ABSTRACT
Objective Using silkworm expression system to express human serum paraoxonase 1 (PON1) fusion protein with protein transduction domain Pll and to study its biological activity of fighting aginst the toxicity of dichlorvos. Methods P11-PON1 fusion gene was constructed in cloning sites of silkworm pFastBac 5B vector, the vector and was transformed to silkworm DH10BmBac competent cells. Virus particles and 5 instar silkworm was infected 96 hours after infection, parasites were collected and lyophilized crushed and preserved at -80 7. The protien was dissolved, sonicated and centrifuged before used. Supernatants were harvested. The fusion protein P11-PON1 proportion of the total protein was analyzed with SDS-PAGE electrophoresis and protein content was calculated. Mouse and zebrafish models were used to evaluate P11-PON1 fusion protein bioactivity. Each mouse was treated with 1 mg P11-PON1 fusion protein via intragastric or rectal administration. 0 and 3 hours after administration, 20 mg/kg dichlorvos were injected intraperitoneally. The status of intoxication was observed and the survival rate was scored. P11-P0N1 fusion protein with concentrations of 1, 2.5, 5, 10, 20 mg/L was dissolved in zebrafish breeding water respectively. 0 and 3 hours after exposing dichlorvos were added with a final concentration of 50 mg/L. Observe their behavioral change.The survival rate of zebrafish was recorded. Results The content of P11-P0N1 fusion protein was 8% of silkworm total protein. In mice experiments, P11-P0N1 fusion protein by intragastric adminstration did not increase the survival of mouse. By intraperitoneal injection with dichlorvos 0h after rectal adminstration with protein,the survival rate of mouse did not significantly increase. In contrast, the mouse intraperitoneally injected with dichlorvos 3h after adminstration with protein, the survival rate increased extremely significantly (P < 0.01). In zebrafish experiments, the zebrafish exposed to dichlorvos 0 h after adminstration with protein, the survival rate was not significantly improved, while exposed to dichlorvos 3h after admindtration, the survival rate significantly increased. The survival rate of 20, 10, 5 mg/L group were 62.5%, 62.5% and 50% respectively at 24 h time point, compared to the control group. The survival rate increased extremely significantly (P < 0.01). 2.5 mg/ L group was 41.7%, with the survival rate increasing significantly (P < 0.05). However, the survival rate of 1 mg/L group was 16.7%, compared to the control group. The increase had no sistatistical significance. Conclusion The PTD-containing P0N1 fusion protein can be expressed in silkworm. Pretreatment with the fusion protein in mice and zebrafish decreased the toxicity of dichlorvos.
ABSTRACT
Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton's jelly derived mesenchymal stem cells (WJ-MSC), and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80°C refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage (PP1). Thus, PP2-PP15 WJ-MSC were obtained by continuous cell subculture. The 1st control passage (CP1) to 15th passage (CP15) represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated cells, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92.6%. There was a statistically significant difference in proliferation index between PP1 and CP1 (P < 0.05), but there were no statistically significant differences between PP2-PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not different. After induced differentiation into adipocyte, osteoblast and neuron, the staining with oil red O, alkaline phosphatase and neuron-specific enolase was performed respectively, and the positive degrees were not clearly different macroscopically between two groups. Relatively high levels of triglyceride, alkaline phosphatase, and neuron-specific enolase in relevant cells could be detected, but had no significant differences between two groups. It is concluded that some WJ-MSC (< 10%) are damaged after cryopreservation, and the biological characteristics of WJ-MSC in cryopreservation group keep constant, as compared with that in non-cryopreservation group.