论文部分内容阅读
目的采用渗透冲击联合超声和去污剂处理新生牛真皮,观察处理前后新生牛真皮基质的组织学和生物活性成分的变化。方法从8头健康雄性新生牛皮肤获取网状层真皮组织。采用反复渗透压改变、超声震荡和去污剂联合法对新生牛网状层真皮进行处理,HE染色、DAPI染色和核酸电泳检测细胞成分残留情况;扫描电镜(SEM)观察胶原束结构和细胞成分;PicoGreen法、DMMB法和BCA法对脱细胞前后真皮基质的DNA、sGAG和蛋白质含量进行定量分析;ELISA法测定脱细胞前后真皮基质内TGF-β1、EGF、bFGF和KGF含量。结果形态学观察可见脱细胞处理后的真皮三维结构完整,胶原纤维束排列较疏松,细胞清除较彻底,未见明显细胞及细胞碎片残留。与脱细胞处理前比较,脱细胞处理后的DNA含量[(2 516.1±324.2)ng/mg vs(249.5±53.8)ng/mg,P=0.000)]、sGAG含量[(5.92±0.50)μg/mg vs(2.48±0.24)μg/mg,P=0.000]、TGF-β1含量[(478.8±196.1)pg/g vs(180.3±111.0)pg/g,P=0.009]和EGF含量[(10.52±2.78)pg/g vs未能检测到EGF含量]均明显减少;而bFGF含量[(788.6±333.8)pg/g vs(364.8±294.8)pg/g,P=0.424]下降了53.7%,KGF含量[(0.033±0.000)pg/g vs(0.033±0.000)pg/g,P=0.433]变化不显著。结论反复渗透冲击联合超声和去污剂处理对新生牛真皮基质明显地清除了细胞及细胞碎片,较好地保留了真皮细胞外基质的三维结构和主要生物活性成分。
Objective To investigate the changes of histology and bioactive components of newborn bovine dermal matrix before and after treatment by osmotic shock combined with ultrasonic and detergent. Methods The dermis of reticular layer was obtained from 8 healthy male newborn cow skin. The dermis of newborn bovine reticular layer was treated with repeated osmotic pressure change, ultrasonic vibration and detergent combination method. The cell residue was detected by HE staining, DAPI staining and nucleic acid electrophoresis. The structure and cell components of collagen bundles were observed by scanning electron microscope (SEM) The DNA, sGAG and protein contents of dermal matrix before and after decellularization were quantified by PicoGreen method, DMMB method and BCA method. The contents of TGF-β1, EGF, bFGF and KGF in dermal matrix were detected by ELISA. Results Morphological observation showed that the three-dimensional structure of the dermis after acellular treatment was intact, the collagen fiber bundles were loosely arranged, and the cell clearance was more thorough. No obvious cell and cell debris remained. Compared with that before decellularization, the content of DNA after decellularization [(2 516.1 ± 324.2) ng / mg vs (249.5 ± 53.8) ng / mg, P = 0.000) (478.8 ± 196.1) pg / g vs (180.3 ± 111.0) pg / g, P = 0.009] and EGF content [(10.52 ± 2.78) pg / g vs. undetectable EGF] were significantly decreased; while bFGF content [(788.6 ± 333.8) pg / g vs (364.8 ± 294.8) pg / g, P = 0.424] [(0.033 ± 0.000) pg / g vs (0.033 ± 0.000) pg / g, P = 0.433]. Conclusion Repeated osmotic shock combined with ultrasonic and detergent treatment of newborn bovine dermal matrix clear cell and cell debris, retained the three-dimensional structure of dermal extracellular matrix and the main bioactive components.