Effect of osteopontin expression level on the growth and development of Echinococcus multilocularis protoscoleces

doi:10.12140/j.issn.1000-7423.2022.03.004

Abstract

Abstract:

Objective To investigate the effect of osteopontin (OPN) expression level on the growth and development of Echinococcus multilocularis protoscoleces. Methods Protoscoleces were isolated from E. multilocularis-infected Meriones unguiculatus and cultured in vitro for 3 days. The E. multilocularis protoscoleces were assigned into four groups: LV-EmOPN-734 (knockdown EmOPN) group, LV3-NC (knockdown control) group, LV-EmOPN-0423 (overexpression EmOPN) group, and LV5-NC (overexpression control) group. Each group was tested in parallel triplicate wells, with 5 000 protoscoleces in each well. LV-EmOPN-734 lentivirus diluent (final concentration 1.4 × 10⁷ TU/ml) was added to the silenced EmOPN group, LV-EmOPN-0423 lentivirus diluent (final concentration 4.83 × 10⁸ copies/ml) was added to the EmOPN overexpression group, and the corresponding control group was added with the same amount of lentivirus diluent (blank plasmid). After 72 hours of lentivirus infection, the fluorescence intensity of each group of protoscoleces was observed under fluorescence microscope, the relative expression of OPN in protoscoleces was detected by Western blotting, Caspase-3 activity was detected by applying cysteine aspartate-3 (Caspase-3) assay kit, and the proliferation ability of protoscoleces was detected by applying EdU Imaging kit (Cy3). 72 hours after infection, the protosegment was co-cultured with rat hepatoma cells for 8-12 weeks, and the morphology of the germinal layer of E. multilocularis vesicles was observed by scanning electron microscopy. Statistical analysis of differences between groups using t-test. Results After 72 hours of lentivirus infection, it can be seen that the E. multilocularis protoscoleces were infected by lentivirus successfully, which is distributed in a circle or dots. Western blotting results showed that the relative expression of EmOPN in the LV-EmOPN-734 group was (0.43 ± 0.04), which was lower than that in LV3-NC group (0.80 ± 0.07) (t = 8.623, P < 0.01); the relative expression of EmOPN in LV-EmOPN-0423 group was (1.18 ± 0.21), which was higher than that in the LV5-NC group (0.73 ± 0.06) (t = 3.333, P < 0.05). Caspase-3 activity assay showed that the Caspase-3 activity was (61.55 ± 1.64) μmol/L in the LV-EmOPN-734 group, higher than (28.20 ± 2.16) μmol/L in the LV3-NC group (t = 24.57, P < 0.01); Caspase-3 activity in the LV-EmOPN-0423 group was (50.11 ± 6.45) μmol/L, which was lower than (78.22 ± 16.43) μmol/L in the LV5-NC group (t = 3.185, P < 0.01). The results of EdU Imaging Kits (Cy3) assay showed that the protoscoleces of EdU+ in the LV-EmOPN-734 group were(0.47 ± 0.06), which was lower than that of the LV3-NC group (0.72 ± 0.10) (t = 3.663, P < 0.05); the protoscoleces of EdU+ in the LV-EmOPN-0423 group had of EdU+ protoscolecesof (0.81 ± 0.09), which was higher than that of the LV5-NC group (0.54 ± 0.06) (t = 4.309, P < 0.05). Scanning electron microscopy observations showed that most of the germinal layer cells of the LV-EmOPN-734 group were collapsed, shrank, shed to a large extent, and lost their normal structure; while the germinal layer cells of the LV-EmOPN-0423 group had intact cell membranes and full morphology, forming germinal layer vesicles and growing small tips to connect with the germinal layer. Conclusion The OPN expression level upon rising-up or lowering-down may promote or inhibit the growth and development of the E. multilocularis protoscoleces.

Key words: Echinococcus multilocularis, Osteopontin, Protoscoleces, Growth and development

CLC Number:

R383.33

ZHUO Yi-cheng, YANG Hai-cheng, LIU Cheng-hao, ZHANG Bao-cai, DUO Xiao-yong, ZHANG Shi-jie. Effect of osteopontin expression level on the growth and development of Echinococcus multilocularis protoscoleces[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(3): 299-304.

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