人Ⅰ型胶原包被培养板不同时间对人原代表皮细胞培养的影响  

Effects of different culture plates coating time on human primary epidermal cells culture with human type Ⅰ collagen

作　　者：徐圣博 王鑫 申传安[1] 郑波 Xu Shengbo;Wang Xin;Shen Chuan′an;Zheng Bo(Department of Burns and Plastic Surgery,Fourth Medical Center of PLA General Hospital,Beijing 100048,China;Department of Emergency,Weinan Central Hospital,Weinan 714000,China)

机构地区：[1]解放军总医院第四医学中心烧伤整形科,北京100048 [2]渭南市中心医院急诊科,714000

出　　处：《中华损伤与修复杂志（电子版）》2019年第3期166-173,共8页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金(81641090);国家重点研发计划项目(2017YFC1103503);北京市自然科学基金(7171009);北京市科技计划项目(Z171100001017146)

摘　　要：目的探索人Ⅰ型胶原包被培养板不同时间对培养的人原代表皮细胞形态、黏附、增殖、迁移率及细胞周期的影响。方法取来源于解放军总医院第四医学中心泌尿外科新鲜人体皮肤组织标本,配置体积分数为1%的人Ⅰ型胶原蛋白溶液并包被培养板,按包被时间不同分为10 s组、1 min组、5 min组、15 min组、30 min组及0 s组,采用胰蛋白酶动态消化法消化分离表皮细胞,接种于不同胶原包被时间的培养板中,加入表皮细胞培养基(CnT-Pr培养基)于37℃、含5%CO 2的细胞培养箱中培养。倒置相差显微镜下观察细胞形态,细胞计数试剂盒8(CCK-8)法检测细胞黏附及增殖,划痕实验观察细胞迁移,流式细胞法检测细胞周期。对数据行方差分析与LSD-t检验。结果(1)细胞数量及形态:随时间推移,各包被组细胞形态未见明显差异,大小较均匀,呈球形或眼型,细胞数量逐渐增多。0 s组细胞数量较其他5组少且未贴壁细胞比例多。(2)细胞黏附:人原代表皮细胞接种后24 h,10 s组、1 min组、5 min组、15 min组、30 min组、0 s组的吸光度值分别为0.28±0.07、0.30±0.05、0.33±0.06、0.34±0.07、0.36±0.05、0.16±0.02,6组间比较,差异有统计学意义(F=4.640,P=0.014);10 s组、1 min组、5 min组、15 min组、30 min组组间两两比较,差异均无统计学意义(P值均大于0.05)。0 s组分别与10 s组、1 min组、5 min组、15 min组、30 min组比较,差异均有统计学意义(t=2.640、3.059、3.584、3.889、4.187,P=0.021、0.010、0.004、0.002、0.001)。(3)细胞增殖:人原代表皮细胞增殖曲线显示胶原包被的各组细胞增殖情况相似,明显高于0 s组细胞增殖情况。细胞接种后2 d,10 s组、1 min组、5 min组、15 min组、30 min组及0 s组的细胞吸光度值分别为0.41±0.05、0.41±0.02、0.46±0.06、0.49±0.08、0.53±0.12、0.09±0.04,6组间比较,差异有统计学意义(F=16.050,P<0.05)。组间两两比较可以发现,10 s组、1 min组�Objective To explore the effects of different culture plates coating time with human typeⅠcollagen on the cell morphology,adhesion,proliferation,migration rate and cell cycle of cultured human primary epidermal cells.Methods Fresh human skin tissue was taken from Department of Urology Surgery,Fourth Medical Center of PLA General Hospital,then the human typeⅠcollagen solution with a volume fraction of 1%was placed.Dishes were coating with collagen solution and divided into 10 s group,1 min group,5 min group,15 min group,30 min group,and 0 s group according to the different coating time.Then epidermal cells were dissociated from skin tissue by dynamic digestion with trypsin,inoculated in culture plates with different collagen coating time and cultured in a cell culture incubator with 5%CO 2 at 37℃with epidermal culture medicum(CnT-Pr medium).Then cell morphology was observed under interted phase contrast microscope.Cell adhesion and proliferation were detected using cell counting kit-8(CCK-8).Cell migration was observed by scratch assay,and cell cycle was detected using flow cytometry.Data were processed with analysis of variance and LSD-t test.Results(1)Cell quantity and morphology:there was no significant difference in the morphology of epidermal cells within each group over time.The size of cells in each group were relatively uniform,spherical or eye-shaped,and the number of cells gradually was increasing.The number of cells in 0 s group was less than that of the other 5 groups and more unattached cells were found in the 0 s group than in the other groups.(2)Cell adhesion:24 hours after human primary epidermal cells were inoculated,the absorbance value of epidermal cells in the 10 s group,1 min group,5 min group,15 min group,30 min group and 0 s group was 0.28±0.07,0.30±0.05,0.33±0.06,0.34±0.07,0.36±0.05,and 0.16±0.02.The difference in absorbance among the 6 groups was statistically significant(F=4.640,P=0.014).There were no significant differences between any 2 of 5 groups(the 10 s group,the 1 min g

关 键 词：胶原Ⅰ型 细胞 培养的 细胞增殖 细胞运动 细胞周期 人表皮细胞 孔板 

分 类 号：R641[医药卫生—外科学]