| The soft-shelled turtle is our precious freshwater aquatic products, their meat is not only delicious, but also high nutritional value, which cater the favorite of consumers, the market prospects is better, its aquaculture development has been concerned over the years. The high level of intensive farming and breeding density make Chinese softshelled turtle easy to breed bacteria and cause disease. Especially, the viral disease can lead to the secondary infection in the production practice, resulting in decreased quality of the soft-shelled turtle products, taste worse, even a large number of deaths, breeding efficiency decreases. However only few cell lines and applications have been reported on P. sinensis. As known to all, cell lines can be used to study the viral diseases of P. sinensis, the function analysis of immune gene and immune mechanism and also can be transfected with foreign genes, and will be a useful systerm in vitro for cell enginerring. The results are as followings.1. The process of establishing fibroblast cell line(PSF) from P. sinensis. The cell line was established by the semi-digestion explant culture technique form heart tissue of P. sinensis, named PSF. In the experiment, we explore the optimum growth temperature, the optimum medium and the optimal serum concentration of the cell line.5% CO2, the serum concentration of 15%, MEM medium, we set 25 ℃, 30 ℃ and 35 ℃ three temperature gradient, the results showed that the optimum growth temperature of the cell line was 30 ℃. under 5% CO2, 30 ℃, three medium MEM, DMEM and RMPI-1640, the results showed that MEM is the most suitable medium. 5% CO2, 30 ℃, 15% fetal bovine serum, MEM medium, 10%, 15% and 20% three serum concentration gradient, the results showed that the concentration of 15% is the optimal serum concentration of the cell lines. EGF is effective to promote cell proliferation in primary cells as well as the first 10 generations of cell line. Growth kinetics of the cell lines were measured, cryopreservation and resuscitation cell survival assay showed that the biological cell line presents a typical "S" type curve and shows population growth of this cell line doubling time is 51.1h, survival rate before freezing/after being thawn were 98.54 ± 4.53% and 96.36 ± 3.77%, statistical analysis showed that the difference was not significant(p> 0.05).2. Identify the source of the fibroblast cell line PSF. The fibroblast cell line was used for comparison of gene sequencing, morphology analysis, karyotype, Isoenzyme, contamination of microbiology, fluorescent protein expression rate. Analysis of mitochondrial cytochrome D-loop sequences revealed 96% identity among other Chinese turtle subspecies, and combine the PCR result we conform the source of the cell line is P. sinensis., hematoxylin-eosin(HE) staining, PSF cell was swirling arrangement with the surrounding boundaries more clearly, more cytoplasm, pink, blue and purple nucleus, obviously, the cells showed the typical spindle-shaped, diamondshaped or irregular polygonal shape typical of fibroblasts. Karyotype analysis showed that the cells were diploid chromosome composition of 2N = 66. Mycoplasma contamination test results showed that the cell line is not infected with mycoplasma. Immunohistochemistry showed PSF cell lines was typical fibroblast morphology. Isoenzyme electrophoresis analysis includes lactate dehydrogenase(LDH) and malate dehydrogenase(MDH) cell lines and showed that there is no cross-contamination of other cells.3. Transgenic technology of PSF cell line. PEGFP-C3 plasmid and liposomes Lipofectamine? 2000, the EGFP, The PSF cell line produced significant fluorescent signals after transfection with plasmid p EGFP-C3, after 48 h, the highest transfection efficiency is 16%In this paper, we establish a fibroblast cell line form P. sinensis heart tissue successful, this newly established cell line will be a valuable tool for transgenic and genetic manipulation studies and will act as an efficient instrument for studies of the viral diseases of the soft-shelled turtle. |
PDF Full Text Request