不同肝癌细胞上清对细粒棘球蚴原头节体外培养活性的影响

Effects of supernatant of different hepatoma cells on the vitality of Echinococcus granulosus protoscoleces in vitro

目的通过比较3种肝癌细胞上清对体外培养细粒棘球蚴原头节的活性影响,探讨较长时间维持细粒棘球蚴原头节体外活性的新模型。方法收集培养48 h后的Huh7、Hepa1-6和HepG-2等3种肝癌细胞无血清上清,分别与细粒棘球蚴原头节(每组约5000个)共培养,对照组采用无血清培养基培养。培养后第1、3、5、7天,取各组混悬液50μl,台盼蓝染色后光镜下观察、计数并计算存活率;培养后第7天,于扫描电镜下观察原头节超微结构,并用ELISA测定4组细粒棘球蚴原头节半胱氨酸蛋白酶-3酶的活性。采用SPSS 20.0统计学软件对计量数据进行方差分析。结果台盼蓝染色可见,随原头节培养时间延长,各组存活率均不断下降,无血清组、Huh7组、Hepa1-6组和HepG-2组原头节在第5天时存活率分别为(46.54±1.11)%、(46.19±3.60)%、(55.90±2.33)%和(68.73±1.06)%;至第7天,Hepa1-6组和HepG2组原头节存活率分别为(36.09±0.95)%和(45.69±0.40)%,与对照组(17.99±1.52)%差异均有统计学意义(P<0.01),Huh7组(19.04±1.7)%与对照组差异无统计学意义(P>0.05)。培养后第7天,光镜下可见对照组和Huh7组原头节蓝染数目多,透光度差,有较多崩解坏死物质;Hepa1-6组也可见较多蓝染原头节,透光度尚可,镜下崩解坏死物质少;HepG-2组原头节蓝染数目少,透光度较好,镜下崩解坏死物质少。扫描电镜下可见对照组和Huh7组原头节形态大体破坏,微毛、吸盘等超微结构紊乱、崩解,Hepa1-6组、HepG2组虫体表面较为饱满且平滑,超微结构也较为清晰完整。半胱氨酸蛋白酶-3酶的活性检测结果显示,Hepa1-6组和HepG2组酶活力单位分别为20.51±0.61和17.51±0.59,与对照组(22.15±1.14)比较差异均有统计学差异(P<0.01);Huh7组为22.67±0.96,与对照组比较差异无统计学意义(P>0.05)。结论肝癌细胞上清可改善无血清体外培养条件下细粒棘球蚴原头节活性,并以HepG2细胞上清作用更明显。

Objective To explore a new model for long-time retainment of the vitality of Echinococcus granulosus protoscoleces in vitro by comparing the effects of supernatant of three hepatocarcinoma cell lines on the vitality of E.granulosus protoscoleces in vitro.Methods Serum-free supernatants of three liver carcinoma cell lines,Huh7,Hepa1-6,and HepG-2,were collected after 48-h culture,and co-cultured with the protoscoleces of E.granulosus(about 5000 protoscoleces in each group).Protoscoleces of the control group were cultured with serum-free medium only.On days 1,3,5 and 7 of culture,50μl of the suspension was taken from each group for trypan blue staining to count dead cells by light microscopy,and the survival rate was calculated.On day 7 after culture,the ultrastructure of protoscoleces was observed under a scanning electron microscope,and ELISA was performed to determine the activity of caspase-3 of the four groups.SPSS 20.0 software was used for variance analysis on the measured data.Results Trypan blue staining showed that the survival rate of the protoscoleces continuously decreased with prolonged culture in each group.On day 5 of culture,the survival rate of protoscoleces in the serum-free group,Huh7 group,Hepa1-6 group,and Hep G-2 group decreased to(46.54±1.11)%,(46.19±3.60)%,(55.90±2.33)%,and(68.73±1.06)%,respectively.On day 7,the survival rate of protoscoleces in the Hepa1-6 and HepG2 group was(36.09±0.95)%and(45.69±0.40)%,respectively,which were significantly lower than that from control group[(17.99±1.52)%,P<0.01],while that of the Huh7 group[(19.04±1.70)%]was not significantly different from the control group(P>0.05).On day 7 of culture,light microscopy indicated more blue-stained protoscoleces with poor light transmittance and more disintegrated and necrotic debris in the control and Huh7 groups;higher numbers of stained protoscoleces but moderate light transmittance and less necrotic debris in the Hepa1-6 group;and small number of stained protoscoleces,high light transmittance and few necrotic debris in the HepG-2 group.Scanning electron microscopy showed that the morphology of the protoscoleces of the control group and Huh7 group was glargely destroyed,and the ultrastructures of microhairs and acetabula were disrupted and disintegrated.The protoscoleces of the Hepa1-6 and HepG2 groups showed a plump and smooth surface as well as clear and intact ultrastructure.The enzyme activity unit of Caspase-3 of the Hepa1-6 group and HepG2 group was(20.51±0.61)and(17.51±0.59)on day 7,which were statistically different from the control group(22.15±1.14)(P<0.05,0.01).There was no statistically significant difference in enzymatic activity between the Huh7 group(22.67±0.96)and the control group(P>0.05).Conclusion The supernatant of hepatocarcinoma cell culture can improve the vitality of E.granulosus protoscoleces under serum-free culture in vitro,and particularly,culture presents more obvious effect.