CIK细胞源外泌体对髓系白血病细胞生物学特性的影响

Exosome is a nanosized membrane microvesicle which is shed from almost all living cell types involved in cell-to-cell substance transporting and signaling. Leukemia is a malignant proliferation hematopoietic stem and progenitor cells as well as a blockade of differentiation. At present it is an incurable disease. CIK cells have strongly proliferation ability with broad spectrum of killing tumor as well as multidrug resistance cells,which is benefitial to myeloid leukemia. However, the cytotoxicity activity of CIK cells derived exosomes still has no evidence, and its promoting tumor proliferation or inducing apoptosis in tumor bearing enviroment remains to be elucidated. The exosomes obtained by ultracentrifuge.Then explored its role in affecting the proliferation, apoptosis and invasion of myeloid leukemia by flow cytometry, qRT-PCR, Western blotting. We examined gene expression profiles of serum samples from mouse bearing myeloid leukemia after incubation with exosomes by genome wide miRNA microarray and quantitative real-time PCR(qRT-PCR), which results in the most significant differntially expressed miRNAs. Then the function of miRNAs are determined by lentiviral vector system and animal model. The project intented to clarify the cytotoxicity effect of CIK cells derived exosomes in tumor bearing environment which contribute to strengthen the immune activities or help immune escape to promote progression? It is important to explore the mechanism of immunoediting and providing theoretical basis for supplementary anti-tumor efficacy of CK cells, as well as its reasonable application and save medical resources.

外泌体是细胞分泌的纳米级别的膜性小泡，是细胞间物质和信息转导的通信分子。白血病是一种造血干/祖细胞恶性增殖并伴有分化受阻的恶性疾病，目前尚不可治愈。CIK细胞增殖能力强，杀瘤谱广，对耐药的肿瘤细胞也敏感，对髓系白血病治疗有效。但对于CIK细胞源外泌体是否有溶瘤作用，在荷瘤环境中是促肿瘤细胞增殖，还是诱导凋亡尚不清楚。本项目拟通过超速离心获取CIK细胞源外泌体，在体外、体内，借助流式、qRT-PCR、Western blotting等检测其对髓系白血病细胞恶性生物学行为的影响；通过基因芯片技术检测两者共同作用后血浆miRNA表达谱的变化，并验证"表达差异最显著"miRNA的功能，回答CIK细胞源外泌体是发挥正向调节的溶瘤作用，还是有利于诱导免疫逃逸促进疾病的发生发展。本项目对阐明肿瘤免疫编辑机制有重要意义，可补充丰富CIK细胞的溶瘤机制，为合理、有效应用CIK细胞及节约医疗资源提供重要信息。

微囊泡（microvesicles, MVs）是活细胞分泌到胞外的脂质双层膜包被的囊性小泡，根据体积大小，分为微粒、外泌体和凋亡小体。微囊泡可近距离和/或经体液流动远距离介导细胞间物质和信息的交流，影响受体细胞的生理和病理活动。细胞因子诱导的杀伤细胞(cytokine-induced killer cells, CIK)是以CD3+CD56+双阳性细胞为主的异质细胞群，可有效清除白血病微小残留病灶，但目前对CIK细胞源微囊泡的生物学特性及其对白血病细胞的作用尚不清楚。本项目通过超速离心/SBI公司的外泌体抽提试剂盒获取微囊泡，借助电镜（扫描/透射）观察微囊泡的形态，用qRT-PCR、Western blotting检测微囊泡的标记物，在细胞水平、分子水平观察到微囊泡对髓系白血病K562细胞增殖无明显影响，但促进其迁移以及抵抗凋亡，该作用可能与CIK细胞源MVs上调IDO-1、MMP-9在K562细胞表达，下调VEGF的表达有关。其次，我们观察到白血病细胞源外泌体上调IL-8在髓系白血病患者骨髓间充质干细胞的表达，而IL-8促进白血病细胞增殖，提示白血病细胞经外泌体驯化肿瘤微环境中的间充质干细胞，为自己的生存创造一个有利条件。另外，我们用高通量测序法获取白血病患者血清外泌体源miRNA分子表达谱，在急性髓系高白细胞白血病组筛选到表达水平有统计学差异的表达上调的15个miRNA，表达下调miRNA11个；在急性髓系非高白细胞白血病组表达上调的miRNA有11个，表达下调的miRNA有8个；对其进行生物信息学分析的结果提示差异表达的miRNA调控的靶基因主要在MAPK、Wnt、NF-κB和hippo信号通路中出现富集。本项目的研究结果为开展免疫细胞治疗以及白血病的分子机制研究提供重要信息。