| The paper studied the separation for giant panda's cell of cutaneous tissue, designed cell culture system in vivo which can be used for cutaneous fibroblast and established preliminarily cutaneous fibroblast line of giant panda,finally, cutaneous fibroblast line was cryopereserved and identified the species of cell line by karyotype.The results are as follow:1.In four tests of enzyme digestion for cutaneous tissue of giant panda:the test which collagenase type I is at the concentration of 200IU/ml under 4℃ being kept 48-72 hours is the best method in four tests.The gaining ratio of living cell reach to 4.604 #106/g, and fibroblast is main cell in the best test;2.When cutaneous tissue of giant panda was cultured in the medium which contain 199/EBSS+20%fetal bovine serum(FBS)+100IU/ml penicillin andstreptomycin+M-Plasmolin 5 u g/ml+AmphtericinB 5 u g/ml+2mmol/L L-Glu, free fibroblast was fonud after 6-8 days for infant giant panda, for mature giant panda ,the same cell was found after 12-14 days;3.Under the base of the difference of adhesion speed between fibroblast and epithelium, fibroblast was purified after continuous three generations.The fibroblast purity reach to 99.84 ±0.563% (n=30) ;4.By testing cell adhesive ratio, growing speed, morphogenesis and variation ratio of chromosome number in four basic medium marked :BCS-K BCS-2-, BCS-3,> BCS-4,the tset had showed BCS-3 was more suitable for giant panda's cutaneous fibroblast culturingthan others. In BCS-3 medium, the average of adhesive ratio was 85.996%, the ratio of diploid at 4* generation was 87.27% and in BCS-3 medium, fibroblast grew quickly and had normal morphogenesis. The variational tendency of adhesive ratio in BCS-3 medium showed the best time of fibroblast being cryopreserved is from 6th generation to 8th generation; 5.Six culturing systems were designed by adding Epidermal Growth Factor.(EGF) and Insulin in different level into BCS-3 medium which were marked as CS-1 > CS-2, CS-3,. CS-4, CS-5 and CS-6. By testing cell growing speed and variation ratio of chromosome number, the effect showed CS-5 was more suitable for giant panda's cutaneous fibroblast culturing than others. In the CS-5 culturing system, the density of fibroblast reached to 6.890 #105/ml according to 1.673 ± 0.185 #105/ml being inoculated after three and a half days, the fibroblast growing speed was the highest in all culturing system and the ratio of diploid at the 5th generation was 75.77%;6.The karyotype analysis of the fibroblast by large-scale culturing in CS-5 culturing system show: the most number of giant panda's cutaneous fibroblastic chromosome is 42 at 5th generation. Relative length and centromere index of chromosome in normal karyotype were analysied ulteriorly. The results showxultured fibroblast is belong to giant panda's somatic cell;7.By analysis for the toxicity of the four kinds of freezing-medium and the resuscitation percent after fibroblast being freezed and thaw, the results show the toxicity of FM-1 was the lowest to fibroblast and resuscitation percent of fibroblast being cryopreserved in FM-1 was the highest. FM-1 was better than FM-1, FM-3 and FM-4 for cryopreserving giant panda's cutaneous fibroblast.The studies demonstrated we could gain giant panda's cutaneous fibroblast line which had the steady heredity if we had the enzyme digestion for giant panda's cutaneous tissue under the condition that collagenase type I is 200IU/ml and temprature is 4℃, then cultured it in CS-5 culturing system, and cryopreserved it in FM-1 at the 6th-8th generation.
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