异种(猪)脱细胞真皮基质粉对表皮细胞和成纤维细胞的促进作用  

Promoting effect of xenogeneic(porcine) acellular dermal matrix powder on epidermal cells and fibroblasts

作　　者：常铭洋 陈志强 柳娟 闫舫 季守平 王韫芳 Chang Mingyang;Chen Zhiqiang;Liu Juan;Yan Fang;Ji Shouping;Wang Yunfang(Tissue Engineering Laboratory,Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院、卫生勤务与血液研究所、组织工程研究室,北京100850

出　　处：《中华损伤与修复杂志（电子版）》2018年第4期289-296,共8页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金(31370990)

摘　　要：目的观察冷冻研磨获得的脱细胞真皮基质粉(ADMP)生物学性状,探索高纯度且保留生物活性的异种(猪)ADMP对表皮细胞和成纤维细胞生长的支持及在皮肤创伤修复中的应用。方法选取幼龄长白猪背部皮肤,经生物酶法脱细胞、冷冻干燥后经过不同冷冻研磨程序得到异种(猪)ADMP,再由60钴辐照获得无菌异种(猪)ADMP;通过扫描电子显微镜和透射电子显微镜观察异种(猪)ADMP颗粒大小及胶原形态,确定适合应用的研磨程序;通过MTT法检测Ha Ca T细胞和BJ人皮肤成纤维细胞在不同浓度异种(猪)ADMP包被下的黏附作用,确定异种(猪)ADMP实现细胞最大黏附的浓度范围;DMEM培养液基添加异种(猪)ADMP为实验组,单纯DMEM培养液基为对照组,使用实时细胞分析技术分析BJ人皮肤成纤维细胞迁移指数,验证异种(猪)ADMP对成纤维细胞的促迁移作用;原代表皮细胞接种在异种(猪)ADMP培养液8 d,绘制增殖曲线,检测异种(猪)ADMP对原代表皮细胞的促增殖作用,同时免疫荧光检测原代表皮细胞表面成熟与分化蛋白的表达。对数据进行Student's t-检验。结果 1、2、3、5、7、10次循环研磨获得异种(猪)ADMP,肉眼可见全部为白色、无块状,其中1、2、3次循环呈较粗颗粒;5、7、10次循环呈现集中均匀趋势,粒径分布在0~12μm之间。其中5次循环粒径(13.00±2.10)μm与7次循环[(6.00±0.96)μm比较,差异有统计学意义(t=6.093,P=0.0002)。异种(猪)ADMP透射电子显微镜下发现,不同循环次数获得的异种(猪)ADMP均能保留大量胶原分子。BJ人成纤维细胞和Ha Ca T细胞随包被浓度的增加,细胞黏附作用不断增强,浓度高于750μg/cm2,无明显变化。实时细胞分析技术检测BJ人皮肤成纤维细胞在DMEM培养液基对照组培养液72 h后的迁移指数为1.21±0.10,而异种(猪)ADMP培养液基试验组的迁移指数为3.66±0.11,两组比较差异有统计学意义(t=22.24,P=0.002)。在异种(猪)ADMP培�Objective To observe the biological characteristics of xenogeneic（porcine） acellular dermal matrix powder（ ADMP）,and explore the support of high purity and bioactive ADMP for the growth of epidermal cells and fibroblasts and its application in skin wound repair. Methods The back skin of Landrace was selected and decellularized by biological enzymatic method and freeze-dried to obtain xenogeneic（ porcine） ADMP after being subjected to different cryogenic grinding procedures. Then,the sterile xenogeneic（ porcine） ADMP was obtained by irradiation with60 cobalt. The particle size and collagen morphology of Ha Ca T cells and BJ human skin fibroblasts with the cover of xenogeneic（ porcine） ADMP were observed by scanning and transmission electron microscope,and the grinding program suitable for application was determined. The MTT was used to detect the adhesion efficiency of xenogeneic（ porcine）ADMP cells in different concentrations,and the concentration range of xenogeneic（ porcine） ADMP to achieve maximum adhesion was determined. DMEM medium was added with xenogeneic（ porcine） ADMP as experimental group,and DMEM medium alone was used as control group. The real-time cellular analysis technology was used to analyze the migration index of BJ human skin fibroblasts,to verify the promotion of xenogeneic（ porcine） ADMP on the fibroblasts. The primary epidermal cells were inoculated in xenogeneic（porcine） ADMP culture for 8 days and the proliferative curve was drawn to detect the proliferative effect of xenogeneic（ porcine） ADMP on the primary epidermal cells. Meanwhile,immunofluorescence was used to detect the expression of the surface maturation and differentiation proteins of the primary epidermal cells. The data were compared with Student＇ s t-test. Results After 1,2,3,5,7 and 10 cycles of the grinding procedure,xenogeneic（ porcine） ADMP was obtained. All of which was white small powder,of which 1,2,3 cycle was large particles,5,7,10 cycles showed a uniform concentrated t

关 键 词：成纤维细胞 猪 创伤和损伤 表皮细胞 脱细胞真皮基质粉 

分 类 号：R622[医药卫生—整形外科]