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
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中国修复重建外科杂志2008年9月第22卷第9期·1113·
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
肖博 郭树忠 潘勇 刘丹【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、200 µg/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0.2 mL，作为实验组（A、B、C、D组）。培养1.5、3.0和4.5 h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0.2 mL，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2 mL，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100 µg/mL浓度时可发挥最大促进作用。【关键词】 釉基质蛋白 成纤维细胞 黏附 增殖 胶原合成中图分类号： R318 Q813 文献标志码：A
EFFECTS OF ENAMEL MATRIX PROTEINS ON ATTACHMENT, PROLIFERATION AND PRE-mRNA OF TYPE I COLLAGEN SYNTHESIS OF HUMAN DERMAL FIBROBLAST CELLS IN VITRO/ XIAO Bo, GUO Shuzhong, PAN Yong, LIU Dan. Department of Plastic Surgery, Xijing Hospital, the Forth Military Medical University, Xi’an Shaanxi, 710032, P.R.China. Corresponding author: GUO Shuzhong, E-mail: shuzhong@ fmmu.edu.cn【Abstract】 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 μg/mL). ① The cell attachment experiment: 0.2 mL cells suspension at the concentration of 1 × 106/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 × 104/mL was added to the pre-coated 96-well plates as the experimental groups (groups A1, 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 × 106/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). ② The cell proliferation experiment: At 2 days, there
基金项目：国家自然科学基金青年科学基金资助项目（30400508）作者单位：西安第四军医大学西京医院全军整形外科研究所（西安，710032）通讯作者：郭树忠，教授，博士导师，研究方向：同种异体组织移植，E-mail: shuzhong@fmmu.edu.cnChinese Journal of Reparative and Reconstructive Surgery, September 2008, Vol. 22, No.9·1114·
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 (0.598 ± 0.020 and 0.639 ± 0.016 ), C1 (0.582 ± 0.017 and 0.641 ± 0.020) and D1 (0.574 ± 0.021 and 0.635 ± 0.021) was significantly higher than that of the control group (0.548 ± 0.021 and 0.605 ± 0.019, P < 0.05); at 8 days, the absorbance of group B1 (0.629 ± 0.012) and group C1 (0.631 ± 0.014) was significantly higher than that of the control group (0.606 ± 0.031, P < 0.05). ③ The synthesis experiment of pre-mRNA: The synthesis of type I collage pre-mRNA of groups B2, C2 and D2 was significantly higher than that of the control group. Conclusion EMPs stimulate human dermal fibroblast cell attachment, proliferation and synthesis of type I collage pre-mRNA, and its maximal effect can be achieved at the concentration of 100 μg /mL.【Key words】 Enamel matrix proteins Fibroblast cell Attachment Proliferation Collagen synthesisFoundation item: National Natural Science Fund for Young Scientists (30400508)
