Establishment And Characteristic Analysis Of Larval And Tissue Cells In Vitro In Chlamys Farreri

It is significant and challengeable field to explore the techniques establishing celllines in marine mulloscs. The culture systems and/or cell lines are urgently need toresearch in pathogenic mechanism of aquaclulture pathogenic microorganisms, celluarand molecular breeding, gene function, etc. However, no practical techniques forestablishing the cell lines were at present available in mulloscs even among all themarine invertebrates. The purpose of this study is try to establish a method of cellpassage culture for marine mulloscs, furthermore, to promote more applications usingcultured cells in mulloscs.In this study, viorious tissues were employed for cell culture in Chlamys farreri, aneconomic shellfish in the northern of China. Subculture system for cells dissociatedfrom trochophores and primary culture system for the adult tissues were establishedby adding various factors and transducting exogenous gene. Furthermore, thecharacterastics and function for passaged cells were analysized. Main results were asfollows:A continous cell culture system which can subculture19passages cells wasestablished from C. farreri trochophores by optimization on cell dissociation andcomplete medium, and the passaged cells were identified as C. farreri cells analysized by PCR with C. farreri ITS sequence. A mechanical method after calcium-free buffertreatment for dissociating cells of the trochophores was more optimal than that ofenzymolysis (collagenase or trypsin) based on the cell viability; FBS in medium canincreased mitogenic and adherent abilibty of the cultured cells in vitro; C. farreriserum in the medium was effective to improve the adherent ability of the cells in vitroand maintein their morphology; yolk extract added in the medium promotedproliferation and adhesion for the late subculture cells. It is vital to add EGF and ofbFGF to medium for maitaining the morphology and activity of cells. Three types ofcells in the continuous culture system in vitro of C. farreri trochophores wereobserved, including round hyaline cells, large round granule cells and small rounddark cells. The round hyaline cells were in the best physiological state based on thecell proliferation and adherent property, for example the round hyalin cells presenteda good adherent and rapid mitogenic ability after the culture was conductedrespectively using the three types of the cells separated by density gradientcentrifugation in Percoll. In general, the proliferative and adhernt status of all the cellswere decreased gradually with the time delay during the culture.Propotion of cell at S stage of cell cycle was analysized by flow cytometry for theprimary cells, passage12cells and passage18cells from C. farreri trochophores, anddecreased gradually when the time of passage culture increased in vitro. Expression ofgenes piwi (a stem cells related gene) and phb2(a inhibtation mitogenic gene) in cellsin vitro were detected using in situ hybraziation technique. Results showed that both piwi and phb2expressed in primary cells and passage16cells. Positive signals of piwiwere more densitive in primary cells than that in passage16cells. While, theexpression of phb2was more densitive in the passage16cells than that in primarycells, indicating a phenomenon of decline gradually in proliferating abiligy with theincrease of the passage mumber.Furthermore, expressions of Myosin (myocyte specific),5HT (neurocyte specific),tubulin or F-action (cytoskeleton) were detected in cultured cells usingimmunohistochemistrical technique. It was found that Myosin expressed moreobvious in passaged16cells than that in primary cells, implying a differentiativetrend of muscle cell in the passage16cells, while it was late in differentiative time inthe cultured cells than in mormal developmental trochophores. No positive signals of5HT were visible in cultured cells, and it was inconsistent with that in normaldevelopmental trochonphores where two sites of expression were observed in mid-and late-trochophores.In this study, primary culture systems from several adult tissues (hemolymph, gills,ovary, testis and heart) were established and the cells survived for more than2.5weeks in vitro. Furthermore, several techniques (Lipo2000, PEI and combinedlentivirus) were tried to transducte or transfecte exogenous gene SV40LT into theprimary cells. The method of lentivius infection was proved to be the most effectivewith an efficiency of up to25%, and the lifetime of the infected cells extended obviously. Moreover, cell division phase was visible occasionally in culture system ofheart cell transducted by SV40LT.In summary, the methods of establishment continuous culture systems for thedissociated cells of trochophore and gene transduction to adult tissue cells in vitro willsupply some basic data to the cell line establishment in molluscs, and the findings inthis paper will be expected to be important in the application of the research on genefunction, molecular breeding, aquarculture disease prevention and environmentaltoxicology.