鼠抗人CXCR3单克隆抗体的研制及生物学功能研究

Mouse anti-human CXCR3 mAb preparation and its biological function

目的:获得持续分泌鼠抗人CXCR3单克隆抗体(mAb)的杂交瘤细胞株;以鼠抗人CXCR3 mAb作为工具,研究人CXCR3分子的表达特性及CXCR3信号转导对L929-huCXCR3和结肠癌细胞株的迁移及增殖的影响。方法:以高表达人CXCR3膜型分子的L929-huCXCR3细胞作为免疫原免疫BALB/c小鼠,采用B淋巴细胞杂交技术,将免疫小鼠的脾脏细胞与同种系小鼠的骨髓瘤细胞Sp2/0进行融合。以L929-huCXCR3作为筛选细胞,转染空载体的L929-mock细胞作为阴性对照细胞,采用间接免疫荧光和流式细胞术,筛选能持续分泌抗人CXCR3 mAb的杂交瘤细胞株。采用Ig亚类快速定性试纸法和间接免疫荧光法对所获得的杂交瘤细胞株和mAb进行鉴定;用间接免疫荧光法分析CXCR3分子在肿瘤细胞表面的表达;Transwell隔离小室检测mAb对L929-huCXCR3和结肠癌细胞株Colo205、HCT116及HT29迁移的影响;MTT法分析mAb对结肠癌细胞株Colo205增殖的影响。结果:获得了1株能持续分泌鼠抗人CXCR3 mAb的杂交瘤细胞株,命名为9B5。经快速定性试纸分析显示,该mAb重链为IgG1亚类,轻链为链;间接免疫荧光和流式细胞术分析显示,mAb 9B5可识别活化T淋巴细胞和结肠癌细胞株Colo205、HCT116及HT29表面的CXCR3分子。通过阻断CXCR3信号转导,mAb 9B5可抑制L929-huCXCR3细胞和结肠癌细胞株Colo205、HCT116和HT29的定向迁移及IP-10对Colo205的促增殖作用。结论:成功获得了1株能持续分泌鼠抗人CXCR3 mAb的杂交瘤细胞株,为研究CXCR3的表达特性及深入探讨CXCR3信号转导在肿瘤生长与转移过程中的作用及机制奠定了物质基础,并且有望为治疗肿瘤转移提供新的思路和新型药物。

AIM： Obtain hybridoma cell line with continuing expression of mouse anti-human CXCR3 mAb, investigate expression characteristics of human CXCR3 and how CXCR3 signal transduction function on L929-huCXCR3 and colon carcinoma cell lines transfer and growth. METHODS： Taking L929-huCXCR3 cell with high expression of human CXCR3 membrane molecule as immunogen to immunize BALB/c mouse, we fused immunized mouse spleen cell with myeloma cell Sp2/0 of its same germ line, then used L929-huCXCR3 as screening cell and empty vector transfected cell L929-mock as negative control. Obtained hybridoma cell line with continuing secretion of anti-human CXCR3 mAb through flow cytometry. We used Ig subclass type rapid qualitation indicator paper method and indirect immunofluorescence to identify obtained hybridoma cell line and mAb, indirect immunofluorescence to analyze CXCR3 expression on tumor cell surface, Transwell isolation cabin to assess effect on L929-huCXCR3 and colon carcinoma cell line Colo205, HCTll6 and HT29 migration by mAb, MTF method to analyze how mAb function on colon carcinoma cell line Colo205 proliferation. RESULTS： Obtained a hybridoma cell line with continuing secretion of mouse anti-human CXCR3 mAb, named 9B5. According to rapid qualitati- on test paper analysis, light chain of the mAb was chain and heavy chain is IgG1 subclass. Indirect immunofluorescence and flow cytometry results show that the mAb can recognize CXCR3 molecules on the surface of activated T lymphocyte and colon carcinoma cell line Colo205, HCT116 and HT29 cell. mAb 9B5 can inhibit oriented migration of L929-huCX- CR3 cells, colon carcinoma cell line Colo205, HCT116 and HT29 cell, it can also inhibit Colo205 growth promotion effect by IP-10. CONCLUSION： Successfully obtain a hybridoma ceil line with continuing secretion of mouse anti-human CXCR3 mAb, which has laid material foundation on investigation of CXCR3 expression characteristics and CXCR3 signal transduction function on tumor growth and migration. It is prospective to create a new way and a new drug for treatment of tumor metastasis.