基质金属蛋白酶对猪脱细胞真皮基质免疫原性的影响

Effect of matrix metalloproteinase-7 on immunogenicity of porcine acellular dermal matrix

目的探讨应用基质金属蛋白酶一7（MMP一7）降低“双断层”异种（猪）脱细胞真皮基质（Xeno—ADM）免疫原性的可行性及对真皮支架组织结构的影响。方法取健康白色家猪边腹部皮肤，机械法制备O．5～0．6i／lnl厚网状层即“双断层”真皮33块，随机分为正常对照组（A组）、脱细胞处理组（B组）和脱细胞＋MMP-7处理组（c组），每组11块。行HE染色，波形蛋白（Vimentin）、层黏连蛋白（LN）、IV型胶原蛋白（ColIV）免疫组化染色和扫描电镜检查，观察“双断层”真皮组织的细胞、细胞碎片、毛发残留情况，LN、Col1V脱除及半定量情况，和真皮胶原支架组织结构变化。结果HE染色、Vimentin免疫组化染色和扫描电镜观察显示，B组毛囊内仍残留毛发及含完整细胞的上皮根鞘，原微血管区域可见细胞核、细胞碎片样结构；C组毛囊内未见残留毛发及上皮根鞘，全部视野均未见完整细胞，偶见细胞核、细胞碎片样结构。LN和Col1V免疫组化染色观察及半定量分析显示，B组LN和Col1V含量明显少于A组（P〈0．01）但多于C组（P〈0．01），C组LN和ColIV基本被完全脱除。扫描电镜及HE染色观察显示，B、C组胶原纤维排列规则近似A组，B组胶原纤维束及胶原纤维之间间隙较A组增大而C组又较B组增大；胶原纤维及胶原原纤维结构完整，未见断裂、融合等破坏性改变。结论MMP一7处理“双断层”Xeno—ADM，能够有效降低其高免疫原性成分，同时保持真皮胶原支架的正常组织结构并相对扩大其三维网络空间，初步显示良好的研究与应用前景。

Objective To investigate the feasibility of matrix metalloproteinase-7 （ MMP-7 ） reducing immunogenicity in xenogenic （porcine） acellular dermal matrix （Xeno-ADM）, which contains only the dermal reticular layer, and its effect on dermal scaffold structures. Methods 33 pieces of porcine dermal reticular layer, 0.5-0.6 mm in thickness, obtained by using a mechanical method, were randomly divided into group A（ normal controls）, group B （decellularized by Dispase 1I and TritonX-100 ） and group C（ treated with MMP-7 after decelluladzation） （ n = 11 in each group）. The samples were observed by HE staining,immunohistochemical staining（ vimentin, laminin and collagen 1V ） and a scan- ning electron microscope （SEM）. Quantities of cells, cell debris and hair, semi-quantities of laminin and collagen IV, along with changes of the dermal scaffold structure were evaluated. Results HE staining, vimentin immunohistochemical staining and SEM revealed that some hair and epithelial root sheath with intact cells were observed in hair follicles ingroup B but not in group C. Some fragments which seemed to be cell nuclei or cell debris were observed in previous areas of blood vessels in group B, but they obviously decreased in group C. Semi-quantitative analysis by immunohistochemical staining showed that contents of laminin and collagen IV in group B were significantly lower than in group A （ P 〈 0.01 ）, while they were obviously higher than in group C（ P 〈 0.01 ）, in which they were almost cleared off. SEM and HE staining also showed that there was no obvious difference in the array of collagen fibers among the three groups. The interspace of collagenous fiber bundles or coUagenous fibers in group B was larger than that in group A, while it was smaller than that in group C. In addition, no obvious fracture or degradation was observed in groups B and C. Conclusions After treatment with MMP-7, high immunogenic components of Xeno-ADMs produced by the porcine dermal reticular layer are significantly cleared away. Also, the dermal scaffold structure is kept, along with relatively larger interspace of collagenous fiber bundles or collagenous fibers. Therefore, MMP-7 preliminarily shows good prospects in the preparation of lower immunogenic Xeno-ADMs.