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

doi:10.12140/j.issn.1000-7423.2020.03.010

Abstract

Abstract:

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 5 000 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, 50 μl 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 HepG-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.

Key words: Hepatocellular carcinoma supernatant, Echinococcus Granulosus, In vitro culture, Activity

CLC Number:

R3838.33

CHEN He-jie, JIANG Hui-jiao, LIANG Qian, Wu Jie, GUI Xian-wei, ZOU Hai-liang, XING Zhi-kun, WANG Er-qiang, CHEN Xue-ling, WU Xiang-wei. Effects of supernatant of different hepatoma cells on the vitality of Echinococcus granulosus protoscoleces in vitro[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 317-323.

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组别Group	培养时间/d Culture duration/d
组别Group	1	3	5	7
无血清组Serum-free group	99.1 ± 1.7	76.3 ± 0.2	46.5 ± 1.1	18.0 ± 1.5
Huh7上清共培养组Huh7 Co-culture group	94.9 ± 1.8	78.0± 1.0	46.2 ± 3.6	19.0 ± 1.7
Hepa1-6上清共培养组Hepa1-6 Co-culture group	97.7 ± 2.0	83.0 ± 2.1	55.9 ± 2.3	36.1 ± 1.0^a
HepG2上清共培养组HepG2 Co-culture group	97.8 ± 2.3	88.2 ± 1.4	68.7 ± 1.1	45.7 ± 0.4^a