，引起小的脂质筏会凝聚成更大的结构，从而增强T细胞活化，提高T淋巴细胞对肿瘤的识别和杀伤能力。首先，分离BALB/C鼠外周血单核细胞(peripheral blood mononuclear cells，PBMC) 后，分离CD8+T细胞，然后成功转染运载pCMV.sport 6-SMS1、SMS1-siRNA的载体，使其高表达或沉默（knockdown）效应细胞-CD8+T细胞的SMS1目的基因，分别建立T-SMS1/high 细胞、T-SMS1/kd细胞，最终成功鉴定出SMS1高表达或沉默情况。其次，测定了上述细胞对卵巢癌细胞的杀伤活性，并比较细胞毒作用。可见与NL、T-Sm+细胞和T-SMS1/high细胞相比T-SMS1/kd细胞生长速度明显缓慢，有显著性差异(P<0.05)。再次，观测注射各种效应细胞后体内卵巢癌裸鼠移植瘤瘤体缩小情况。肿瘤体积变化可见与NL、T-Sm+细胞和T-SMS1/high细胞相比T-SMS1/kd细胞生长速度明显缓慢，与NL相比，T-SMS1/kd细胞有显著性差异(P<0.05)。最后， 解析高表达SMS1的T淋巴细胞对卵巢癌细胞增强杀伤作用的机制。本研究为临床肿瘤免疫治疗提供了一种新的方法和途径。