摘要
目的构建hIFN-α-2B-BCG重组体，探讨其对膀胱肿瘤细胞的生物学效应。方法对重组BCG与常规BCG生长情况和形态学进行比较；生长10代后，ELISA法测定菌体内、外表达hIFN-α-2B量。重组BCG体外作用于膀胱肿瘤细胞EJ、MB49后行电镜和MTY检测细胞抑制率。结果常规BCG与重组BCG生长差异无统计学意义：1～7d为近似对数生长期，600nm处吸光度（A）值从0．1升到1．2左右，然后进入平台期。重组BCG和常规BCG抗酸染色均为阳性，均保持相互连接的特点，但重组BCG稍大，余无明显异常。1ml重组BCG分泌IFN—α-2B为997．2pg，菌内含量为99.3pg。连续传10代的重组BCG分泌量为990．3pg，与第一代无明显差别，二者形态学也未见明显差异。重组BCG作用于肿瘤细胞后可见细胞表面微绒毛减少，胞质内线粒体肿胀，空泡样变性，核染色质溶解，细胞质坏死。重组BCG对两种细胞的抑制率都显著高于野生BCG（P〈0．05）。结论重组hIFN-α-2B-BCG的分泌性表达持续稳定，其生长和形态特征与野生型基本一致。重组BCG体外对肿瘤细胞的抑制杀伤作用强于野生BCG。
Objective To investigate the biological characteristics of recombinant hIFN-α-2B-BCG and its direct effect to bladder tumor ceils in vitro. Methods BCG and recombinant BCG（rBCG） wild-type growth and morphology were compared. After 10 generations, hIFN-α-2B content was analyzed by ELISA, of bacteria in vivo and in vitro. The effects of rBCG on bladder cancer cells EJ, MB49, were detected by electron microscopy and cell inhibitory rate from MTr. Results The normal BCG and rBCG had no significant difference in growth phase. They were both positive for acid-fast stain, maintaining the characteristics of cell＇s connection. While rBCG slightly larger than normal BCG, no else abnormality was obvious between them. IFN-α-2B was 997.2 pg/ml in secretions, 99.3 pg. In bacteria in vivo, 990.3 pg/ml in 10th generation＇s sections, of rBCG. Compared with 1st generation, rBCG had no significant differences in morphology and interferon expression. Normal BCG and rBCG both had, anti-proliferation directly on bladder tumor cell in vitro, the rBCG is most effective in all. After rBCG cultivated with bladder tumor cell together, tumor cell＇s slow proliferation, detach, quantity decreasing and death were observed under microscope. Different degeneration in degree on most of tumor cells, disorganization on organelle, aggregation on chromatin, pyenosis on nucleolus, and cytoplasm lysis were on tumor cell under transmission electro microscopy. MTT assay showed rBCG inhibited the proliferation of bladder caner cell and more active than normal BCG. Conclusion These results suggest that the rBCG have the same characteristics in growth phase as normal BCG, and stable properties in interferon expression and morphology by generations, rBCG has more anti-tumor effects in vitro than normal BCG.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2010年第1期46-50,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助（30700834）
天津市科技支撑计划重大项目（07ZCKFSH03200）
国家科技部“十一五”重大专项新药创建课题（2009ZX091034-699）
