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Objective To investigate the expression of miRNA associated with hepatic fibrosis induced by Schistosoma ja-ponicum soluble egg antigen stimulation in mouse hepatocytes(AML12),so as to lay the foundation for clarifying the mecha-nism of schistosome infection leading to hepatic fibrosis. Methods The expressions of miR-122,miR-182,miR-23b,miR-27b and KSRP in AML12 cells treated with SEA were measured by q-PCR. KSRP protein in cell lyses was measured by Western blotting. AML12 cells were transfected with miR-27b precursor or anti-miR-27b for 24 h,then q-PCR was adopted to determine KSRP mRNA,and KSRP protein was detected by Western blotting. Results The expressions of miR-182,miR-23b and miR-27b were decreased and miR-122 was increased in AML12 cells following SEA treatment(all P<0.05). An increase of mRNA and protein of KSRP expression was also observed in AML12 cells after SEA stimulation(both P<0.05). In addition,KSRP mRNA expression was not changed significantly in AML12 cells transfected with anti-miR-27b or miR-27b precursor,and miR-27b precursor reduced KSRP protein expression as compared with the control. In contrast,the expression of KSRP protein was increased in the anti-miR-27b group and decreased in the miR-27b precursor group. Conclusions After the stimulation of SEA,the expressions of a variety of liver fibrosis-related miRNAs and KSRP change in murine hepatocytes,including miR-27b. And miR-27b can regulate the expression of KSRP. These findings might lay a foundation for further study on the molecular mechanism of fibrosis induced by schistosome infection.
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Objective To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. Methods The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentra-tion of A1E3-HRP. Under the optimal conditions,the serum samples of 20 acute schistosomiasis cases,46 chronic schistosomiasis cases,and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum sam-ples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit,to evaluate the detection effects of this method. Results The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88 000 and 52 000 respectively and had the same light chain with molecular weight of 20 000;while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schis-tosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1∶105 and 1∶30 000,respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8%and 43.1%respectively,and there was no significant difference between the results of the two methods. Conclusion A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.
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Objective To investigate and compare the different effects of soluble adult wornl antigen(SWA)and soluble egg antigen(SEA)of Schistosoma japonicum on the apoptosis and cell-cycle of routine CD4~+T cells.Methods Purified CD4~+T ceUs from normal C57BL/6 mice were cultured with CFSE labeled antigen presenting clls in the presence of different stimuli for 36 h.Flow cytometry(FCM)was used to detect the apoptosis of CD4~+T cells by fluorescence conjugated caspase-3 antibodie staining.The flow cytometry was used to analyze the cell-cycle of CD4~+T cells cultured as described above for 96 h by propidium iodide staining.Results Compared with the apoptosis percentage of CD4~+T cells[(1.24±0.29)%]in the SEA stimulated group,that in the SWA stimulated group[(1.52±0.38)%]did not show statistically significant difference(P>0.05).Compared with the cell percentages in G1 phase[(78.91±2.98)%],S phase[(7.39±0.85)%]and G2/M phase[(10.69±1.05)%] in the SWA stimulated group,that of the G1 phase[(59.42±1.32)%]was significantly lower,but those in the S phase[(21.07±O.88)%] and G2/M phase[(18.88±1.21)%]were significantly increased in the SEA stimulated group(P<0.01).Conclusions There is no statistically significant difference between the apoptosis levels of CD4~+T ceHs stimulated by SWA and SEA.However,SEA significantly promotes the progression of the cell-cycle of CD4~+T cells compared with SWA.
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0. 05) and the specificity is higher than that of the SEA-ELISA (P