新生仔猪皮肤干细胞的分离培养

Isolation and Culture of Stem Cells from New Born Porcine Skin

为大量获得出生后仔猪皮来源干细胞,以出生24h内仔猪皮肤为试验材料,首先对新生仔猪背部皮肤组织石蜡切片进行皮肤干细胞标记蛋白Integrinβ1和Cytokeratin 15(K15)双重间接免疫荧光染色,随后利用酶消化法分离新生仔猪皮肤干细胞,以DMEM/F12为基础培养基并添加B-27、EGF、bFGF等细胞因子,采用悬浮培养方法培养分离的干细胞,对细胞滴片进行Integrinβ1、K15、SOX2和OCT4免疫荧光染色,最后对获得的皮肤干细胞进行核型分析。皮肤组织石蜡切片染色结果显示K15阳性信号主要在毛囊外根鞘外侧表达,Integrinβ1阳性信号主要集中于毛囊的外根鞘基部内侧。在细胞培养阶段,随着培养时间延长,细胞克隆团数目不断增加,内部细胞更加紧凑,克隆团边缘清晰且折光性变强。免疫荧光染色显示干细胞克隆团中的细胞同时表达Integrinβ1和K15,且为SOX2、OCT4阳性,核型分析显示该细胞为正常核型2n=38。结果说明成功建立了培养新生仔猪皮肤干细胞的技术方法,为仔猪皮肤干细胞多能性及多向分化潜能的后续研究提供试验依据和技术支持。

To establish a method for isolation and culture of porcine dermal stem cells,the skin of newly born pigs are used as experimental material.First,the stem cell in the porcine skin tissue are located,the double indirect immunefluoresce staining of skin stem cell marker proteins Integrinβ1 and Cytokeratin 15(K15)is then performed on paraffin sections of newborn pigs'back skin.Second,Enzyme digestion is adopted to isolate dermal stem cells of new born porcine,DMEM/F12 is used as the base culture and added cytokines such as B-27,EGF and bFGF,afterwards stem cell clone clusters are acquired with suspension culture.Results show the positive signals of K15 occurred in the bulge area and the dermal sheath of the hair follicle.With the extension of time,the cluster count continuously increases,the internal cell becomes more compact,with clear edge and strong refractivity.Digest stem cell clone clusters into signal cell and immunefluoresce staining of Integrinβ1,K15,SOX2 and OCT4 is performed on drop sections,which shows the cells in stem cell clone clusters simultaneously expressed Integrinβ1 and K15,in addition,SOX2 and OCT4 are also positive.Karyotyping analysis reveals the cell is the regular karyotype 2n=38.The results demonstrates that a method to obtain porcine skin-derived stem cells has been established and these results also provide an experimental basis for the further study on in vitro multiplication capability and multidirectional differentiation potential of porcine dermal stem cells.