绒山羊毛囊毛乳头细胞体外培养条件的优化  

Optimization of Dermal Papilla Cell Cultivation Condition In vitro in Cashmere Goat(Capra hircus)

作　　者：周雅婷 马森[1] 李金朋 高旭红 陈玉林[1] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100

出　　处：《农业生物技术学报》2018年第2期319-329,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31372279;No.31402038和No.31572369);公益性行业(农业)科研专项(No.201303059);国家绒毛羊产业技术体系(No.CARS-39-12)

摘　　要：毛乳头细胞(dermal papilla cells,DPCs)是毛囊(hair follicle,HF)底部一种特化的具有诱导毛囊再生能力的细胞,是诱导毛囊重建、传导毛发生长信号的重要结构。本研究通过采集陕北白绒山羊(Capra hircus)生长期皮肤组织样本,使用显微解剖法剥离毛乳头(dermal papilla,DP)并进行体外原代培养,利用细胞免疫荧光染色法检测DPCs中α-平滑肌激动蛋白(α-smooth muscle actin,α-SMA)和CD133/prominin-1(5_transmembrane,5_TM)的表达,然后采用细胞计数、MTT(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide)比色法、碱性磷酸酶(alkaline phosphatas,AKP)的测定等方法比较DPCs在不同血清比例下的常规培养DMEM/F12(dulbecco's modified eagle media/nutrient mixture F-12)(1∶1)与DMEM/F-12 Gluta MAX、减血清培养基Advanced DMEM/F-12与Advanced RPMI 1640(roswell park memorial institute 1640)的体外培养效果。结果显示:(1)细胞免疫组化染色证实所培养的细胞为DPCs,在供试的4种培养基中加入不同比例的血清后,均可成功培养DPCs并传代;(2)体外培养DPCs的4种培养基中,添加的血清比例越高,细胞的生长、增殖速度越快,细胞活力、细胞诱导能力越强;(3)当添加10%血清时,减血清培养基培养的DPCs的生长增殖速率及生物学功能均明显高于常规培养基(P<0.05);(4)添加4%~6%血清的减血清培养基与添加10%血清的常规培养基的培养效果无显著性差异(P>0.05)。由此可知,减血清培养基相较于常规培养基,含有对DPCs生长增殖、细胞活力、细胞诱导能力维持或加强的有效因子,因此添加4%~6%血清的减血清培养基更适合于DPCs培养。本研究首次提出利用减血清培养基体外培养DPCs,并探究出较适宜陕北白绒山羊DPCs体外培养的最佳培养基类型及血清浓度,为进一步研究毛乳头在毛囊周期发育中的分子机制提供了理想细胞模型参考。Dermal papilla cells（DPCs） are specialized mesenchymal-originated fibroblasts with prominent ability to induce hair follicle morphogenesis and development in mammals. They are situated at the bottom of hair follicle and direct hair follicle remodeling by sending various growth factors to follicular keratinocytes.Isolation and culture of DPCs from hair follicle is laborious and time-consuming job, thus present study wasdesigned to optimize the culture condition of goat DPCs in order to acquire enough DPCs for other studies. In this study, DP was separated and cultured from goat hair follicle under stereoscope by microdissection, and confirmed the identity of cultured cell by using antibodies specific to their marker genes α-smooth muscle actin and CD133/prominin-1, then cell counting,（3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-diphenytetrazoliumromide assay, alkaline phosphatas analysis and other methods were used to compare the combination of serum concentration and medium types. The results indicated that（1） goat DPCs shared the expression of specific markers as previously reported, and they were successfully cultured in four tested culture medium supplemented with different proportions of serum respectively;（2） Growth of goat DPCs depends on the nutritional supplement： the higher proportion of serum added in culture medium resulted faster proliferation rate and stronger ability to migrate and induce hair growth;（3） Advanced medium does contain effective factors to maintain or strengthen goat DPCs proliferation and inducing ability, thus it is more suitable for propagating goat DPCs than conventional medium, otherwise when the serum addition proportion is 10%,the growth and proliferation of goat DPCs cultured advanced medium are significantly higher than that in conventional media（P0.05）;（4） the effect of Advanced media added with 4% ~6% serum for gaot DPCs propagation is as good as the normal medium added with 10% serum（P0.05）. In conclusion, we successfully cultured goat DPCs

关 键 词：绒山羊 毛囊 毛乳头细胞 培养基 血清比例 

分 类 号：S8[农业科学—畜牧兽医]