人bFGF单克隆抗体的制备及MabF7对黑色素瘤B16的体外抗瘤效应

Production preparation of human bFGF monoclonal antibodies and their antitumor activities with MabF7 on melanoma cells B16 in vitro

目的:制备抗人碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)单克隆抗体,并探讨其体外对黑色素瘤细胞B16的抗肿瘤效应。方法:以rhbFGF免疫Balb/C小鼠,用杂交瘤技术制备单克隆抗体;间接ELISA法筛选阳性杂交瘤细胞株,测定腹水型单抗的效价及其Ig亚类;用RT-PCR、细胞免疫组化和双抗体夹心等方法检测黑色素瘤B16细胞中bFGF的表达;用MTT法检测单抗MabF7对B16细胞的体外抑瘤效应;用流式细胞术及DNA琼脂糖电泳技术检测细胞凋亡。结果:共获得19株稳定分泌抗bFGF单抗的杂交瘤细胞株;ELISA效价>105;除MabF8、MabF9为IgG2a外,其余为IgG1;黑色素瘤细胞B16中有bFGFmRNA及其蛋白的表达,且可在细胞培养液中检测到bFGF;经蛋白A纯化的MabF7对B16细胞的抑制作用呈剂量及时间累计效应,抑制率为(39±4.12)%,P<0.01。流式细胞术结果显示,随抗体浓度增加及作用时间延长,肿瘤细胞在G1期前出现明显的亚二倍体期;DNA电泳结果显示,抗体作用后的细胞DNA呈明显的梯度条带。结论:MabF7具有体外抑制黑色素瘤B16细胞增殖作用,其机制与诱导细胞凋亡有关。

OBJECTIVE：To produce monoclonal antibodies against recombinant human bFGF and explore their antitumor effects on melanoma cells B16 in vitro. METHODS： Hybridomas were obtained according to the routine procedure with Balb/c mice immunized with rhbFGF; indirect ELISA was used to screen positive hybridoma clone and conform the titrations and subtype of the monoclonal antibodies; expression of bFGF mRNA and bFGF were detected by RT-PCR, immunocytochemistry and the double antibody sandwich ELISA assay; MTT assay was used to investigate the inhibitory effect of MabF7 on B16 Cells in vitro. Cell apoptosis was aseessed by flow cytometry and DNA electrophoresis. RESULTS： Nineteen hybridoma clones designed as MabF1-19 were generated; the titers of most of the ascite fluids were above 105 ; MabF8 and MabF9 were IgG2a isotype, and the others were IgG1 isotype; bFGF mRNA and bFGF could be detected in B16 cells, also in cell culture. In vitro MTT test showed that the inhibitory effect of purified MabF7 by proteinA was in a dose-and timedependent manner, and the killing rate was （39 ± 4.12）%, P〈0.01. The technique of flow cytometry showed that the Sub-G1 peak was more and more obviously with the exposure time and dose increase; DNA electrophoresis exhibited a DNA ladder pattern. CONCLUSION.. MabF7 has the inhibitory effect on B16 cells in vitro and the possible mechanism is cell apoptosis.