The Isolation And Culture Of The Skin Fibroblast Of Elaphursu Davidianus And The Study Of Cell Cycle Synchronization

In this paper, the skin tissue of Elaphursu davidianus was used for the culture of skin fibroblast. The biological characteristics of the fibroblast were investigated, and the regulation of cell cycle was studied for the preparation as donor cell of inter-species somatic cell nuclear transfer. The studies provide references for the engineering of tissue and the protection of animals that is in severe danger. The results are as follow: 1 The Dissociation and Culture of Skin Fibroblast of Elaphursu davidianus in VitroThe skin fibroblast was obtained from skin tissue of Elaphursu davidianus by primary culture, and was purified by low-grade trypsia digestion after 3-4 passages. Cutaneous fibroblast line was established by cryopreservation. As shown in the growth curve of F4 passage, the population doubling value of the 5^th day is 5.50 and the population doubling time is 39.03h, and the growth rate of the 6^th day steps down. As shown in the cleavage curve, the cleavage index appeared highest at the 5th day, and descent dramatically at the 6th day. The growth and cleavage curve shown that the skin fibroblast of Elaphursu davidianus in vitro is normal somatic cells as which has delayed growth in earlier and growth inhibiting in later. The chromosome number of Elaphursu davidianus is 2n=68, and the formula of karyotype is 2(M)+64(A), XY(A, M). The growth of cell is influenced by the serum concentration. The series of 10% and 15% NCS have apparent exponential phase of growth, but have more apoptosis and died cells at the telo-exponential phase of growth; the series of 20% NCS has no apparent exponential phase of growth, and the cell number is less than the series of 10% and 15% NCS before the 6^th day. So the fibroblast was cultured with 10% NCS, and the medium was changed every 3days, and fibroblast was passaged before the inhibition by density and contaction, in order to avoiding the nutrition exhaustion and the accumulation of metabolin. Fibroblast was cryopreserved with 10% DMSO as the cryoprotectant. Before cryopreserved, Fibroblast was at 4℃ for 30min, and then at -20℃...