Establishment And Biological Characteristics Of Fibroblast Cell Lines From Three Kinds Of Tissues Of Tibetan Mastiff

Tibetan Mastiff is a Rare Breed of Qinghai-Tibet Plateau. In order to permanently preserved its genetic resources at the cellular level, 2 of one month old Tibetan baby mastiffs were as the research object, to built Tibetan Mastiff kidney and myocardial tissue fibroblast cell line by trypsin hot digestion; to built Tibetan Mastiff ear marginal tissue fibroblast cell line by tissue culture. According to the specific requirements for cell lines established of ATCC, the fibroblasts cell lines from three tissues of Tibetan mastiffs were studied about the culture conditions and biological characteristics. Results: the cultured cells of Tibetan Mastiff kidney (TMK), ear marginal cells (TME) and myocardial cells (TMH), were typical fibroblast; In accordance with conventional cryopreservation method of Bio-engineering laboratory, the three cell lines were cryopreservated and recovery. The cell survival rate were maintained at 90%, cells grew well, showed a growth curve "S" type when they were recoveried. Every generation has experienced growth latency, exponential phase of growth and stagnation period, consistented with in vitro cell growth pattern. The cell morphologies were no significant difference from 1 to 7 passages when cultured in DMEM containing 10% fetal bovine serum. The test results of bacteria, fungi, mycoplasma and viral contamination of exogenous factor were negative; Karyotype analysis: the normal chromosome of cultured fibroblasts from three tissues of Tibetan Mastiff were 2n = 78, including 38 pairs of autosomes, all telocentric chromosome; one pair of sex chromosomes, X chromosome was metacentric, Y is telocentric chromosomes. Counted 100 of cells in metaphase chromosome for three cell lines, the result showed 2n = 78 were 90%, which indicates the cell lines were diploid cells in the main and genetic performance were stability. Isoenzyme electrophoresis for three cell lines of Tibetan Mastiff kidney, cardiac muscle, ear marginal fibroblast showed that lactate dehydrogenase (LDH) have three bands with LDH1, LDH2, LDH3. The transfection of Liposome-mediated plasmid with green fluorescent protein showed that three tissue fibroblasts of Tibetan Mastiff were transfected respectively 50%, 51%, 50% for Kidney, cardiac and ear fibroblast after transfection 24 hours, that means the established cell lines can effectively transfer and expression of foreign genes. Conclusion: the three tissue fibroblast cell lines of Tibetan Mastiff meet the requirements of cell line construction of ATCC.