摘要
目的观察基质金属蛋白酶7（MMP-7）、京尼平及真空冷冻干燥处理对猪脱细胞网状层真皮基质组织结构及细胞相窬性的影响。方法取清洁级健康大约克夏猪边腹部皮肤，采用机械法制作54块网状层真皮，面秋10．0mm×5．0mm，厚0．5～0．6mm，按随机数字表法分为正常对照绀（A。组，小仃处娌）、脱细胞组（B组，行脱细胞处理）、脱细胞＋MMP-7组（C组，行脱细胞和MMP-7处理）、脱细胞＋MMP-7＋京尼平组（D组，脱细胞处理后用MMP-7和京尼平处理）和脱细胞＋MMP-7＋康皑平＋真窄冷冻干燥组（E组，除行D组处理外，加真空冷冻了：燥处理），A，组6块，其余每组均为12块、，乃取6块卡相同面积人ADM没为人ADM组（A，组，不行处理），即细胞相容性实验中的对照组、采用HE染色、扫描电镜检测A1、B～E组真皮支架中细胞数量及组织结构变化，采用免疫组织化学染色法俭测A1、B、C组样本中波形蛋白、层粘连蛋白（LN）、Ⅳ型胶原蛋白残留情况，采用细胞毒性试验榆测B～E组浸提液的细胞毒性。接种人Fb于B～E组和A，组真皮支架表面培养，观察培养3、7、14d时Fb生长情况，采用ELISA法检测培养3、7d上清液中IL-6、IL-8含量。对数据进行双因素办差分忻及LSD-t检验。结果A1组可见含毛囊的浅黄色颗粒状结构，B组标本毛囊内仍有少艟毛发、上皮根鞘、细胞核、细胞碎片样结构及波形蛋白、LN、1V型胶原蛋白残留，C～E组经MMP-7处理后上述物质被完个去除。大体观察显示，D、E组真皮支架韧性较B、C组增加。C～E组胶原纤维结构亢褴，排列与A．组卞日似儿胶原纤维之间间隙增大，其中C、D组间隙卡H近但均大于B组，E组最大．B～E组真皮支架浸捉液细胞毒性为0或1级。F11于A2和B～E组真皮支架上均能增殖，并且随培养时问延长逐渐由单层变成复层生长进而向标本内部生长。D、E组真皮表面细胞密度较高，A1、C组细胞则主要存在于组织内邦。培养7d，A1、B～E组培养上清液中IL-6含请分别为（132±14）、（104±9）、（122±14）、（120±12）、（128±17）pg／mL，IL-8含量分别为（135±18）、（102±17）、（127±18）、（134±23）、（141±24）pg／mL，分别较培养3d的（55±13）、（34±8）、（48±8）、（50±13）、（49±12）pg／mL和（93±19）、（63±11）、（82±15）、（82±16）、（89±16）pg／mL明显增高（，值分别为98．869、184．038、125．531、93．237、87．265和15．694、23．451、22．801、19．607、18．808，P值均小于0．05）；M-时相点A1、B～E纽纽间IL-6、IL-8含髓比较，差异均有统计学意义（F值分别为2．809、3．301±3．757、3．266，P值均小于0．05）；LSD-t检验结果表明，A1、C～E组组间IL-6、IL-8含避比较差异尤统计学意义（t值分别为0．058～1．905、0．034～1．295，P值均大于0．05），但均较B组高（t值分别为3．707～5．612、2．785～4．079，P值均小于0．05）。结论脱细胞联合MMP．7、京尼平及真宅冷冻干燥方法制备的低免疫原性猪真皮支架具有较好的细胞相容性，细胞长人组织内部较少可能与京尼平增加组织韧性有关。
Objective To observe the changes in the structure and cytocornpatibility of porcine a-cellular dermal matrix, which was prepared with dermal reticular layer, treated with matrix metalloproteinase 7 （MMP-7） , genipin, and vacuum freeze-drying. Methods Fifty-four pieces of porcine dermal reticular layer, prepared with lateral abdominal skin were obtained from healthy large Yorkshire pig with mechanical method under sanitary condition, each 10.0 mm ± 5.0 mm in size and 0.5-0.6 mm in thickness. They were divided into normal control group （ A1 , without treatment, n = 6） , decellularization group （ B, decellulized, n = 12）, decellularization ＋ MMP-7 group （C, treated with MMP-7 after decellularization, n = 12）, decel- lularization ＋ MMP-7 ＋ genipin group （ D, treated with MMP-7 and genipin after decellularization, n = 12） , and decellularization ＋ MMP-7 ＋ genipin ＋ vacuum freeze-drying group （ E, treated with MMP-7, genipin, and vacuum freeze-drying after decellularization, n = 12） according to the random number table. Mean- while, 6 pieces of human acellular dermal matrix, with the same size and thickness as listed above, were taken as control group （A2 , without treatment） in the cytocompatibility tests. HE staining and scanning e- lectron microscope were used to detect the cell number and the change in tissue structure in dermal scaffold in groups AI and B-E. Immunohistochemical staining was used to determine residual vimentin, laminin and collagen 1V in groups A1 , B, and C. Cytotoxicity tests were employed to test the cytotoxicity of the leaching solutions of groups B-E. Human fibroblasts were seeded on the surface of dermal scaffold in groups A2 and B-E. The proliferation of fibroblasts were determined on post culture day （PCD） 3, 7, and 14, and the con- tent of IL-6 and IL-8 in the supernatant were determined on PCD 3 and 7 with enzyme-linked immunosorbent assay. Data were processed with two-way analysis of variance and LSD- t test. Results Granular structure with hair follicle in pale yellow color was observed in group A1. Small amount of hair, epithelial root sheath, nuclei, cell debris-like structure, vimentin, laminin, and collagen IV were observed in group B but not in group C, D, or E, which had been treated with MMP-7. The toughness of dermal scaffold was stronger in groups D, E than in groups B and C as observed in gross condition observation. The collagen fibers of dermal scaffold in groups C-E maintained their structural integrity with similar arrange as that of group A1. The in- terspaces among collagen fibers in groups C-E were all increased, while those of groups C and D were similar but larger than that in group B ; the interspace in group E was the largest. Groups B-E scored level 0 or 1 in the cytotoxicity test. Fibroblasts could proliferate on the surface of dermal scaffold in groups A2 and B-E. Furthermore, with the extension of culture time, fibroblasts gradually became to be stratified to form muhiple layers, and they proliferated toward the dermis. High density of fibroblasts was observed on the surface in groups D and E and in the deep layer in groups A2 and C. On PCD7, the contents of IL-6 [（132＋14）, （104±9）, （122±14）, （120±12）, （128±17） pg/mLl and IL-8 [（135 ±18）, （102±17）, （127 ± 18） , （134 ± 23 ） , （141 ± 24） pg/mLl in the supernatant in groups A2 and B-E were significantly higher than those on PCD 3 [（55 ±13）, （34 ±8）, （48 ±8）, （50±13）, （49 ±12） pg/mL] and [（93 ±19）, （63 ±11）, （82±15）, （82± 16）, （89 ± 16） pg/mL], with Fvalues respectively 98. 869, 184. 038, 125. 531, 93. 237, 87. 265 and 15. 694, 23. 451, 22. 801, 19. 607, 18. 808, P values below 0.05. The differences among groups A2 and B-E in the levels of IL-6 and IL-8 at each time point were statistically sig- nificant （ with F values respectively 2. 809, 3. 301 and 3. 757, 3. 266, P values below 0.05 ）. The differ- ences among groups A2 , C, D, and E in amount of IL-6 and IL-8 at each time point were not statistically significant （ with t values respectively 0. 058-1. 905 and 0. 034-1. 295, P values above 0.05 ） , but they were all higher than those in group B （with t values respectively 3. 707-5. 612 and 2. 785-4. 079, P values below 0.05）. Conclusions The low immunogenic porcine dermal scaffold treated with MMP-7, genipin, and vacuum freeze-drying after deeellularization, has good cytocompatibility. The growth of only a few fibroblasts in the dermal scaffold may be correlated with genipin, which increases tissue toughness.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2012年第5期329-335,共7页
Chinese Journal of Burns
基金
山东省医药卫生科技发展计划（2011HZ008）
山东大学留学人员创业计划（20080405）
