The Primary Culture Of Gecko Neural Cells And Establishment Of Immortalized Cell Lines Of Gekko Japonicus

Objective: The aim of the study is to develop the standard cell culture systems for gecko neural cells, to set up the primary culture of the adult spinal cord cells from gecko, glial cells and neurons from embryonic gecko cerebral cortex; and to establish immortalized gecko neural cell lines. This study provides an in vitro model to investigate the neural cells and biofunctions of gecko nervous system, and to study the function of the genes related to spinal cord regeneration of Gekko japonicus. Methods:(1) Direct tissue explant method was used to culture the gecko cells from cerebral cortex and spinal cord. By screening a variety of parameters, including the osmotic pressure, pH, temperature, CO2 concentration, the gecko neural cells culture medium and condition were determined regarding to the cells growth state and adherent rate.(2) The adult gecko spinal cord was dissociated and digested with 0.25% Trypsin, and the cells were seeded in culture flask and incubated in DMEM/F12 (osmotic pressure, 200~260 mmol/kg, pH 7.2) supplemented with 10% fetal calf serum in 5%CO2, 100% humidity incubator at 30°C. The cells were consecutive passaged and idenfied by immuncytochemistry.(3) The Gecko embryonic (E15) cerebral cortex was dissociated and digested with Trypsin. The glial cells were purified by using adhesive differences combined with successive passage, and neurons were obtained by using serum-free Neurobasal culture medium supplemented with B27. The purified cells were identified by immuncytochemistry assay.(4) Embryo gecko cerebral cortex cells were cultured, and the cells from the second passage were transfected with the recombinant pcD2-SV40 T plasmid mediated by liposome. G418 was used to select positive colonies. The expression and incorporation of SV40 T antigen gene were detected by RT-PCR, genome DNA-PCR, Dot blot and Southern blot. G418-resistant colonies were characterized by the following: light and electron microscopy for the morphological studies, freezing-downs and thawing-ups for the research of cell survival rates, MTT for cell activity, FACS analysis for cell cycle, the rates of BrdU incorporation for the cell proliferation study. The characteristics of stably tranfected cell lines were demonstrated by using karyotype analysis, colony formation assay, serum dependency assay, contact inhibition assay. The types of transformed cells were identified by immunocytochemistical staining using various antibodies (cell marker).Results:(1) The condition as follows: DMEM/F12 (1:1) supplemented with 10% fetal calf serum, osmotic pressure 200~260 mmol/kg, pH 7.2, 30°C, 5% CO2 and 100% humidity, is the optimization for gecko neural cells culture. Under such condition, the best results were obtained regarding to cell growth state and cell adherent rate.(2) The methods of isolation, cultivation and purification of adult Gekko japonicus spinal cord cells were developed and eight types of cells were observed. The results of immunocytochemistry assay showed NF was positively stained in neuron like cells,whereas with GFAP positively in glia like cells.(3) The methods of isolation, cultivation and purification for embryonic Gekko japonicus cortical neurons and glial cells were established. More than 95% of GFAP positive cells were obtained after 4 passages. After being cultured with defined medium for 10 days, the purity percentage of the neurons were more than 95%, which were positively immunostained with NF and MAPⅡ.(4) After G418 selection, 49 immortalized gecko cell lines were obtained. Two stably transfected cell lines, identified as Gsn1 and Gsn3 (Gecko-SV40T-Neural cells 1 and 3) have been cultured for 50 passages. The results of RT-PCR, genome DNA-PCR, Dot blot, Southern blot all showed that the SV40T gene has been incorporated into the genome DNA of the two gecko neural cell lines. As compared to those of untransfected Gecko cortical neural cells, Gsn1 and Gsn3 cells shared not only most normal features but also partial immortalized characteristics: no suspended growth capability, aneuploid and polyploid karyotype, faster cell growth and proliferation rate, higher colony formation rate, decrease of serum dependency, disappearance of contact inhibition. The immunostaining assay showed that the Gsn1 was GFAP-positive cells and Gsn3 was GC-positive cells.Conclusion: In the study, the optimal culture medium and condition for gecko neural cells were established. The primary culture methods of the spinal cord cells from adult gecko, glial cells and neurons from embryonic gecko cerebral cortex were established. It was found that there were 8 cell lines with distinct morphologies from spinal cord cells including GFAP positive glial cell like and NF/MAPⅡpositive neuron like cells. Gecko embryonic cortical gial cells and neurons were isolated and purification efficiency is higher than 95%. Immortalized gecko neural cell lines (Gsn1 and Gsn3) were established and exhibited some characteristics of transformation. A GFAP-positive cell line and a GC-positive cell line were obtained. This study could provide in vitro model systems for future investigations of the gecko neural cells and the biological functions of some specific gene products related to the regeneration of the central nervous system.