猪急性创面不同部位组织匀浆液对体外培养的成纤维细胞凋亡的影响及其机制  

THE PROAPOPTOTIC EFFECT OF THE HOMOGENATE OF THE TISSUE OF DIFFERENT PARTS OF PIG'S FULL THICKNESS DERMAL WOUNDS ON CULTURED FIBROBLASTS

作　　者：李金清[1] 付小兵[1] 孙同柱[1] 顾绍峰[1] 都义日[1] 

机构地区：[1]解放军总医院304临床部全军创伤修复重点实验室

出　　处：《中国修复重建外科杂志》2005年第1期6-9,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家重点基础研究规划资助项目 (G1 9990 542 0 4 ) ;国家自然科学基金面上资助项目 (30 1 70 966) ;国家自然科学基金重点资助项目(30 2 30 370 )~~

摘　　要：目的　探讨猪急性创面不同部位组织匀浆液对体外培养的成纤维细胞 (fibroblast,FB)凋亡的影响。　方法　在 6只小型猪的背部造成直径 4 cm的圆形全层皮肤缺损创面 ,用取下的真皮组织进行 FB原代培养。在创面形成后第 15天 ,取创面中心及边缘新生上皮下的组织匀浆 ,离心后将上清液 - 70℃冰箱保存备用。用第 4代 FB进行实验 ,根据培养液的不同分成 7组 : 组 :DMEM+5 %胎牛血清 ; 组 :DMEM+5 %创面中心组织匀浆液 ; 组 :DMEM+5 %创面边缘组织匀浆液 ; 组 :DMEM+5 %创面中心组织匀浆液 +10μg/ ml广谱基质金属蛋白酶抑制剂 (GM6 0 0 1) ; 组 :DMEM+5 %创面边缘组织匀浆液 +10μg/ ml GM6 0 0 1; 组 :DMEM+5 %创面中心组织匀浆液 +10 ng/ ml酸性成纤维细胞生长因子 (acid fibroblast growth factor,a FGF) ; 组 :DMEM+5 %创面边缘组织匀浆液 +10 ng/ ml a FGF。在上述培养液中培养 16 h,均采用同一动物的匀浆液处理该动物来源的 FB(n=6 )。采用 PI和标记 FITC的 Annexin 作为探针 ,利用流式细胞仪检测细胞凋亡。　结果　各组细胞凋亡率分别为 : 组 4 .39%± 0 .4 1% , 组 10 .98%±1.4 2 % , 组 13.4 7%± 1.4 4 % , 组 7.2 %± 0 .4 6 % , 组 12 .1%± 0 .85 % , 组 3.9%± 0 .6 3% , 组 9.8%± 0 .5 0 % ;Objective To observe the proapoptotic effect of the homogenate of different parts of pig's full thickness dermal wounds on cultured fibroblasts. Methods The tissues were dissected from the wound center and sub neoepithelium separately 15 days after homogenization and sterilization, the specimens stored at -70℃. The forth passage of the fibroblasts were cultured for 16 hours in different culture solutions and were grouped into 7 groups: DMEM containing 5% fetal bovine serum as Group Ⅰ, DMEM containing 5% homogenate of tissue from wound center as GroupⅡ, DMEM containing 5% homogenate of tissue from subneoepithelium as Group Ⅲ, the culture solution of Group Ⅱmixed with 10 μg/ml GM6001 in Group Ⅳ, with the culturing medium of Group Ⅲ plus 10 μg/ml GM6001 as Group Ⅴ, the culture solution of Group Ⅱ mixed with 10 ng/ml aFGF as Group Ⅵ, and the culture solution of Group Ⅲ mixed with 10 ng/ml aFGF as Group Ⅶ. In all groups except Group Ⅰ, the fibroblasts of the 6 pigs were treated with the homogenate derived from the same animal respectively. After being incubated in Annexin Ⅴ FITC and PI, cells were analyzed by Flow Cytometry and the rate of apoptotic cells was acquired. The data were analyzed by SPSS 11 0 using Least significant Difference test(LSD). Results The apoptotic rate of the 7 groups were as follows: 4 39%±0 41% in Group Ⅰ,10 98%±1 42% in Group Ⅱ,13 47%±1 44% in Group Ⅲ,7 2%±0 46% in Group Ⅳ, 12 1%±0 85% in Group Ⅴ,3 9%±0 63% in Group Ⅵ,9 8%±0 50% in Group Ⅶ; there were significant differences between every two groups except Group Ⅰand Group Ⅵ. Conclusion Homogenate of the tissue derived from the subneoepithelium has greater proapoptotic effect than that from the wound center; the proapoptotic effect of homogenate of the tissue both under neoepithelium and in wound center can be significantly alleviated by acid fibroblast growth factor, partly because of MMPs.

关 键 词：创面 组织匀浆液 心组织 FB DMEM 凋亡 体外培养 FGF 培养液 成纤维细胞 

分 类 号：Q813[生物学—生物工程]