Establishment Of Fibroblast-like Cell Lines From The Caudal Fins Of Fishes With Different Ploidy And The Primary Study Based On Them

The allotetraploid (AT), the red crucian carp (RCC) and their hybrid—the triploid crucian carp (TCC) form a polyploid system which is characterized by the clear ploidy and the sibship. What is more, this polyploidy system offers a unique research platform for molecular immunology and genetics. In this manuscript, the allotetraploid, the red crucian carp and the triploid crucian carp were chosen for tissue primary culture and cell lines establishment, which was aimed at the innate immunity study of this polyploidy system. The fibroblast-like cell lines were successfully obtained separately from the primary cutured tissues of the caudal fins of the AT/RCC/TCC. The mitochondrial antiviral signaling protein (MAVS) gene cDNAs for AT/RCC/TCC were cloned from the fibroblast-like cell lines separately and bioinformatic study were performed based on the sequence results. The experimental results of this study are listed as the followings:1. The in vitro cultured fibroblast-like cells of caudal fins of AT/RCC/TCC have been passaged more than100generations. The spindle shape of the AT/RCC/TCC caudal fin cells reflects the typical morphologic characteristic of fibroblast.2. We analysised the chromosome number of the fibroblast-like cells of AT/RCC/TCC (2N-FCF/3N-FCF/4N-FCF) independently through Giemsa staining and cell drop technique. The results showed that the chromosome number of2N-FCF is majorly distributed between90and98, the chromosome number of3N-FCF is majorly distributed between120and145, and that of4N-FCF is majorly distributed between151and190. The generated data suggested that the in vitro cultured fibroblast cell losed part of their chromosomes during the subculturing and immortalizing process, comparing with the choromosomes of AT/RCC/TCC.3. The doubling time of the2N-FCF/3N-FCF/4N-FCF increased with the increase of ploidy. The doubling time of fibroblast cells of2N-FCF is36.6hours, that of3N-FCF is38.9hours, and that of4N-FCF is40.3hours.4. The DNA contents of the three fibroblast cell lines were studied through flow cytometry. DNA content of2N-FCF is1.04times of that of RCC blood cells. The DNA content of3N-FCF was1.69times of that of RCC blood cells. The DNA content of4N-FCF was2.04times of that of RCC blood cells.5. Three different means (lipofectamine2000, calcium phosphate and lenti-virus) were used to transfect the three cell lines with GFP construct. The transfect efficiency based on all the three medium was very low. Among the three transfection methods, the transfection efficiency of lipofectamine2000method was higher than the other two, however, the transfection efficiency was still as low as10%. 6. The total RNA were extracted from the2N-FCF/3N-FCF/4N-FCF respectively and RACE method was used to amplify MAVS cDNAs of AT/RCC/TCC. The cloning and sequencing results demonstrated that the particle MAVS cDNA for RCC and the full length MAVS cDNAs for TCC and AT were obtained.7. The bioinfomatic study of MAVS genes for RCC/TCC/AT revealed that putative RCC MAVS contains584amino acids, the putative TCC MAVS contains582amino acids, and the putative AT MAVS584amino acids. All the three MAVS contain three typical domains, which include a N-terminal CARD, a proline-rich region, and a C-terminal mitochondrial transmembrane sequence(TM). Comparison among the deduced amino acid sequences of RCC/TCC/AT MAVS showed that the MAVS of RCC exhibited70.9%identities to other fish MAVS,and the homology between RCC and TCC was99.3%.In this manuscript, fibroblast cell lines of RCC/TCC/AT had been established from the primarily cultured caudal fin cells of RCC/TCC/AT respectively. MAVS genes of the three speices were cloned from the three cell lines. This study had built a solid foundation for elucidating how MAVS functions in innate immune antiviral response in RCC/TCC/AT.