河曲马睾丸组织成纤维细胞系的建立及生物学特性研究

采集河曲马睾丸组织,用热消化法制备原代细胞,通过差速消化和差速贴壁法纯化细胞,扩大培养至第3代用液氮保存,细胞复苏后进行活力、形态、生长曲线、微生物污染、核型、乳酸脱氢同工酶谱及荧光蛋白质粒转染表达等特性研究。结果显示,河曲马睾丸组织原代和传代细胞呈成纤维型,生长良好,最大增殖数量为4.32×105 mL-1,倍增时间为34.58h;细菌、真菌、病毒、支原体检测呈阴性;染色体2 N=64,二倍体为85%,占主体;乳酸脱氢酶同工酶电泳图谱有明显特征,共有4条谱带,其中LDH3浓度较高,活性较强;外源质粒在该细胞中能进行复制和表达。表明已成功建立河曲马睾丸组织成纤维细胞系,使河曲马这一重要种质资源在细胞水平上得以保存。

Establishment of Testicle Fibroblast Line and Its Biological Characteristics in Hequ Horse

The testical explants of Hequ horse were cultured with trypsinization.The primary cells were purified and preserved with liquid nitrogen after three generations.Observations on morphology,dynamic growth,microbial contamination,analysis of karyotype,isoenzyme of lactate dehydrogenase and fluorescin plasmid transfection and expression were carried out.The results showed that the cell line was fibroblast and grew well.The maximum proliferation density was 4.32×105 mL-1 and population doubling time of cells(PDT) was 34.58 h;the tests for bacteria,fungi,virus and mycoplasma were all negative;the chromosomes were 64 and diploid cells were dominant of 85%.The banding patterns of the isoenzyme of lactate dehydrogenase were significant characteristics.There were four bands in all.As follow,the LDH3 was higher and stronger.The exogenous plasmid could copy and express in the cell.Those results showed that the cell line was established successfully,which makes the Hequ horse,a national important genetic resource preserved in cell level,as well as will provide an ideal experimental material for genetic studies.