两种人包皮成纤维细胞分离培养方法的比较  

Comparison of two methods for isolation and culture of human foreskin fibroblasts

作　　者：曹卉[1] 王薇[2] 肖敬川[1] 黄邓高[1] 高元慧[1] 朱丹 CAO Hui;WANGWei;XIAO Jingchuan;HUANG Denggao;GAO Yuanhui;ZHU Dan(Central Laboratory,Affiliated Haikou Hospital of Xiangya School of Medicine,Central South University,Haikou 570208;Department of Dermatology,Affiliated Haikou Hospital of Xiangya School of Medicine,Central South University,Haikou 570208,China)

机构地区：[1]中南大学湘雅医学院附属海口医院中心实验室,海口570208 [2]中南大学湘雅医学院附属海口医院皮肤科,海口570208

出　　处：《中南大学学报（医学版）》2021年第8期800-808,共9页Journal of Central South University ：Medical Science

基　　金：海南省自然科学基金(818QN325);海南省卫生健康行业科研项目(20A200475)。

摘　　要：目的:作为理想的种子细胞,成纤维细胞的高效获取及纯化尤为重要。本研究通过比较改良组织块培养法(improved tissue culture method,ITCM)及酶消化培养法(enzyme digestion method,EDM)分离培养人包皮成纤维细胞(human foreskin fibroblasts,HFF),以探讨两种方法的优缺点。方法:ITCM是将剪碎的皮肤组织置0.1%Ⅱ型胶原酶中于4℃下消化过夜,分离表皮与真皮,对真皮组织采用0.25%胰酶于室温下再次消化15 min,待组织块贴壁于培养皿后进行培养。EDM是将剪碎的皮肤组织置0.25%胰酶中于4℃下消化过夜,分离表皮与真皮,将真皮组织再次于4℃下用0.1%Ⅱ型胶原酶消化过夜,然后将组织块与酶的混合物一起过滤、挤压,用含有胎牛血清的培养基冲洗滤网,对得到的细胞悬液进行培养。ITCM与EDM均采用两种酶进行消化,但两种酶的使用顺序、消化时间及温度不同,最终接种于培养皿中进行后续培养的分别是组织块和细胞悬液。本研究利用ITCM和EDM分离、培养HFF,观察ITCM组和EDM组HFF的原代至第3代(Passage 0~Passage 3,P0~P3)的细胞形态;染色波形蛋白、CD68和角蛋白以鉴定细胞纯度;绘制P3生长曲线对比两组细胞的增殖能力。采取120 mJ/cm^(2)的中波紫外线(medium-wave ultraviolet,UVB)照射P3 ITCM组和EDM组HFF,建立光损伤模型。根据有无照射紫外线,实验分为UVB组和未照射对照组(Control)。采取二次离心法提取贫血小板血浆(platelet-poor plasma,PPP),用含不同浓度(0,2.5%,5.0%及10.0%)PPP的完全培养基对ITCM组和EDM组的HFF进行分组培养,加入PPP 24 h后用CCK-8试剂盒检测损伤细胞的增殖情况。结果:ITCM组培养至第3天时可观察到大量HFF,受杂质影响小;EDM组HFF形态的观察受到较多杂质影响;第9天时,两组细胞均可传代;两种方法体外分离培养的HFF均呈长梭形、旋涡状生长。HFF培养至P2时ITCM组和EDM组波形蛋白的阳性率比较差异有统计学意义[(97.36±0.Objective:The efficient acquisition and purification of fibroblasts as ideal seed cells are very important.For optimization of the isolation and culture of human foreskin fibroblasts(HFF),we compared the improved tissue culture method(ITCM)and the enzyme digestion method(EDM).Methods:In ITCM,the skin tissue was digested with 0.1%TypeⅡcollagenase overnight at 4℃,the epidermis was separated from the dermis and digested again with 0.25%trypsin at room temperature for 15 min,and then the tissue block was attached to the culture dish.In EDM,the skin tissue was digested with 0.25%trypsin overnight at 4℃,the epidermis was separated from the dermis and digested with 0.1%TypeⅡcollagenase overnight at4℃,the tissue block was filtered and squeezed together with the enzyme mixture,the filter was rinsed with medium containing fetal bovine serum,and the cell suspension was cultured.Both ITCM and EDM used 2 digestion enzymes,but the order,digestion time,and temperature of the 2 enzymes were different.The final inoculations of ITCM and EDM in the dishes for subsequent culture were tissue blocks and cell suspensions,respectively.In this study,HFF cells were isolated and cultured with ITCM and EDM,and the cell morphology was observed from Passage 0 to Passage 3 in the ITCM and EDM groups.The cell purity was identified by staining for vimentin,CD68,and Pan-keratin.The growth curves of Passage 3 were plotted to compare the proliferation ability of the 2 groups.Passage 3 HFF cells in the ITCM and EDM groups were irradiated with medium-wave ultraviolet(UVB)at an energy value of 120 m J/cm^(2)to establish a light damage model.The experiments were grouped into an UVB group and a control group(Control)according to the presence or absence of UVB irradiation.Platelet-poor plasma(PPP)was extracted by secondary centrifugation,and the HFF cells of ITCM and EDM groups were cultured in groups using complete medium containing different concentrations(0,2.5%,5.0%,and10.0%)of PPP,and the proliferation of damaged cells was detected by cell

关 键 词：人包皮成纤维细胞 改良组织块培养法 酶消化培养法 细胞鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]