Study On Six Aquaporins Genes Of Echinococcus Granulosus

Objective1.To search for a suitable culture system for transforming of protoscoleces of Echinococcus granulosus into hydatid cysts in vitro,and supply the hydatid cysts for researches on echinococcosis.2.To study the relationship between the expression status of EgAQPs genes and the formation of Echinococcus granulosus hydatid fluid,the dynamic expression of EgAQPs genes of protoscoleces and the germinal cells of hydatids developed from protoscoleces in vivo.3.Six kinds of aquaporin genes were cloned from Echinococcus granulosus,and Eg AQPs protein was expressed heterologously in Xenopus laevis oocytes to detect the function of water permeability,which may provide a theoretical basis for elucidating the formation process of hydatid cyst.Methods1.Based on the two basic cell culture media?RPMI1640 or DMEM?,and two types of fetal calf sera or one type of sheep serum as nutrients,and human liver cell line L02 as server cells,the culture systems for transformations of protoscoleces into hydatid cysts in vitro were established.According the different ingredients of culture media,sera,and sever cells,the culture systems were divided into two main groups,and ten subgroups.The protoscoleces were cultured in the ten groups for 45 days in order to select the most suitable medium for transformations of protoscoleces into hydatid cysts in vitro,in which the evaluation criteria were the ratios of evaginated or encysted protoscoleces,and the sizes of protoscoleces.In the selected best culture system,the encysted protoscoleces were kept cultivating up to 180days.2.The hydatid tissues in vitro and in mice during the development of hydatid from protoscoleces were collected from different stages and designed them into specific primers to obtain target sequences of six EgAQPs genes.The expression of the above mentioned genes in vitro protoscoleces and in the germinal cells of hydatids developed from protoscoleces in vivo were tested by Real-time PCR.3.According to the six AQPs gene sequences of Echinococcus granulosus in GenBank,the coding region of EgAQP and EgAQP1 genes were amplified by RT-PCR using the cDNA of Echinococcus granulosus as template.The method of chemical synthesis was used to expand the coding region of four EgAQPs?EgAQP4-1,EgAQP9-2,EgAQPAnG,EgAQP FA-CHIP?;and these six EgAQPs proteins were expressed in X.laevis oocytes to verify their water channel functions.Results1.Within the first period of 45 days,it demonstrated that the subgroup G?DMEM medium+fetal calf serum+human liver cell line L02?was the best culture system for transformations of protoscoleces into hydatid cysts in vitro,according to the data such as the fastest growth speed of protoscoleces,the maximun ratio of encystation of protoscoleces since 20 d?P<0.05?,the biggest size of hydatid cysts since 25 d?P<0.05?,and 100%percentage of encystation of protoscoleces at 45 d.In the following culture days?45-180d?,the hydatid cysts were keeping growing up,and the transparent hydatid cysts were visible to the naked eyes at 80 days,the biggest cyst size reached to 2.2 mm.2.Morphologically,it was found that the volume of hydatid and the transparent hydatid fluid increased with the time.The transcriptional level of EgAQP9-1 gene reached the highest on the first day of culture?P<0.01?,then the expression decreased rapidly on the 10th day?P<0.01?,and there was a trend of decreasing with the time.There were statistically significant differences in the amount of transcriptional expression at different culture times.However,the transcriptional levels of EgAQP?Eg AQPAnG,EgAQP1,EgAQPFA-CHIP,Eg AQP4-1 and EgAQP9-2 genes in the different stages of the hydatid tissues in vitro and in mice during the development of hydatid from protoscoleces were very low and undetectable.3.The difference of volume changes rate?V/V₀?between oocytes injected with six kinds of EgAQPs genes?EgAQP,EgAQPAnG,EgAQP1,EgAQP FA-CHIP,EgAQP4-1,Eg AQP9-2?and control oocytes injected with DEPC water was not statistically significant?P>0.05?,but the difference of osmotic coefficient was statistically significant?P<0.05?.The results of western blotting showed that the total oocytes protein,cytoplasmic protein and membrane protein of X.laevis injected with these six Eg AQPs cRNAs were not recognized by the Anti-Flag-tagged antibody.These six EgAQPs genes did not exhibit water channel functions in X.laevis oocytes.Conclusion1.The culture medium of DMEM,added with fetal calf serum and human liver cell line L02,was a suitable culture system for transformations of protoscoleces into hydatid cysts in vitro.2.The transcriptional expression of EgAQP9-1 genes gradually decreased in hydatids developed from protoscoleces,which might have less effect on the formation of hydatid fluid after echinococcosis.Also,the expression of EgAQP?EgAQPAnG,EgAQP1,EgAQPFA-CHIP,EgAQP4-1,and Eg AQP9-2 genes were very low in the cultured cells of Echinococcus granulosus and vesicle germinal cells in vitro,which might have little effect on the formation of hydatid fluid.3.In this study,the water channel function was not observed in the six EgAQPs genes with the verification of gene cloning,transcriptional expression analysis and oocyte function,indicating that the six Eg AQPs genes were not expressed or trace expression in X.laevis oocytes,and their functions of water channel were not detected by western blotting,which indicated that these six genes might play little roles in the formation or accumulation of hydatid fluid.