CD100阳性NK细胞上调Plexin-B1影响胶质瘤细胞凋亡和侵袭中的分子信号机制

Glioma is the common type of intracranial malignant tumor. Inflamation induced-cytotoxicity to tumor cell is one of the new strategy for glioma immunotherapy. CD100 positive natural killer (NK) cells are one of the effector lymphocytes of the innate immune system bearing strong adhension effect that control several types of tumors and microbial infections. Previously, we found that CD100 positive NK cells were the major lymphocytes appearing in the cerebrospinal fluid from glioma patients undergoing the intracranial infection after the operation. Meanwhile, the soluble form of CD100 (sCD100) were greatly increased in the cerebrospinal fluid, and the recurrence of glioma in 15 months of patients were greatly reduced .Further studies showed that Plexin-B1, receptor for CD100, expressed on glioma cells was upregulated when binding with CD100+ NK cells during the intracranial inflammation, and regulated the expression of Survivin and MMP9, which involved in the apoptosis and invasion of glioma cells. However, the mechanisms underlying the anti-glioma effect of this subset is still unknown. In the present study, we will first detect the roles of CD100 expression on NK cells and sCD100 during the the intracranial inflammation after the operation in glioma patients. Then we will establish coculture systems containing both NK cells and glioma cells, and modle of NOD/SCID mice bearing glioma .After that we will detect the molecular mechanisms of CD100 positive NK cell in regulation of molecules involved in Plexin-B1-Survivin/MMP9 pathway on the apoptosis and invasion of glioma cells both in vitro and in vivo. This study may declare the mechanisms of CD100 positive NK cells to glioma cells during the intracranial inflamation after the craniocerebral operation and provide the new experimental basis to improve the success of glioma immunotherapy.

胶质瘤治疗是当今医学界亟需解决的难题。文献报道，炎症应答诱导免疫系统杀伤胶质瘤细胞是重要的治疗手段。CD100阳性NK细胞（NKCCD100+）是黏附性最强的NKC亚群之一，既参与炎症免疫应答，又拮抗肿瘤，但分子机制尚不清楚。我们前期研究发现，发生颅内感染的胶质瘤患者脑脊液中，NKCCD100+亚群数量最多，患者远期肿瘤复发率显著降低。进一步发现，NKCCD100+黏附于胶质瘤细胞上，上调其受体Plexin-B1，进而调控肿瘤相关分子Survivin和MMP9表达，强烈提示NKCCD100+在抑制胶质瘤增殖和侵袭中发挥重要作用。本课题拟搞清NKCCD100+亚群等在患者脑脊液中的表达规律。深入探讨并明确NKCCD100+调控胶质瘤细胞Plexin-B1-Survivin/MMP9信号通路,进而影响胶质瘤细胞侵袭和转归的功能特点和分子机制，为临床应用炎症应答治疗胶质瘤提供新的理论和实验依据。

胶质瘤是中枢神经系统最常见的恶性肿瘤，以往认为大脑是一个“免疫缺陷”器官，且胶质瘤具有只在颅内侵袭，不转移到其他器官的生物学特性，导致人们忽视了免疫系统拮抗胶质瘤的作用。近年来，文献和我们临床发现，部分颅内感染的胶质瘤患者，生存期明显延长，生存治疗显著提高，甚至治愈。但由于机制不清、疗效不佳及诱发炎症的不可控性等缺点，免疫应答调节剂应用受到很大的限制。如何提高免疫应答治疗胶质瘤的效果是是临床亟待解决的问题。本课题在上述工作基础上，利用前期建立的细胞共培养的胶质瘤模型和NOD/SCID荷瘤小鼠颅内感染模型，① 研究脑组织和脑脊液中CD100分子在NKC上的表达规律及sCD100的产生，及胶质瘤细胞中CD100受体Plexin-B1的表达，及其与临床疗效的关系；② 研究调控Plexin-B1表达后胶质瘤细胞系体外生物学性状的变化；及其调控Survivin表达，影响肿瘤细胞凋亡和调控MMP9表达，影响肿瘤细胞侵袭的分子信号机制；③构建胶质瘤细胞系和NOD/SCID荷瘤小鼠颅内感染模型，明确上调CD100和sCD100后肿瘤细胞侵袭和凋亡的变化；及两者上调其受体Plexin-B1表达的信号机制研究。我们首先在成功培养U87胶质瘤细胞系和共培养U87和NK细胞；同时，我们利用 CD100基因敲除小鼠等，成功构建了NOD/SCID荷瘤小鼠颅内感染模型；其次，研究发现伴发颅内感染的胶质瘤患者的脑脊液和脑组织中，NKCCD100+、sCD100和Plexin-B1高表达，阳性率高；再次，研究发现NKCCD100+、sCD100和Plexin-B1在NKC和胶质瘤细胞上的表达变化规律，与正常对照组和胶质瘤细胞组比较，合并感染的胶质瘤细胞组中，CD100、sCD100和Plexin-B1蛋白及其mRNA均高表达（P<0.05）。再次，筛选并构建了CD100、sCD100和Plexin-B1基因的慢病毒过表达载体和培育CD100基因敲除小鼠，上调CD100的受体Plexin-B1表达，不仅降低Survivin表达，促进胶质瘤细胞凋亡，而且下调MMP9表达，抑制肿瘤细胞的侵袭；最后，研究发现过表达CD100，可上调NKC上的Plexin-B1表达，胶质瘤细胞上Plexin-B1表达无变化；而上调sCD100, 则下调NKC上Plexin-B1表达，胶质瘤细胞上Plexin-B1表达增高，进