急性淋巴细胞白血病（ALL）逃逸NK细胞杀伤的机制研究

项目名称： 急性淋巴细胞白血病（ALL）逃逸NK细胞杀伤的机制研究

项目编号： No.30873014

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 段连宁

作者单位： 中国人民解放军空军总医院

项目金额： 30万元

中文摘要： 通常，急性淋巴细胞白血病（ALL）比急性髓系白血病（AML）有更好的预后效果，但有趣的是NK细胞抗ALL的作用反而更弱，具体机制仍不清楚。本研究拟通过PCR技术大规模检测ALL和AML细胞系的NK配体，探讨NK配体表达差异，构建能增强NK细胞活性的嵌合受体并稳定转染NK-92细胞株，并在细胞水平和动物模型进行验证。经3年的研究，已获部分成功，主要结果：1、通过半定量PCR发现NK-92不敏感的白血病细胞系低表达激活性配体PVR而高表达抑制性配体HLA-F；2、成功建立26种NK配体的实时定量PCR检测方法，发现ALL的ULBP-2高于AML，而ALL的PVR低于AML；3、流式细胞术检测临床标本，发现ALL患者白血病细胞的MICA/B和HLA-E阳性率均高于AML；4、成功克隆6种NK野生型受体，并构建5种嵌合受体，然而转染NK-92细胞均未获成功；5、课题组适当调整技术路线，研究NK细胞发挥效应时的自身凋亡机制，证明白血病细胞可通过FasL/Fas途径诱导NK凋亡；6、为进一步研究白血病的致病机制，建立小鼠单倍体相合骨髓移植模型，并进行移植耐受的初步探讨，目前仍在继续相关研究。

中文关键词： 白血病；自然杀伤细胞；配体；嵌合受体；凋亡

英文摘要： In general, acute lymphoblastic leukemia (ALL) has a better prognosis than acute myeloid leukemia (AML). But, interestingly, ALL is much more resistant to NK cells than AML and the specific mechanism remains unclear. Our study plans to detect large-scale NK ligands by PCR technology and finds out the different expressions between ALL and AML. On this basis, we will construct several chimeric receptors which could enhance NK cytotoxicities effectively. Then, screen stably transfect NK-92 cell lines and test their effectiveness in vitro and in animal models. After 3 years of study, we have achieved some important results as follows: 1. Through semi-quantitative RT-PCR method and flow cytometry, we found that PVR of NK insensitive leukemia cell lines was the lowest while HLA-F of which was the highest. 2. We have established real-time PCR methods which can detect 26 NK ligands availably. The results suggest that ULBP-2 of ALL is higher than AML, while PVR is lower. 3. Also, we tested some clinical specimens by FCM and found that MICA/B and HLA-E of ALL patients' leukemia cells are higher than AML patients'. 4. We have cloned 6 wild-type NK receptors and constructed 5 chimeric receptors. However, NK-92 is difficult to be transfected and we haven't screen the stable cell line yet. 5. Then, we made some adjustments accordingly. We focused on the apoptosis of NK cell during its cytotoxic effect and proved that leukemia cell lines could induce apparent apoptosis in NK cells through FasL/Fas pathway. 6. We have established a haploidentical bone marrow transplantation mouse model in non-myeloablative conditioning in order to further study the pathogenesis of leukemia. We found that transfusion of donor spleen cells before BMT can promote implantation of hemopoietic stem cells.

英文关键词： Leukemia; Natural killer cell; Ligand; Chimeric receptor; Apoptosis