β-榄香烯对CD8+树突状细胞交叉抗原提呈的抗恶性胶质瘤免疫效应研究

Different from routine chemotherapeutic durgs that control tumor growth by dirct killing of individual tumor cells, antitumor Chinese Traditional Medicine could boost the immune response of the patient with the exact mechnism remains unclear. β-elemene, a compound that derived from RhizomaZedoariae, is capable of passing the brain blood barrier, and has already been used in the treatment of several malignancies including gliomas. We have previously showed β-elemene was capable of killing glioma cells through activating apoptotic pathway and, more recently, inducing glioma cells autophagy. On the other hand, we also has discovered the autophagosom derived from alpha-tocopheryloxyacetic acid treated tumor cells could stimulate efficient antigen cross-presentation and lead to activation of anti-tumor immune response. Therefore, we hypothesize the induction of autophagy in glioma cells by β-elemene could activate the host immune system via antigen cross-presentaiton to CD8+ T cells. To test this hypothesis, we designed the following studies: 1) to elucidate the mechanism by which β-elemene could induce autophagy in glioma cells in vitro; 2) to determine the in vitro and in vivo efficacy of autophagosomes-pulsed dendrite cells on CD8+ T cells activations; 3) to test the in vivo antitumor efficacy of dendrite cells vaccination. We believe these efforts would be valuable for illuminations of the mechnisms by which β-elemene strengthens the host immune system and supporting its clincial appplicaitons in the field of anti-glioma treatments.

一般西医抗癌药物在杀伤肿瘤细胞同时也会对肝、肾、血液系统等产生不同程度的毒副作用。中药莪术提取物β-榄香烯与前者不同，其毒副作用小而抗瘤效应强，且能穿透血脑屏障，是一种较为理想的脑癌治疗药物。我们前期研究发现β-榄香烯除对恶性胶质瘤细胞有较强杀伤作用外，还能诱导产生自噬，同时可以调节机体T细胞亚群功能；另外，我们在对维生素E类似物α-TEA的研究中发现，自噬介导的抗原交叉提呈在抗肿瘤免疫反应中存在重要作用。由此，我们提出如下假说：榄香烯诱导的自噬可通过抗原交叉提呈从而产生整体抗肿瘤免疫效应。为证实此假说，本项目拟：1、探讨榄香烯诱导胶质瘤细胞产生自噬的机制；2、探究树突状细胞负载自噬体后在体外／体内条件下激活CD8+ T细胞产生免疫应答的效果；3、研究树突状细胞负载自噬体后的整体抗瘤活性及其机制。上述研究旨在阐明榄香烯的抗肿瘤免疫效应机制，为进一步拓展其抗胶质瘤的临床应用提供科学依据。

脑恶性胶质瘤是最常见的神经系统肿瘤，治疗手段局限，预后差。免疫治疗是近年肿瘤治疗的热点，其中包括各种树突状疫苗的制备。本项目中我们首先运用CCK-8和LDH释放检测β-榄香烯处理小鼠胶质瘤细胞系GL261 72小时后的细胞活性与毒性，发现小于20 ug/ml的β-榄香烯无明显的细胞毒性，而大于20 ug/ml的β-榄香烯作用后具备随剂量增加而增加的细胞毒性。随后我们进行了自噬相关实验，WB实验发现β-榄香烯与雷帕霉素作用类似，都增加了GL261细胞LC3Ⅱ蛋白表达与LC3Ⅰ蛋白表达之间的比值,同时增加了p62蛋白表达，表明β-榄香烯与雷帕霉素作用相似，都能促进GL261细胞自噬，并增加自噬流。随后我们检测了β-榄香烯诱导的自噬是保护性自噬还是损伤性自噬。CCK-8试验检测发现自噬抑制（3-MA, Bafilomycin A1与atg12-siRNA）后，β-榄香烯作用于GL261细胞后细胞活性更低。另外，流式Annexin V/PI双染色发现自噬抑制后，增加了β-榄香烯诱导的细胞凋亡，表明β-榄香烯促进小鼠恶性胶质瘤细胞系GL261保护性自噬。随后，β-榄香烯处理后募集的带有模式抗原OVA的自噬小体-DCs疫苗能在体外刺激OT-1 CD8阳性细胞活化，增殖细胞增加。最后我们进行了荷瘤小鼠的抗肿瘤检测，我们发现制备的DCs疫苗能减缓原位种植瘤的生长，并促进小鼠存活。另外，我们发现低剂量β-榄香烯具备神经保护的作用。20 ug/ml的β-榄香烯促进大鼠脊髓运动神经元细胞系VSC4.1的细胞活性，并减缓氯化钴的损伤。WB检测发现β-榄香烯减少氯化钴诱导的内质网应激相关蛋白（p-PERK，p-eif2a，ATF4,CHOP,GRP78,Cleaved-Caspase12,Bax,Cleaved-caspase3），增加抗凋亡蛋白Bcl2的表达。BBB评分发现β-榄香烯治疗促进了脊髓损伤后大鼠后肢功能的恢复，表明β-榄香烯可能通过减轻内质网应激减少脊髓损伤后大鼠神经元损伤，从而促进运动功能恢复。