Fura-2/AM荧光法测定日本血吸虫培养细胞内游离钙离子浓度

DETERMINATION OF INTERCELLULAR FREE Ca^(2+) CONCENTRATION IN TH E CULTURED CELLS FROM SCHISTOSOMA JAPONICUM BY USING FURA-2/A M

目的 研究用钙荧光探剂 (Fura- 2 / AM)检测静息状态下日本血吸虫培养细胞内的游离钙离子浓度 ([Ca2 + ]i) ,以及β-巯基乙醇对培养细胞 [Ca2 + ]i的影响。方法 将 18d虫龄的日本血吸虫童虫制成细胞悬液 ,贴壁法接种于 30 ml培养瓶中 ,培养液为 RPMI- 16 4 0含 2 0 %小牛血清及常量抗生素。在培养的 0 - 3d,制备日本血吸虫细胞悬液 ,采用 Fura- 2 / AM钙荧光探剂测定正常静息状态下及加入β-巯基乙醇后日本血吸虫培养细胞内的 [Ca2 + ]i。结果 正常静息状态下 ,培养 0 d的日本血吸虫培养细胞内的 [Ca2 + ]i为 188.2 nmol/ L ;培养 1- 3d的 [Ca2 + ]i两两比较差异无显著性(P>0 .0 5 ) ,它们的平均值为 (187.0± 10 .7) nm ol/ L ,与培养 0 d的 [Ca2 + ]i比较 ,差异也无显著性(P>0 .0 5 )。β-巯基乙醇可使日本血吸虫培养细胞内 [Ca2 + ]i明显升高 (P<0 .0 1) ,并随浓度的增加而升高。结论 培养 1- 3d,静息状态下日本血吸虫培养细胞内的 [Ca2 + ]i比较稳定 ,β-巯基乙醇能影响日本血吸虫培养细胞内的 [Ca2 +

ObjectivesTo determine intra ce llular free Ca 2+ concentration ([Ca2+ ]i ) in the cultured cells f rom Schistosoma japonicum by using Fura 2/AM, and to evaluate the eff ect of the β Mercaptoethanol on [Ca 2+ ]i of the cultured cells. MethodsThe cell suspension from 18 day old Schis t osoma japonicum was inoculated in 30 ml flasks by adherence. RPMI 1640 with 20% calf serum and nomal amount of antibiotics was used as the culture medi a. Zero to three days after culture, cells were collected and cell suspension wa s prepare d. Free Ca 2+ concentrations in the cultured cells of rest stage and after tr eated by β Mercaptoethanol were detected by using Fura2 /AM. Re sultsIn rest stage, [Ca 2+ ]i in the cells cultured at day 0 was 188.2 nmol/L. [Ca 2+ ]i was not different significantly betwee n the cells cultured for 1, 2 and 3 days(P>0 05), being ( 187 0±10 7) nmol/L in average. Moreo ver, it was not significantly different from the cells cultured at day 0, ei ther . However, [Ca 2+ ]i increased significantly(P<0 01) af ter t he cultured cells were treated by β Mercaptoethanol, and it correlated positi vely with the concentration of β Mercaptoethanol. Conclusion In rest stage, [Ca 2+ ]i in the cells cultured for 1-3 d ays is very stable.β Mercaptoethanol can increase [Ca 2+ ]i in the cul ture d cells.