脱细胞处理对小肠黏膜下层细胞残留及生长因子含量影响的实验研究  

EFFECT OF ACELLULAR PROCESS ON SMALL INTESTINAL SUBMUCOSA CELL RESIDUE AND GROWTH FACTOR CONTENT

作　　者：陈薇[1] 李次会[2] 武术[2] 解慧琪[1] 罗静聪[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041 [2]北京德得创业科技有限公司

出　　处：《中国修复重建外科杂志》2010年第1期94-99,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：四川省科技攻关资助项目(05SG022-021);四川省科技重点资助项目(2006Z09-019;05SG022-005)

摘　　要：目的探讨机械-酶消化法对小肠黏膜下层(small intestinal submucosa,SIS)细胞残留量及生长因子含量的影响。方法取4h内市售新鲜猪空肠,采用机械处理(去除浆膜层、黏膜层及肌层)、脱脂、胰蛋白酶消化、去垢剂处理及冻干5个步骤制备SIS。每步操作后留取样品(n=4),分别作为A、B、C、D、E组;以新鲜猪空肠作为对照(n=4,F组)。HE染色及扫描电镜观察组织学变化;采用Nest-PCR技术测定死亡相关蛋白12(death associated protein12,DAP12)含量;ELISA法检测VEGF、bFGF、TGF-β和TNF-α的含量。结果组织学及扫描电镜观察显示A、B组有细胞残留,C、D、E组未见细胞残留。Nest-PCR检测示各组均含有DAP12;A～F组DAP12拷贝数分别为(18.01±9.53)、(11.87±2.35)、(0.59±0.27)、(0.29±0.05)、(0.19±0.04)、(183.50±120.13)copy×106/cm2。F组DAP12含量显著高于A～E组(P<0.05),A、B组高于C、D、E组(P<0.05),C、D、E组间比较差异有统计学意义(P<0.05),A、B组间差异无统计学意义(P>0.05)。ELISA法检测示A组VEGF、bFGF、TGF-β及TNF-α含量均显著高于其他各组(P<0.05);B、C、D、E组间比较差异无统计学意义(P>0.05)。结论单纯采用机械法制备SIS有较多细胞残留;机械-酶消化法能有效去除细胞,显著降低DAP12含量,生长因子含量无明显改变。Objective To investigate the effect of machine-enzyme digestion method on the residual quantity of small intestinal submucosa（SIS）cell and the content of growth factors.Methods Fresh jejunum of pig within 4 hours after harvesting was prepared into SIS after machine digestion（removing placenta percreta,mucosa,and muscular layer）,degrease, trypsinization,abstergent processing,and freeze drying.Samples were kept after every preparation step serving as groups A,B,C, D,and E,respectively（n=4 per group）.And the fresh jejunum served as control group（group F,n=4）.The histological alteration in each preparation process was reviewed with HE staining and scanning electron microscope（SEM）.Nest-polymerase chain reaction（PCR）was used to determine the content of death associated protein 12（DAP12）,and enzyme-linked immunosorbent assay（ELISA）was applied to detect the content of vascular endothelial growth factor（VEGF）,basic fibroblast growth factor（bFGF）, transforming growth factorβ（TGF-β）,tumor necrosis factorα（TNF-α）.Results HE staining and SEM observation showed that there were residual cells in groups A and B,and there were no residual cells in groups C,D,and E.Nest-PCR test revealed the occurrence of DAP12 in each group.The contents of DAP12 in groups A,B,C,D,E,and F were（18.01±9.53）,（11.87±2.35）, （0.59±0.27）,（0.29±0.05）,（0.19±0.04）,and（183.50±120.13）copy×106/cm2.The content of DAP12 in group F was significant higher than that of other groups（P0.05）,groups A and B was higher than groups C,D,and E（P0.05）,there were significant differences among groups C,D,and E（P0.05）,and there was no significant difference between groups A and B（P0.05）.The ELISA test showed the content of VEGF,bFGF,TGF-β,and TNF-αin group A was significantly higher than that of groups B,C, D,and E（P0.05）,and there was no significant difference among groups B,C,D,and E（P0.05）.Conclusion SIS prepared by simple mechanical method has more residual cells,w

关 键 词：小肠黏膜下层 脱细胞处理 机械-酶消化法 生长因子 死亡相关蛋白12 

分 类 号：R318.08[医药卫生—生物医学工程] Q813.1[医药卫生—基础医学]