B-CLL患者瘤细胞膜表面Id-ScFv和人Hsp70的制备及其抗瘤效应

背景与目的:恶性B型淋巴细胞表面免疫球蛋白(surface membrane immunoglobulin,SmIg)表达的独特型决定簇(Idiotypic determinant,Id)不仅是该类肿瘤特异性标记,也是该类肿瘤的特异性抗原,可诱导机体对其产生特异性免疫应答,但由于Id是自体成分,分子量小,免疫原性较弱。人热休克蛋白70(heat shock protein,Hsp70)是一类重要的抗原提呈分子,能有效加强抗原肽的免疫原性。本研究通过制备慢性B型淋巴细胞性白血病(B-cell chronic lymphatic leukemia,B-CLL)患者瘤细胞膜表面独特型单链抗体(Idiotypic determinant single-chain antibody,Id-ScFv)和Hsp70两种蛋白,在体外研究两者联合抗瘤作用并初步探讨其机制。方法:分别在大肠杆菌中表达Id-ScFv和Hsp70两种蛋白,表达产物经SDS-PAGE电泳(sodium dodecyl sulfate polyacrylamide gel electropheresis)及ELISA(enzyme-linked immunosorbentassay)检测鉴定后,分别用金属螯合层析和离子交换层析纯化并在体外将这两种蛋白结合成复合物(Hsp70-Id)。用MTT法及检测Id-ScFv组、人Hsp70组及Hsp70-Id组刺激外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的增殖作用,以ELISA法检测各组培养上清中IL-12和TNF-α水平,并用流式细胞术检测各组PBMC亚群的变化。用活细胞计数法检测各组被激活的PBMC对慢性B细胞白血病细胞株Daudi、慢性髓性白血病细胞株K562和肝癌细胞株HepG2的杀伤作用。结果:经SDS-PAGE电泳分析纯化后表达产物的分子量大约为30ku(Id-ScFv蛋白)和70ku(Hsp70蛋白),分别与其理论预期值相符。PBMC的增殖作用、培养上清中IL-12和TNF-琢水平,Hsp70-Id组明显强于Id-ScFv组和人Hsp70组(P<0.05),而Id-ScFv组和人Hsp70组明显强于阴性对照组(P<0.05)。流式细胞术检测显示在Hsp70-Id组、Id-ScFv组和人Hsp70组PBMC中CD8 T细胞亚群的百分率均有增加,其中Hsp70-Id组最明显。在Id-ScFv组和Hsp70-Id组激活的PBMC对Daudi细胞的杀伤作用较K562、HepG2细胞强(P<0.05),且Hsp70-Id组激活的PBMC对Daudi细胞的杀伤作用明显强于Id-ScFv组(P<0.001)。结论:成功获取B-CLL患者瘤细胞膜表面Id-ScFv和人Hsp70两种纯化蛋白;Hsp70-Id在体外可增强PBMC的特异性杀瘤作用,其机制可能与促进PBMC的增殖、活化CD8 T细胞并诱导具有抗肿瘤作用的Th1型细胞因子的分泌有关。

Preparation of heat shock protein 70 (Hsp70) and idiotypic determinant single-chain antibody (Id-ScFv) in a patient with B-cell chronic lymphatic leukemia (B-CLL) and antitumor effect of peptide complex Hsp70-Id

BACKGROUND & OBJECTIVE: Idiotypic determinant (Id) of malignant B lymphocyte surface membrane immunoglobulin (SmIg) is not only a specific marker for chronic B-cell lymphatic leukemia (B-CLL) but also a specific antigen as an attractive target for active immunotherapy. However, as a small self antigen, it has poor immunogenicity. As an important molecule for antigen presentation, heat shock protein 70 (Hsp70) can efficiently strengthen the immunogenicity of antigen. In this study, we prepared Hsp70 and idiotypic determinant single-chain antibody (Id-ScFv) fragment against SmIg in patient with B-CLL and explored the in vitro antitumor effect of peptide complex Hsp70-Id and the possible immune mechanism. METHODS: Id-ScFv and human Hsp70 proteins were expressed in E.coli and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA), and purified by metal chelate chromatography and DEAE ion-exchange chromatography. The purified Hsp70 was combined with the prepared Id-ScFv to produce peptide complex Hsp70-Id in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy human were stimulated with Id-ScFv, HSP70 and HSP70-Id, separately. The proliferation rate of PBMCs was determined by MTT assay; the changes of T lymphocyte subsets were assessed by flow cytometry (FCM)? the levels of interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) in supernatant were detected by ELISA. Killing efficiency of activated PBMCs to chronic B-cell leukemia cell line Daudi, chronic myelogenous leukemia cell line K562 and hepatoma carcinoma cell line HepG2 were tested by cell counting. RESULTS: Id-ScFv and Hsp70 were expressed successfully in E.coli. The proliferation rate of PBMCs and secreting levels of IL-12 and TNF-alpha of lymphocytes in Hsp70-Id group were significantly higher than those in Id-ScFv and HSP70 groups (P<0.05), which were significantly higher than that in negative control group (P<0.05). The proportion of CD8+ T cells was increased in Hsp70-Id, Id-ScFv and Hsp70 groups, and the most significant increase was observed in Hsp70-Id group. The activated PBMCs in both Hsp70-Id and Id-ScFv groups had stronger cytotoxic activities to Daudi cells than to K562 or HepG2 cells (P<0.05), and the effect was significantly stronger in Hsp70-Id group than in Id-ScFv group (P<0.001). CONCLUSION: Purified Hsp70 and Id-ScFv proteins in a B-CLL patient were prepared successfully. Hsp70-Id complex could significantly enhance specific antitumor effect of PBMCs in vitro through improving the proliferation of PBMCs, activating specific CD8+ T cells, and inducing secretion of Th1 type cytokines.