bulge区毛囊组织块培养结合酶消化法分离大鼠毛囊隆突部细胞的生物学特征

Biological characteristics of rat follicular bulge cells isolated by explant culture and enzyme digestion

背景：目前毛囊隆突部细胞的分离培养方法多存在细胞分离纯度不高、活力差等缺点，采用组织块法和酶消化法分离培养大鼠毛囊隆突部细胞能否克服上述缺点？目的：本实验采用组织块法和酶消化法分离培养大鼠毛囊隆突部细胞并对其进行鉴定。设计、时间及单位：单一样本设计，体外细胞培养实验。于2007—03／08在中国医科大学组织工程学实验室完成。材料：选用15只雌性Wistar大鼠，由中国医科大学实验动物中心提供。实验用表皮生长因子、Hoechst33342购白美国Sigma公司，SAB-小鼠IgG抗体免疫组化试剂盒、SABC—Cy3免疫荧光组织化学试剂盒、DAB显色剂、SABC—FITC免疫荧光组化试剂盒均购白武汉博士德公司。方法：利用显微分离的方法将大鼠触须部皮肤分离，剪开真皮和皮下组织，将毛囊与周围组织钝性分离取出毛囊，剪开结缔组织鞘，用显微外科镊取出毛囊外根鞘bulge区，移入培养板中进行组织块培养，应用0．25％胰酶＋0．02％EDTA消化分离大鼠毛囊bulge区细胞，倒置相差显微镜下观察其增殖能力，免疫荧光法检测毛囊隆突部细胞K15、β1整合索和P63的表达情况。主要观察指标：①大鼠隆突部细胞增殖能力。②毛囊隆突部细胞K15、β1整合索和P63的表达。结果：①大鼠隆突部细胞胞体积较大，胞核大，核仁明显，细胞生长迅速，活力好，3-4d即可传代，具有增殖能力强、细胞纯度高等特性。②细胞免疫荧光结果显示培养的细胞胞浆角蛋白15阳性，细胞的胞膜β1整合索阳性，细胞的胞核P63阳性。结论：组织块法结合酶消化法可成功分离培养毛囊隆突部细胞，细胞可同时表达K15、β1整合索和P63，纯度高，活力好。

BACKGROUND： At present, the method of isolating and culturing the follicular bulge cells is short of purity and vigor, can we isolate and culture rat bulge cells by explants culture and enzyme digestion to overcome the defects？ OBJECTIVE： To isolate and culture rat bulge cells by means of explants culture and enzyme digestion, and identify cells. DESIGN, TIME AND SETTING： A study based on single sample of cell culture in vitro was carded out in the Department of Tissue Engineering, Basic Medical College, China Medical University between March to August in 2007. MATERIALS： Fifteen female Wistar rats were provided by the Laboratory Animal Center in China Medical University. Epidermal growth factor and Hoechst33342 were bought from Sigma. SABC （mouse IgG）-AP kit, SABC （mouse IgG）-FITC kit, DAB color developing reagent and SABC （mouse IgG）-Cy3 kit were bought from Boster. METHOSD： The whisker skin was separated from rats by micro-isolation method, the dermis and subcutaneous tissue were cut, and then hair follicles were took out from surrounding. We sheared the connective tissue sheath and separated the bulge with micro-surgical forceps. Next, the bulge was put into the tissue culture plate for culture, and were separated with 0.25% enzyme and 0.02% EDTA. The proliferation of bulge cells was identified by inverted phase contrast microscope. Immunofluorescence staining was used to detect expression of K15, β1 integrin and P63 in the cultured cells. MAIN OUTCOME MEASURES： The reproductive activity of rat bulge cells;The expression of K15, β 1 integrin and P63 in bulge cells. RESULTS： The cell body of bulge cells was big, cell nuclei was large and the nucleoli was distinct. The cells grew fast and the vigor of the cells was good. We could passage the cells after 3-4 days. The cells maintained higher reproductive activity and higher purity. K15,β 1 integrin and P63 were detected to be positive for immunofluorescence staining. CONCLUSION： Follicular bulge cells can be successfully isolated and cultured by explants culture and enzyme digestion, meanwhile the expression of K15, β 1 integrin and P63 is positive in bulge cells. The bulge cells have high purity and good vigor.