摘要
目的 筛选耐热 T2 4细胞亚克隆 ,研究其生物学特性变化 .方法 用热暴露筛选 ,联合应用免疫荧光化学方法及流式细胞仪筛选高表达 HSP70的 T2 4细胞亚克隆 ,计数法测定其细胞生长曲线及细胞倍增时间 ,流式细胞仪分析其细胞周期的变化 .分子生物学方法及细胞化学方法观察有无凋亡出现 .结果 有限稀释法筛选获取耐热 T2 4单克隆细胞株(Hr T2 4)共 2 6株 ,应用免疫荧光法筛选出 HSP70阳性克隆 12株 ,取荧光较强的两株命名为 Hr T2 4e,Hr T2 4j,FCM(平均荧光强度为 0 . 2 9)检测 HSPs分子的阳性表达率 T2 4,Hr T2 4e,Hr T2 4j分别为 2 .4% ,37.6 % ,6 5 .8% .细胞周期分析显示 T2 4,Hr T2 4e,Hr T2 4j增殖指数 (proliferation index,PI分别为44 .3% ,39.0 % ,38.5 % ,Hr T2 4e与 T2 4相比差异有显著意义(Yates corrected,χ2 =5 .5 6 ,P=0 .0 183) ,Hr T2 4e与 T2 4j相比差异无显著意义 (Yates corrected,χ2 =0 .0 3,P=0 .85 43) .Hr T2 4e与 Hr T2 4j凋亡率分别为 4.7% ,5 .9% .分子生物学方法观察到 DNA凋亡梯带出现 ,细胞化学方法观察到凋亡小体 .结论 Hr T2 4细胞 HSP70表达增加 ,出现了一定的增殖抑制现象 ,同时观察到细胞凋亡 。
AIM To isolate T 24 cells with high expression of HSP70 and to investigate their biological characteristics change after being exposed to the hyperthermia. METHODS Hyperthermia resistant T 24 cells clone (HrT 24 ) was isolated with immune fluorescence chemistry techniques and flow cytometer (FCM). The growth curves and double proliferation time of HrT 24 were evaluated with cytometre. The diploid cycle and apoptotic peak were analyzed with FCM. Culture curve analysis was studied with cytometre and FCM. The apoptosis was also evaluated with molecular biology (DNA ladder) and fluorescence cell chemistry techniques. RESULTS The percentage of the HSP70 positive T 24 , HrT 24e and HrT 24j were 2.4, 37.6 and 65.8 respectively. Proliferation of HrT 24 was slightly lagged. Double proliferation time (Dt) of HrT 24j was 26.65 h while the Dt of T 24 was 24.7 h. With FCM assay, the proliferation index (%) of the T 24 , HrT 24e and HrT 24j were 44.3, 39.0 and 38.5 respectively. The difference between HrT 24e and T 24 was significant (Yates corrected, χ 2 =5.56, P =0.0183), while the difference between HrT 24e and HrT 24j was slight (Yates corrected, χ 2 = 0.03, P =0.8543). The apoptotic body was found only in HrT 24e and T 24j with fluorescence cell chemistry techniques, the apoptotic DNA ladders were proved with molecular biochemical techniques. CONCLUSION HrT 24 cells may obtain better immunogenicity by expressing more HSP70 and providing more apoptotic body.
出处
《第四军医大学学报》
北大核心
2001年第20期1838-1841,共4页
Journal of the Fourth Military Medical University
