条件培养基对日本血吸虫童虫培养细胞LDH及AgNORs的影响

Effects of conditioned medium on LDH and AgNORs in cultured schistosomulum cells from Schistosoma japonicum

目的以乳酸脱氢酶(LDH)及核仁组织区相关嗜银蛋白(AgNORs)为评价指标,观察条件培养基对日本血吸虫童虫培养细胞的影响。方法灌注法获取21d虫龄的日本血吸虫虫体,冷消化法制备细胞悬液,调细胞为1×106个/ml,联合法接种细胞。将接种于小盖玻片上的血吸虫细胞随机分为对照(0h)组和24、48、72h3个实验组共4组。对照组用常规培养基即RPMI-1640含20%小牛血清附加常量抗生素(青霉素1×105U/L,链霉素100mg/L)培养,实验组培养基为常规培养基分别与培养24、48、72h的鼻咽癌细胞上清液的条件培养基1∶1的混合液。培养第7天,对培养细胞进行LDH及AgNORs染色;每组随机选取300个LDH染色细胞、50个AgNORs染色细胞输入HPIAS-2000图像分析仪测定培养细胞的吸光度(A)值,并作统计分析。结果日本血吸虫童虫细胞在不同条件培养基作用下,其LDH及AgNORs着色均比对照组深,其中72h组细胞着色最深,其次是48h组,再是24h组,对照组细胞着色最浅,条件培养基培养的各组细胞,LDH含量及AgNORsA值显著大于对照组细胞(q24=8.5287,q48=15.4253,q72=27.6207,P均<0.01;q24=13.5690,q48=18.7288,q72=27.0356,P均<0.01)。72h的条件培养基作用日本血吸虫童虫细胞后,培养细胞的LDH含量及AgNORsA值明显较对照组高(q0/72=27.6207,P<0.01;q0/72=27.0356,P<0.01),亦高于其他组(q24/72=19.0919,q48/72=11.8426,P均<0.01;q24/72=13.5690,q48/72=18.7288,P均<0.01)。结论条件培养基可明显增强日本血吸虫童虫培养细胞LDH的活性及增加AgNORs的含量,且72h的条件培养基对童虫培养细胞的影响最大。

Objective To study the effects of conditioned medium on the growth and proliferation of cultured schistosomulum cells of Schistosoma japonicum by observing the content of silver-stained nucleolar organizer regions（AgNORs）and lactate dehydrogenase（LDH）.Methods The 21 days schistosomula of S.japonicum were harvested by perfusion,schistosomulum cells were made by cold digestion and diluted concentration of 1×106 cells/ml,and then were inoculated on the small cover glass.The cultured cells were randomly divided into a control group（0 h）and three test groups（24,48,72 h）,the control group was cultured by the routine medium,the test groups were cultured by the different conditioned media（the routine media mixed with different culture time of nasopharyngeal carcinoma cell（CNE）supernatant liquid with1∶1）.LDH and AgNORs in the cultured cells were stained when they were cultured for 7 days.The contents of LDH and AgNORs in the cultured cells of each group were measured by the photometer-image analytical instrument（HPIAS-2000）.Results The contents of LDH and AgNORs in the cells cultured by the conditioned media were higher than the control group（q24=8.528 7,q48=15.425 3,q72=27.620 7,P0.01;q24=13.569 0,q48=18.728 8,q72=27.035 6,P0.01）;the contents of LDH and AgNORs were the strongest in the group of 72 h（q0/72 =27.620 7,q24/72 = 19.091 9,q48/72=11.842 6,P0.01;q0/72=27.035 6,q24/72=13.569 0,q48/72=18.728/8,P0.01）.Conclusions The conditioned medium could promote and increase the proliferation and metabolic activity of the schistosomulum cells from S.japonicum,and the effects of conditioned medium of 72 h are the most significant.