Studies on the establishment of a co-culture system of lung stage Schistosoma japonicum with host cells

Influence of Schistosoma japonicum programmed cell death protein 10 on the growth and development of schistosomula

“…(30 min, 3 days, 10 days, 14 days, 18 days and 21 days after infection). For skin-type schistosomula (30 min), infected skin was directly removed, cut into pieces and then incubated with 10 ml phosphate-buffered saline (PBS) at 37 °C for 2 h. The incubated mixture was filtered through a filter screen (140-mesh) (Wuhan Kerui Biological Technology Co. Ltd., Wuhan, China) [ 10 ]. The other schistosomula were collected by perfusion of mice according to methods previously described [ 15 ].…”

“…Cercariae were collected as described, mechanically transformed into schistosomula by syringe passage under sterile conditions [ 10 ], and cultured in vitro with “841” medium [a mixture of RPMI1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (Gibco), 1 μM 5-hydroxytryptamine, 1 μM L-hydrocortisone, 0.5 μM hypoxanthine, 0.2 U/ml insulin, 100 U/ml penicillin, 100 μg/ml streptomycin [10], and a concentration gradient of 0.5, 1, 2, 4, 8, 16, 32 or 64 × 10 −5 mg/ml Sjpcdp 10 dsRNA, with no dsRNA as a blank control and 4 × 10 −5 mg/mL egfp dsRNA (same as the optimal concentration of Sjpcdp 10 dsRNA) used as a negative control. Schistosomula were cultured at 37 °C in an incubator with 5% CO 2 for 1 to 7 days, with the medium and dsRNA replaced every other day.…”

“…To measure protein levels after RNAi, western blot analysis was performed after RNAi under optimal conditions. To compare the parasite sizes of the control and RNAi groups, images were obtained under a stereomicroscope (Olympus SZX7; OLYMPUS), and the lengths (L) and widths (W) of schistosomula were analyzed by Image-pro plus 6.0 software (Media Cybernetics), followed by L: W ratio calculations [ 10 ]. Routine dehydration, desiccation and spray gold were performed, and SEM was used to observe the surface morphology using a VEGA 3 LMU scanning electron microscope (TESCAN, Brno, Czech Republic).…”

“…Furthermore, RNAi-mediated knockdown of the 26S proteasome subunit SmRPN11/POH1 affects schistosomula development and survival [ 9 ]. Our laboratory screened for genes, such as orthologues of adenylate kinase 1 , programmed cell death protein 10 ( pcdp 10), and heat-shock protein 70, that might play an important role in the growth and development of S. japonicum schistosomula [ 10 , 11 ]. Among these, PCDP10 is an important molecule involved in apoptosis regulation and was originally named TF-1 cell apoptosis-related factor-15 based on its cloning from human erythroleukemia TF-1 cell lines [ 12 ].…”

“…Although there are many reports regarding the function of human PCDP10, few focused on parasite PCDP10 proteins, including that of S. japonicum ( Sj PCDP10). Sjpcdp 10 was also found to be differentially expressed between schistosomula cultured in vitro and those derived from hosts [ 10 ], and it was hypothesized that Sj PCDP10 might be involved in schistosomula growth and development. Therefore, in this study, the sequence characteristics, localization, and expression levels of Sj PCDP10 were analyzed throughout the life-cycle of S. japonicum .…”