关键词: 豆状囊尾蚴, 体外培养, 长期, 分泌产物, 免疫电镜

Abstract:

Objective To develop a long-term in vitro cultivation method for Cysticercus pisiformis and identify its secretory products in the culture. Methods New Zealand white rabbits were infected with Taenia pisiformis eggs by gavage at a dose of 2 000 eggs per rabbit. Three months after infection, C. pisiformis were collected from the abdominal cavities and randomly assigned to four groups (10 larvae/group), including PBS group, RPMI 1640 group, bile group (RPMI 1640 + 10% rabbit bile) and serum group (RPMI 1640 + 10% exosome-free fetal bovine serum). The cysticerci were cultured at 37 ℃ in a 5% CO₂ incubator and were observed continuously at 5 min, 1 h, 2 h, 12 h, 24 h, 36 h, 48 h and daily for subsequent days to record the scolex evagination, activity, survival status and morphological changes of cysticerci in each group, for screening optimal culture condition. Under the optimized conditions, the cysticerci were cultured in cell culture bottle (25 cm²), 50 larvae each, and the culture medium was collected every 48 h for centrifugal concentration, from which the supernatants were used to detect the expression of enolase (Tpeno) and 14-3-3 (Tp14-3-3) proteins by Western blotting. The exosomes were also extracted from the culture supernatants and incubated with negative rabbit serum, anti-CD63, monoclonal anti-Tpeno 1D7 and polyclonal anti-Tp14-3-3 antibodies for 45 min, followed by adding colloidal gold labelled IgG to observe the exosome morphology by transmission electron microscopy (TEM), and examine the expression of labeled proteins of CD63, Tpeno and Tp14-3-3 by immunoelectron microscopy (IEM). The data analysis was using SPSS 20.0 software, and one-way ANOVA was applied for comparison of group data. Results Before cultivation, the larvae were oval in shape, containing concaved dot-like white scolices. The larvae in the PBS group could survive for 7 days, while in RPMI 1640 group lasted for 2 months. However, the survival time of cysticerci in the bile group was the shortest of 48 h only, while that was longest in the serum group, which was over 3 months, and there was a significant difference between the groups (P < 0.05). While the parasites were viable, the length of cysticerci vesicle in the PBS group, RPMI 1640 group, bile group and serum group was (1.38 ± 0.39), (1.30 ± 0.12), (1.18 ± 0.59) and (1.83 ± 0.10) cm, respectively. Meanwhile the vesicle width for cysticerci was (0.45 ± 0.10), (0.68 ± 0.05), (0.25 ± 0.06) and (0.85 ± 0.05) cm, respectively. Among these, the vesicle length and width of cysticerci in the serum group were significantly different from those in the other three groups (F_length = 7.58, 65.93, 7.11; F_width = 73.85, 29.41, 308.57, P < 0.05 or P < 0.01). In addition, at 12 h, the scolex evagination rates of cysticerci in PBS group, RPMI 1640 group, bile group and serum group were (33.3 ± 5.8) %, (20.0 ± 0.0) %, (100.0 ± 0.0) % and (13.3 ± 5.8) %, respectively, The differences among the serum group, the PBS group and the bile group were statistically significant (F = 18.00, 676.00; P < 0.01). No statistically significant difference was found between the serum group and the RPMI 1640 group (F = 4.00, P > 0.05). Compared with the culture results of the other three groups, RPMI 1640 + 10% exosome-free fetal bovine serum was used as the optimal culture solution. SDS-PAGE analysis showed that the larvae-released products and exosome proteins could be successfully extracted from the culture supernatants with RPMI 1640 + 10% exosome-free fetal bovine serum. Using specific antibodies, two cysticerci-derived proteins, Tpeno and Tp14-3-3, were detected with the expected molecular weight (M_r) of 47 000 and 28 000, respectively. TEM showed cysticerci-derived exosomes are circular or elliptical, nano-sized vesicles (50-150 nm diameter) with disc-shaped morphology. IEM analysis demonstrated that the exosome proteins were not recognized by negative rabbit serum and did not show colloidal gold particle label. In contrast, the biomarker protein (CD63) and two parasite-specific proteins (Tpeno and Tp14-3-3) were positively expressed in exosomes with significant colloidal gold particle label. Conclusion This study established an in vitro culture system for C. pisiformis, being able to consecutively cultivate for at least 3 months. With this system, C. pisiformis-derived exosomes could be extracted from the culture supernatant.

Key words: Cysticercus pisiformis, In vitro cultivation, Long-term, Released products, Immuno-electron microscopic analysis