Establishment Of Fibroblast Cell Lines In Vitro Culture System From Chinese Zokor And Its Biological Characteristics Analysis

Chinese Zokor(Myospalax fontanieri)is an animal of Spalacidae Myospalacinae Mospalax.At present there is no research about the establishment of its fibroblast cell lines and cell biological characteristics.As a representative animal genetic resource in the Mongolian Plateau,in this study,we selected 9 tissues of Chinese Zokor for the experiment.Three tissues,including trachea,lung and xiphoid cartilage,were successfully established fibroblast cell lines and then we analyzed the biological characteristics of these cells.The main experimental method are to calculate the attachment rate of cells,the survival rate of the cell which is before cryopreservation and after resuscitation,and also include drawing and analysising of chromosome karyotypes.To evaluate its evolutionary status,we determine Cyt-b,D-loop,and ND4 gene sequences of mitochondrial DNA in Chinese Zokor.The experimental results showed that fibroblast-like cells derived from primary cultured of trachea,lung and xiphoid cartilage tissues were found in the third to fourth days,and there was more than 90%confluence of adherent cells when cultured in the 11th,16th and 17th days,respectively.The morphology of three kinds somatic cells is same as fibroblast characteristics.The attachment ability of the trachea fibroblast was the strongest,and the attachment rate was 98.10%then cultured for 24 h,and the attachment ability of the lung fibroblast and xiphoid cartilage were 95.28%and 94.88%respectively.The survival rate of cryopreserved fibroblasts from three different tissue sources decreased significantly.The survival rate of the trachea fibroblast from P4 to P8 was 98.92%to 93.20%before cryopreservation,and 91.57%to 73.25%after resuscitation,the survival rates of the lung fibroblast from P4 to P8 was 98.45%to 89.33%before cryopreservation,and89.97%to 70.21%after resuscitation,the survival rates of the xiphoid cartilage fibroblast from P4 to P6 was 98.52%to 82.91%before cryopreservation,and 88.23%to 69.06%after resuscitation.The growth curves of three fibroblasts are all in"S"type.The multiplication capacity of the trachea fibroblast was the strongest,the maximum proliferation number was 2.435×10~4 in well of a 24-well plate,the maximum proliferation number of lung fibroblast and xiphoid cartilage fibroblast were 1.813×10~4 and 1.521×10~4,respectively.The results of karyotype analysis showed that the chromosome number of fibroblast was 2n=62 in Chinese Zokor,thirty pairs were autosomes with a morphological type of 2st+28sm.The largest pair of chromosomes is the mesochromic chromosome.The other 28 pairs chromosomes are all submetacentric chromosomes,and sex chromosome X was a centromere chromosome.The results of analyzed phylogenetic tree showed that zokors come from two different maternal ancestors,and the phylogenetic relationship between Myospalax fontanieri and Eospalax baileyi was the closest.In this study,we established the culture system for the Chinese Zokor fibroblast cell lineage,and also analysis its basic biological characteristics of the fibroblast.The evaluation of the phylogenetic status of the Chinese Zokor provided an important experimental material and reference for the further study of its genetic and species evolution.