Selective Cultivation Of Schistosoma Japonicum Cells With Selective Media And Their Transfection With Adenovirus Containing SV40Large Antigen Gene

Part OneSelective cultivation of Schistosoma japonicum cells with selective culture media and evaluation of cultural effects[Objective] To study on cultivation of S.japonicum cells with three selective culture media, evaluation the cultural effects on the basis of their biological identification and explore the possibility of sorting S.japonicum cells cultivated with selective culture media in vitro.[Methods] Sj-I2d schistosomulum cells and S.J-42d adult worm cells were prepared as described before. Then both cells were selectively cultured with modified human primordial germ cells (HPGC) culture medium. The S.j-12d schistosomulum cells were also cultured with epithelial cell (EC) culture medium and1640-40comprehensive culture medium simultaneously. General morphology and growth state of S.japonicum cells cultured with three culture media were observed. The characters of S.japonicum cells were identified by chromosomal karyotype analysis, ultrastructure observation and alkaline phosphatase (AKP) staining after culturing for4to8-week in three selective media. A part of S.japonicum cell proliferation was detected by BrdU-ELISA assay or3H-TdR incorporation. Antigenicity of cultured cells was measured by Dot-ELISA method.[Results] Biological characterization of cultured S.japonicum cells with three selective media exhibited good cell viability and obvious cell division phases. The identification results of S.japonicum cells cultured with modified HPGC medium for3weeks showed that the cultured cells possessed accumulative semi-floating growth pattern in culture early-stage and had8diploids and8haploids chromosome karyotypes of the blood-fluke. AKP staining of the cultured cells exhibited strong positive reaction. Cell ultrastructure observation displayed that there were a lot of vacuoles in cytoplasm, a feature of germinal cell. The results of BrdU-ELISA revealed that the cultured cells could synthesize DNA, suggesting cell proliferation. The S.japonicum cells cultured with EC medium possessed long fusiform shape, a typical shape of epithelial cell. The result of Dot-ELISA revealed that cultured cells were recognized by portal vein serum of mouse immunized with S.japonicum cells (SjCelMS). The cultured cell ultrastructure showed the normal morphology with complete cellular membrane and distinct nucleolus. The chromosome karyotypes of the cultured cells were8diploids. The characterization results of S.japonicum cells cultured with1640-40comprehensive medium exhibited various cell division phases, DNA synthesis and8diploid chromosome karyotypes. The cultured cell activity reached70%and the ultrastructure of cultured cells showed normal morphology.[Conclusions] Some types of cells similar to germinal cell or epithelial cell were preferentially sorted from S. japonicum cell mixture with modified HPGC or EC selective culture media. These cells had part of specific characteristics of germinal cell or epithelial cell. The1640-40comprehensive culture medium could promote S. japonicum cell growth and prolong cell life duration. Part TwoObservation of transfection effects of schistosomula andschistosomulum cells of S. japonicum with recombinant adenovirus vector carrying with SV40large T antigen gene[Objective] To construct recombinant adenovirus vector carrying SV40large T antigen gene (AdHu5-SV40LT), study the effects of schistosomules and schistosomulum cells of S.japonicum transfected by recombinant adenovirus, detect SV40LT gene expression in schistosomula and schistosomulum cells after adenovirus transduction and observe cell multiplication condition.[Methods]The recombinant adenovirus plasmid AdHu5-SV40LT was re-transformed into Stb12competent cells and was identified by restriction enzyme digestion and polymerase chain reaction (PCR). Recombinant adenovirus plasmid AdHu5-SV40LT was transfected into293A packaging cells by lipofectamine2000and the replication deficient recombinant adenovirus Ad-SV40LT was generated efficiently. The recombinant adenovirus was identified by PCR, Western blotting and immunohistochemistry. The Ad-SV40LT recombinant adenovirus was efficiently duplicated in293A cells and was purified by CsCl density centrifugation and the titer was measured by50%cell culture infectious dose (CCID50). S.j-12d schistosomula and schistosomulum cells were prepared and transfected with recombinant adenovirus. Their transfection effects were detected by PCR, RT-PCR, Western blotting and immunohistochemistry.[Results] The results of restriction endonuclease analysis and PCR showed that AdHu5-SV40LT plasmid was correctly extracted after re-transformation. The recombinant adenovirus was obtained from293A packaging cell lysate after AdHu5-SV40LT plasmid transfection. The result of PCR confirmed the presence of recombinant adenovirus DNA in the transfected293A cell lysate. There was SV40LT protein expression in the transfected293A cells according to Western blotting and immunohistochemistry analyses. The viral titer was2.6×109cfu/ml. The transcription and protein expression of exogenous genes SV40LT within transduced S.j-12d schistosomula and schistosomulum cells could be detected by PCR, RT-PCR, Western blot and immunohistochemistry, respectively. The activity of S.j-12d schistosomula did not affected following recombinant adenovirus transfection. Although SV40LT gene could be successfully expressed in transfected S.j-12d schistosomulum cells, growth condition of cells did not obvious improve.[Conclusions] The recombinant adenovirus encoding SV40LT gene was successfully obtained by293A package and could transfect schistosomula and schistosomulum cells. But transduced schistosomulum cells did not showed immortalization character. The present study showed that recombinant adenovirus has promise for transferring a foreign gene to blood fluke species, which paves a way for studying the function of interest genes in schistosome development and metabolism.