摘要
为建立豚鼠成纤维细胞系,分离培养豚鼠胎儿成纤维细胞,并对其进行细胞活力、生长曲线、细胞周期及基因转染等分析。结果表明,豚鼠胎儿成纤维细胞在10代以内时形态良好,随着传代次数的增加,梭型细胞有拉长趋势,至第20代时梭形明显拉长,单位面积细胞数目大量减少。同时,细胞冻存后复苏贴壁情况良好。EGFP基因转染的成纤维细胞形态特征及生长状态与未转染细胞相似,转基因细胞和未转基因细胞的生长曲线均为S形,符合体外培养细胞正常生长增殖规律。以流式细胞仪分析逆转录病毒介导的EGFP基因转染细胞,可见染色体核型仍为二倍体,说明感染的细胞保持其遗传稳定性。流式细胞仪检测转染后不同时间点收集的病毒液感染的细胞效率,结果显示,病毒转染48h、72h收集病毒液组感染效率分别为16.9%和11.6%,显著高于24h收集病毒液组(0.9%)。
To obtain the highquality fetal fibroblasts cells from guinea pig, in this study, guinea pig fetus fibroblasts were isolated and characterized in vitro, meanwhile, the EGFP gene mediated by ret rovirus was infected. In vitro, EGFP mediated by retroviral vector （named pLEGFPN1） was trans fected into 293 T cells by liposome. Virus were collected after transfection 24 h, 48 h, 72 h later sepa rately, then filtered through 0.45 /am filter. Fibroblasts were infected by retrovirus with 8 mg/L po lybrene. Results showed that guinea pig fetal fibroblast cells within 10 passage maintained good mor phology by morphological observation, with the increasing of passages, the spindle cells were elonga ted, obviously elongated spindle in passage 20, the numbers of cells per unit area of reduced signifi cantly. At the same time, recovery of adherent cells after cryopreservation was in good condition. EGFP gene transfected fibroblasts morphological characteristics and their growth state were similar with nontransfected cells. The results showed that the growth of ceils curve in vitro accorded with normal cell growth proliferation rule, with Sform curve. The flow cytometric analysis showed that the transfected cells maintained normal diploid, which indicated that infected cells maintain the stability of the genetic. were 16.9% and er than after 24h The flow cytometry ana 11.6%respectdively after after virus transfection lysis assay showed the percentages of EGFP positive cells 48 h and 72 h of virus transfection, were significantly higher than after 24h after virus transfection（0. 90%）.
出处
《西北农业学报》
CAS
CSCD
北大核心
2013年第12期174-180,共7页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家重点基础研究发展规划(973)(2012CB126300
2012CB518801)
国家自然科学基金(31260287
31201867)
