消减免疫法制备抗人肝癌凋亡细胞抗体

Preparation of anti-apoptotic human hepatoma cells antibody with subtractive immunization method

目的 分析原发性肝细胞癌 (简称肝癌 )细胞凋亡过程中凋亡相关抗原的表达情况 ,探讨制备抗凋亡细胞抗体的方法。方法 用 6%乙醇作用 6h诱导肝癌细胞系HCC 92 0 4细胞凋亡 ,透射电镜观察其形态学变化 ,流式细胞仪分析细胞DNA含量。消减免疫法免疫 10只Balb/c小鼠。分别于初次免疫后第 14d、2 1d、和 2 8d取鼠尾血。免疫细胞化学法检测抗体产生情况及其特异性。结果 透射电镜可见大部分细胞出现凋亡形态学变化。流式细胞仪分析可见明显的亚二倍体凋亡峰。初次免疫后第 2 8d ,有 2只鼠的尾血在凋亡细胞呈强阳性 ,而与非凋亡细胞的反应很弱。结论 肝癌细胞凋亡过程中有凋亡相关抗原的表达升高或新抗原的产生。消减免疫法有助于制备抗在凋亡细胞高表达 ,而在非凋亡细胞低表达或不表达的凋亡相关抗原的抗体。

Objective To analyze the expression of apoptosis-associated antigens of hepatocellular carcinoma(HCC) during the process of apoptosis,and probe into the method to prepare antibodies against apoptosis-associated antigens.Methods Apoptosis of HCC cell line HCC-9204 cells was induced with 6% ethanol fr 6h.The morphological changes were observed with transmission electron microscope.The DNA content of the cells was analyzed with flow cytometer.10 Balb/c mouse were immunized with subtractive immunization method.The tail blood of the mice was taken on 14th,21st and 28th day after first immunization.The production and specialty of antibodies were detected with immunocytochemistry.Results Most cells showed apoptotic morphological changes under transmission electron microscope.Typical apoptotic sub-G1 peek was seen under flow cytometer.There were 2 mouse whose tail blood reacted strongly with apoptotic cells,but very weakly with non-apoptotic cells in the 28th day after first immunization.Conclusions There exists apoptosis-associated antigens during the process of HCC apoptosis.Subtractive immunization is helpful for preparing the antibodies against the apoptosis-associated antigens which expresses highly in apoptotic cells,but lowly or barely in non-apoptotic cells.