釉基质蛋白对人皮肤成纤维细胞黏附、增殖及Ⅰ型胶原前mRNA合成影响的体外研究  

EFFECTS OF ENAMEL MATRIX PROTEINS ON ATTACHMENT,PROLIFERATION AND PRE-mRNA OF TYPE I COLLAGEN SYNTHESIS OF HUMAN DERMAL FIBROBLAST CELLS IN VITRO

作　　者：肖博[1] 郭树忠[1] 潘勇[1] 刘丹[1] 

机构地区：[1]西安第四军医大学西京医院全军整形外科研究所,西安710032

出　　处：《中国修复重建外科杂志》2008年第9期1113-1116,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金青年科学基金资助项目(30400508)~~

摘　　要：目的观察釉基质蛋白(enamel matrix proteins,EMPs)对体外培养的人正常皮肤成纤维细胞黏附、增殖及Ⅰ型胶原前mRNA合成的影响。方法取自愿捐赠包皮环切术后包皮组织,采用组织块法培养人皮肤成纤维细胞,取第3～6代细胞进行实验。以终浓度为50、100、150、200μg/mL的EMPs工作液预铺培养板。①细胞黏附实验:取细胞以1×106个/mL置于预铺EMPs的96孔培养板,每孔0.2mL,作为实验组(A、B、C、D组)。培养1.5、3.0和4.5h后MTT比色法测定黏附细胞量。②细胞增殖实验:取细胞以5×104个/mL置于预铺EMPs的培养板,每孔0.2mL,作为实验组(A1、B1、C1、D1组)。于第2、4、6、8天以MTT比色法测定增殖细胞量。③细胞Ⅰ型胶原前mRNA合成实验:取细胞以1×106个/mL置于预铺EMPs培养板,每孔2mL,作为实验组(A2、B2、C2、D2组)。于培养第5天RT-PCR法测定Ⅰ型胶原前mRNA合成的量。以上实验均以相同浓度细胞悬液加入未预铺EMPs的板孔作为对照组,每组均3个复孔。结果①细胞黏附实验中,各时间点B、C、D组与A组及对照组比较,差异均有统计学意义(P<0.05);A组与对照组间及B、C、D组间比较差异均无统计学意义(P>0.05)。②细胞增殖实验中,第2天各组间差异均无统计学意义(P>0.05);第4、6天,B1、C1、D1组吸光度(A)值分别为0.598±0.020、0.582±0.017、0.574±0.021及0.639±0.016、0.641±0.020、0.635±0.021,均高于对照组的0.548±0.021及0.605±0.019(P<0.05);A1组A值分别为0.545±0.023及0.603±0.016,与对照组差异无统计学意义(P>0.05)。第8天,B1、C1组A值分别为0.629±0.012、0.631±0.014,高于对照组的0.606±0.031(P<0.05),A1、D1组与对照组比较,差异无统计学意义(P>0.05)。③细胞Ⅰ型胶原前mRNA合成实验中,B2、C2、D2组Ⅰ型胶原前mRNA合成量高于对照组。结论EMPs促进人正常皮肤成纤维细胞黏附、增殖及Ⅰ型胶原前mRNA合成,且EMPs在100μg/mL浓度时可发挥最大促进作用。Objective To investigate the influence of enamel matrix proteins （EMPs） on the attachment, proliferation and pre-mRNA of type Ⅰ collagen synthesis of cultured human dermal fibroblast cells. Methods Human dermal fibroblast cells were obtained from human acrobystia and cultured in DMEM medium with 10% FBS. The 3rd to 6th passage cells were used. Ninety-six-well plates and 6-well plates were pre-coated with different concentrations of EMPs （50, 100, 150 and 200 ug/mL）.① The cell attachment experiment： 0.2 mL cells suspension at the concentration of 1 × 10^6/mL was added to the pre-coated 96-well plates as the experimental groups （groups A, B, C and D based on different concentrations of EMPs）. At 1.5, 3.0, and 4.5 hours after inoculation, the attached cells were measured by MTT method. ② The cell proliferation experiment： 0.2 mL cells suspension at the concentration of 5 × 10^4/mL was added to the pre-coated 96-well plates as the experimental groups （groups AI, B1, C1 and D1 based on the different concentrations of EMPs）. At 2, 4, 6 and 8 days after inoculation, the cells were measured by MTT method. ③ The synthesis experiment of pre-mRNA： 2 mL cells at the concentration of 1 × 10^6/mL was added to the pre-coated 6-well plates as the experimental groups （groups A2, B2, C2 and D2 based on different concentrations of EMPs）. At 5 days after inoculation, the synthesis of pre-mRNA was measured by RT-PCR method. Human dermal fibroblast cells were added to the un-coated plates as the control groups. Results ① The cell attachment experiment： There were significant differences in attachment cells between the control group, group A and the groups B, C and D （P 〈 0.05）. There were no significant difference between group A and control group （P 〈 0.05）. ②The cell proliferation experiment： At 2 days, there were no significant differences in absorbance between the control group and the experimental groups （P 〉 0.05）; at 4 days and 6 days, the absorbance of groups B1 �

关 键 词：釉基质蛋白 成纤维细胞 黏附 增殖 胶原合成 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]