论文部分内容阅读
目的 :建立用 EBV转化长寿人群外周血淋巴细胞永生细胞株 (L CL)的方法。方法 :用 EBV转染 8份百岁以上健康老人 ,2 0份 89～ 99岁健康老人及 8份 6 6岁以下健康人的血样共 36份。采用 2 0 % FBS RPMI16 4 0培养液 ,配合环孢菌素 A、植物血凝素 (PHA) ,置 37℃ 5 % CO2 培养箱内培养。结果 :成功地获得 2 4份永生细胞株 (其中 ,百岁老人 6份 ,89～ 99岁老人 12份 ,6 6岁以下 6份 )。结论 :采血后 2 4～ 4 8h之间接种 ,转化成功率高。转染后 2～ 3周 ,大部分标本出现细胞分裂、变形 ,标志着转化成功。超过 5周仍无细胞分裂者即发生细胞凋亡。PHA有缓解细胞凋亡的作用 ,但不能使细胞分裂。污染是引起转染细胞株凋亡的主要因素。严格的无菌操作、保持培养细胞的适当密度、适时换液、打散较大的细胞克隆团是培养 L CL 的关键
Objective: To establish a method to transform immortalized human peripheral blood lymphocyte cell line (L CL) by EBV in longevity population. Methods: A total of 36 blood samples were obtained from 8 healthy people over 100, 20 healthy elders aged 89-99, and 8 healthy people under 6 years old. The cells were cultured in 20% FBS RPMI1640 medium supplemented with cyclosporine A and phytohemagglutinin (PHA) and incubated in a 37 ° C 5% CO 2 incubator. RESULTS: Twenty-four immortalized cell lines were successfully obtained (of which 6 were centenarians, 12 were 89-99 years old and 6 were under 6 years old). Conclusion: After the blood collection 24 ~ 48h inoculation, high conversion rate. 2 to 3 weeks after transfection, the majority of specimens appeared cell division, deformation, marking the successful conversion. Apoptosis occurs when there is no cell division for more than 5 weeks. PHA has the effect of ameliorating apoptosis, but does not divide the cells. Pollution is the main factor that causes apoptosis in transfected cell lines. Strict aseptic operation, to maintain the appropriate density of cultured cells, timely liquid change, break up the larger cell cloning group is the key to the cultivation of L CL