α-galactosylceramide增强SOCS1基因沉默AAV2/IL-12DC诱导的特异性CTL过继治疗肺癌的动物实验研究

Because of specifity and low toxicity, CTL immunotherapy for lung cancer were clinically acceptable. However, it's different to provoke an effective CTL response to antigens expressed by lung cancer and survival of transferred CTL in recipient is still a problem. Silencing of suppressor of cytokine signaling -1 (SOCS1) in Dendritic cell (DC) enhances the activities of antigen presenting ability and AAV2/IL-12 gene delivery into DC enhances CTL stimulation. α-galactosylceramide (α-GC) at high concentrations can directly activate memory T cells and inhibit apoptosis in our early experiments. On this basis, the project intends to induce more specific CTL dy DC silenced of suppressor of SOCS1 and confirm the specific CTL by α-GC treatment can improve therapeutic effect by enhancing the cellular cytotoxicity and antiapoptosis in animal model of lung cancer. α-GC treatment efficacy can be evaluated through tumor growth curves and animal survival curve. It will lay the foundation for the CTL bio-therapeutic applications for lung cancer of α-GC as an adjuvant in the future after comprehensively understanding of the mechanism in which α-GC augments the therapeutic effect by adoptive transfer of antigen-specific CTLs in animal model of lung cancer.

尽管肺癌的CTL过继疗法因其特异性好，毒副作用少而被临床接受，但由于目前CTL的产生效率较低，同时CTL在患者体内存活时间短，需反复回输来提高治疗效果，因而限制了其临床应用。既往研究表明沉默SOCS1后的树突状细胞（DC）抗原递呈能力显著提高。AAV2/IL-12转染的DC能明显刺激CTL的增殖和杀伤能力。我们的前期研究发现a-半乳糖神经酰胺（α-GC）在高浓度时能直接活化记忆T细胞，抑制细胞凋亡。本项目拟通过沉默AAV2/IL-12DC的SOCS1基因来提高CTL产生效率，进一步动物实验研究α-GC增强特异性CTL对肺癌细胞杀伤效果。通过检测α-GC处理后肿瘤生长曲线和荷瘤动物生存曲线评价α-GC对特异性CTL治疗肺癌的疗效，为α-GC作为肺癌CTL过继治疗佐剂的临床应用奠定基础。

肺癌的CTL过继疗法因其特异性好，毒副作用少而被临床接受，但由于目前CTL的产生效率较低，同时CTL在患者体内存活时间短，需反复回输来提高治疗效果，因而限制了其临床应用。本项目拟通过动物实验初步探讨解决上述难题的方法。课题组成功构建了高表达腺病毒载体pAd/CMV/V5-DEST/IL-2并转染DC细胞株DC2.4，通过siRNA沉默其SOCS1基因，进一步增加其递呈Lewis肺癌肿瘤抗原的能力。与未处理DC2.4及Lewis肺癌细胞株相比，经上述处理过的DC刺激同种T淋巴细胞增殖能力明显增强，CD8/CD4比例，CD69+/CD8+活化(记忆)细胞的比例明显增加，而CD25+/CD4+Treg的量明显降少，CTL对靶细胞杀伤能力更强。与用vehicle处理作为对照组相比， 经α-GC处理的活化CD3细胞下调了FAS和FASL的表达， annexinV阳性细胞比例明显降少，caspase3活性形式明显下降。经α-GC处理激活的Lewis肺癌肿瘤抗原特异性的CD8+T细胞过继转移到荷瘤小鼠体内与对照组用vehicle处理组相比，过继转移细胞在受鼠体内的存活时间明显延长，在脾脏和淋巴结中vehicle处理组10天后就检测不到GFP+细胞了，而α-GC处理组却存有大量的GFP+细胞。在治疗效果方面，α-GC处理组CD8+过继转移荷瘤小鼠后，小鼠的肿瘤生长明显放缓，生存时间明显延长。α-GC能抑制活化T细胞的AICD，具有明显的抗凋亡作用。高表达腺病毒载体pAd/CMV/V5-DEST/IL-2转染DC后能促进IL-12的表达，联合siRNA沉默SOCS1基因后明显提高了肿瘤特异性CTL的产生效率；α-GC是一种非常好的免疫佐剂，在肿瘤免疫治疗中将会有潜在的应用价值。本项目较好的解决了特异性CTL产生效率低下及过继细胞在受鼠体内存活时间较短的难题。