敖汉细毛羊毛囊细胞系的建立及鉴定

Establishment and identification of hair follicle cell line of Aohan Fine-wool sheep

为了研究敖汉细毛羊毛囊细胞的培养方法,建立敖汉细毛羊毛囊细胞系,保存毛囊细胞便于后续细胞水平毛囊相关基因的深入研究,试验采用胰蛋白酶消化法对初生40日龄以内的敖汉细毛羊皮肤组织进行原代培养,通过原代培养、细胞传代、冷冻保存、复苏等方式成功分离并纯化出敖汉细毛羊毛囊细胞,然后对细胞进行了形态学观察,冻存、复苏活力检测,生长动力学分析,核型分析和微生物污染检测等分析。结果表明:原代毛囊细胞经胰蛋白酶消化及差速离心分离培养出毛囊细胞,冻存前细胞活力为94.19%,冻存1个月复苏后细胞活力为91.96%。细胞生长呈S型,经历潜伏期、指数生长期和平台期三个阶段。细胞的细菌、真菌、病毒和支原体检测均为阴性。试验成功分离了敖汉细毛羊毛囊细胞并建立了敖汉细毛羊毛囊细胞系。

To study the culture method of hair follicle cells of Aohan Fine- wool sheep and to establish hair follicle cell line of Aohan Fine - wool sheep and the preservation method of hair follicle cells to facilitate the subsequent intensive research on hair follicle - related genes at a cel- lular level, trypsin digestion method was conducted in this experiment for primary culture of the cells derived from skin tissue of 40 day -old Ao- han Fine - wool sheep. Hair follicle cells of Aohan Fine - wool sheep were successfully isolated and purified by primary culture, passage cul- ture, cell cryopreservation and thawing. Then several testing indexes on the cell line were carried out containing morphological observation, cryo- preservation, thawing cell viability, growth kinetics, karyotype analysis and microbiological contamination detection. The results showed that pri- mary hair follicle cells were isolated and cultured by trypsin digestion and differential centrifugation. Cell viability of 94.19% and 91.96% was determined before and one month after cryopreservation respectively. S type of cell growth kinetics on the cell line was calculated,including la- tent stage, exponential growth stage and platform stage. Negative results were obtained in the cell line＇ s microbiological contamination detection including bacteria,fungi,viruses and mycoplasma. Hair follicle cells of Aohan Fine - wool sheep were successfully isolated,and hair follicle cell line of Aohan Fine - wool sheep was established in this experiment.