一种新型树突状细胞疫苗靶向抑制肾癌干细胞的机制研究

Renal cancer stem cell(RCSC) is the key point of the recurrence and metastasis of kidney cancer. Transforming growth factor-β(TGF-β)is an important role in tumor metastasis and anti-immunotherapy. Our previous research showed blockade the pathway of TGF-β in dendritic cell(DC) could efficiently suppress the pulmonary metastasis of renal cancer. But whether this type of DC vaccine can also have the same effect on the RCSC and what the mechanism is are still not known. According to our previous researches, we presumed that targeting blocked the signal patheway of TGF-β, DC vaccines can efficiently migrate to the niche of RCSC and arouse the immune cell to destroy the RCSC, suppress the proliferation and metastasis of the tumor. In this research, we are planning to isolate the RCSC from patients with renal cancer and to implant these cells to SCID mice to form metastatic tumor. Then we isolate the DCs and CTLs from the same patient. By infected by retrovirus containing gene of pseudo-receptor of TGF-β, the DCs will be targetingly blocked the signal pathway of TGF-β and become TGF-βinsensitive. After transfusing this type of DCs and CTLs back to the tumor forming SCID mice, we can imitate the immune enviorment of human body to observe the situation of tumor growth. By series of observaion and test iv-vivo and in-vitro, eg. tumor weights, survival periods, cytokine levels, we can finally determin the effects of TGF-β insensitive DCs on RCSC and its niche, and find the possible mechanisms of it. Our research is the further investigation of the previous study. But it further close to clinical application and may provide a new thoughts and means to individualized treatment and immunotherapy of metastatic RCC patients.

肾癌干细胞（RCSC）是肾癌复发转移的关键，转化生长因子-β（TGF-β）在肿瘤细胞转移和对抗免疫治疗时具有重要作用。我们前期研究发现，阻断树突状细胞（DC）疫苗的TGF-β信号通路可以有效的抑制肾癌肺转移。但此种DC疫苗对RCSC是否具有相同的抑制作用，作用机制如何，尚不得而知。本课题根据前期研究基础，认为"通过靶向性阻断TGF-β信号通路可以使DC细胞有效迁移至RCSC微环境，动员免疫细胞杀伤RCSC，抑制RCSC的增殖和转移"。本研究拟通过分离肾癌患者的RCSC使SCID小鼠成瘤，再分离患者的DC及杀伤性T细胞（CTL），应用逆转录病毒来靶向性阻断DC细胞TGF-β信号通路，将此DC疫苗及CTL细胞回输小鼠，模拟人体内免疫系统，观察DC疫苗对RCSC体内体外的抑制作用情况，了解其可能机制。本研究是对既往研究的深入，更接近临床应用，有望为转移性肾癌个体化及免疫治疗提供新的思路和方法。

晚期肾癌尚无良好的治疗方法。小分子靶向药物治疗可能因为肿瘤信号通路的变化而出现耐药，而肿瘤免疫治疗可能在晚期肿瘤治疗中取得突破，但肿瘤分泌的免疫抑制因子如转化生长因子-β在肾癌细胞转移和抑制免疫细胞功能中起到重要作用。肾癌干细胞是肾癌发生发展复发转移的重要因素，我们应用免疫磁珠方法从肾癌组织中分离出CD105+肾癌细胞，进行培养扩增并进行肿瘤干细胞相关检测，通过球状聚集生长及相关蛋白表达证实CD105+肾癌细胞即为肾癌干细胞。我们应用磁珠将人外周血DC细胞及单核细胞移植进行分类并移植至SCID-beige鼠，通过ELISA检测小鼠体内IgG水平证实成功获得免疫系统人源化的免疫缺陷小鼠。应用慢病毒基因转染技术通过shRNA将DC细胞及CD8+T细胞转化生长因子-β进行敲减，通过Western-blot检测发现免疫细胞转化生长因子-β明显降低，应用肿瘤干细胞裂解物冲击DC细胞并致敏CD8+T细胞获得肿瘤特异性转化生长因子-β不敏感的免疫细胞体系。体外实验证实将获取的转化生长因子-β不敏感的免疫细胞体系与CD105+肾癌干细胞共同孵育，对比未经干预的免疫细胞体系，应用51Cr释放实验证实转化生长因子-β不敏感肿瘤特异性免疫细胞具有特异性杀伤作用。将CD105+肾癌干细胞经SCID-beige鼠尾静脉注入形成肾癌肺转移模型，将转化生长因子-β不敏感肿瘤特异性免疫细胞，体内实验证实这种免疫细胞可以阻断肿瘤干细胞的EMT作用，抑制荷瘤动物肿瘤生长，抑制肺转移病灶。本课题以既往研究为基础，通过靶向性阻断转化生长因子-β信号通路，使DC及CD8+T细胞对CD105+肾癌干细胞分泌的转化生长因子-β的抑制作用不敏感，而有效迁移至RCSC微环境，阻断肾癌干细胞的EMT并直接杀伤肾癌干细胞，抑制肾癌干细胞的增殖和转移。本研究通过对既往研究的深入，更接近临床应用，有望为转移性肾癌个体化及免疫治疗提供新的思路和方法。