A Preliminary Study Of Wnt2 And Wnt4 Signal Transduction Protein Function In Schistosoma Japonicum

Schistosoma japonicum is one of the most serious and widely parasitic disease,which infect humans and animals.Wnt signaling pathway widely exists in various organisms and involved in cell differentiation, embryo development, the establishment of the body axis, controling growth and reproduction of the organism.Wnt signaling pathway is closely related to the secretion of Wnt protein which be secreted extracellular.It will combine with corresponding transmembrane receptor Frizzled and activate the downstream target genes and a series of biological effects. To further understand the functions of Wnt gene family development in schistosoma japonicum, this study evaluate Wnt family gene m RNA in the different stages of schistosoma japonicum and we utilize Sj Wnt2, Sj Wnt4 si RNA inhibition of target gene expression in vivo and vitro by two ways,it preliminary explore development of Wnt genes in Schistosoma japonicum.At the same time, identify Wnt, Frizzled eukaryotic vector expression by immunofluorescence and Western blot.1.Evaluate Wnt family genes m RNA level respectively in different development stages normal and 25 d dysplasia Schistosoma japonicumComparing Schistosoma japonicum Wnt1, Wnt2, Wnt4, Wnt5, Wnt11 m RNA the relative expression level in different gender in different development stage by relative quantitative PCR, the results showed that in the early developmental stage Sj Wnt1 highest expression level in 7 d, Sj Wnt2 at the egg stage is in a relatively high level, reach the highest Sj Wnt4, Sj Wnt5 expressed in 18 days, Sj Wnt11 with 18 days in 14 days the two stage cosco is higher than the egg stage, prompt Wnt family gene function and development of eggs to sexual maturity this important physiological processes are closely related.Analysis for single compound different host of male and female schistosome infection Wnt family gene m RNA expression level by fluorescence quantitative PCR.The results showed that growth is not normal male and female individuals the Wnt gene expression levels higher than normal male and female individuals, and speculated that Wnt signaling pathways excessive expression could inhibit the growth of schistosomiasis, the development of reproductive organs.Single sex infected BALB/c mice sexual female reproductive organs is in a state of stunting, and Wnt family genes are expressed abnormally high, may inhibit the development of the reproductive organs.2. Evaluate schistosoma japonicum Wnt2 and Wnt4 siRNAUtilize Sj Wnt2 si RNA, the family of Wnt genes and downstream part in vitro interference of evaluation by Real-time quantitative PCR, the result shows that filter to get the best inhibiting effect of small molecule Sj Wnt2-615, the family of Wnt gene expression level were analyzed, and the other is the target.To three at the same time signaling pathways of seven key molecular analysis of m RNA expression level, after Sj Wnt2 genes of Wnt/beta catenin pathway, Wnt/Ca2 + pathway, Wnt/PCP have different degree of activation.Sj Wnt4 si RNA interference in vitro, small molecules can achieve good inhibitory effect at 48 h.The body don’t jamming effectiveness evaluation, according to the results of different period for 14 days, 42 days suppression effect is better and more stable, about 25 days inhibition effect, slightly higher than the male to female worm inhibition effect.3 The identification and expression of Wnt signaling pathway receptor ligands in 293 T cellsDetect Liposome transfection of plasmid by fluorescence microscope, the results show that positive expression, Wnt2, Wnt4 and Frizzled5, Frizzled8 transfection group, showed a total transfection plasmid can be expressed in the same 293 T cell.By Western blot appraisal eukaryotic carrier protein molecular weight is consistent with the theoretical molecular weight size.In comclusion,this study by using real-time quantitative method to evaluate the normal worm at every stage of the development of family of Wnt gene m RNA level, at the same time analyzes the single compound infection 25 d in the family of Wnt gene m RNA transcription level.Select inhibition effect best Sj Wnt2 si RNA, and determine the concentration of the best.At the same time of Wnt homologous genes and downstream interference effect evaluation.For Sj Wnt4 evaluate the jamming effect of different time periods in vivo and in vitro, grope for the interference of the best conditions.In addition, to build a good ligand, p3 x FLAG Wnts, p RK5 Frizzleds- myc for validation.For the next step of Wnt signaling pathways in the growth and development experiment foundation for further function researches of schistosomiasis.