脂肪干细胞联合几丁糖对兔扩张皮肤组织即时回缩率的影响

EFFECT OF ADIPOSE-DERIVED STEM CELLS COMBINED WITH CHITOSAN ON IMMEDIATE RETRACTION RATE OF EXPANDED SKIN IN RABBIT

目的 探讨脂肪干细胞（adipose-derived stem cells,ADSCs）联合几丁糖对兔扩张皮肤组织即时回缩率的影响。方法 取新西兰大白兔腹股沟脂肪,利用酶消化法体外分离培养ADSCs并传代。取第3代细胞鉴别其表面特异性标记物;行成软骨及成表皮诱导,鉴定其多向分化能力。取40只2~3月龄新西兰大白兔,随机分为4组（n=10）,分别为空白对照组（A组）、ADSCs组（B组）、几丁糖组（C组）、ADSCs＋几丁糖组（D组）。各组均建立背部皮肤扩张模型;扩张器埋置前,C、D组于扩张器表面涂抹5 m L 2%医用几丁糖,B、D组在埋置部位皮下多点注射1 m L浓度为5×10~6个/m L的第3代ADSCs细胞悬液。埋置后常规注射扩张,4周后达预期容量。原位维持1周后,取材测量扩张皮肤组织即时回缩率,HE染色测量皮肤全层和表皮以及纤维包膜厚度,Masson染色观察纤维包膜中胶原纤维情况,CD31免疫组织化学染色检测皮肤组织中微血管形成情况,并计算微血管密度（microvessel density,MVD）。结果 细胞鉴定示分离培养细胞为ADSCs,且具有多向分化能力。各组动物均存活至实验完成。D组扩张皮肤组织即时回缩率显著小于其他组,B、C组小于A组,B组小于C组,比较差异均有统计学意义（P〈0.05）。组织学染色示A、B组成熟期成纤维细胞较多,胶原纤维粗大且排列规则;而C、D组幼稚期成纤维细胞较多,胶原纤维纤细且稀疏。B、D组皮肤全层及表皮厚度、MVD均显著高于A、C组,C、D组纤维包膜厚度显著低于A、B组,比较差异均有统计学意义（P〈0.05）;其余组间以上指标比较,差异均无统计学意义（P〉0.05）。结论 ADSCs移植可促进兔扩张皮肤组织再生以及血管新生,但对于纤维包膜无显著影响,而几丁糖可抑制纤维包膜的生成,两者联合应用对于抑制扩张皮肤组织的即时回缩具有协同作用。

Objective To investigate the effect of adipose-derived stem cells （ADSCs） combined with chitosan on the immediate retraction rate of rabbit expanded skin. Methods ADSCs were isolated from rabbit fresh fat under sterile conditions and cultured to the 3rd generation by methods of enzymatic digestion; the specific surface markers and the differentiation into epidermal cells and cartilage cells were identified. Forty New Zealand white rabbits （aged, 2-3 months） were randomly divided into 4 groups （n=10）： control group （group A）, ADSCs group （group B）, chitosan group （group C）, and ADSCs＋chitosan group （group D）. ADSCs cell suspension with the concentration of 5× 10^6 cells/mL was prepared. The skin expansion model was made by embedding 30 mL dilator into the back of rabbit. Chitosan （2%, 5 mL） was coated on the surface of the dilator in groups C and D, and ADSCs cell suspension （1 mL） was injected into the skin in groups B and D. Conventional tissue expansion was performed to expected capacity at 4 weeks, and maintained for 1 week. The expanded skin was harvested to measure the immediate retraction rate, and the thickness of skin, epidermis, and fibrous capsule with HE staining. Masson staining was used to observe the characteristics of collagen in the fibrous capsule, and immunohistochemical staining for CD31 to determine the microvessel density （MVD）. Results ADSCs were successfully isolated, and had multiple differentiation ability. All the animals survived to the end of the experiment. The immediate retraction rate of group D was significantly lower than that of the other groups （P〈0.05）, groups B and C were significantly lower than group A （P〈0.05）, and group B was significantly lower than group C （P〈0.05）. The histological staining revealed that there were more mature fibroblasts and coarse collagen fibers with regular arrangement in groups A and B; there were more naive fibroblasts and tiny and sparse collagen fibers in groups C and D. The thickness of skin and epidermis, and MVD of groups B and D were significantly larger than those of groups A and C （P〈0.05）; the thickness of fibrous capsule of groups C and D was significantly less than that of groups A and B （P〈0.05）; but no significant difference was found in the above indexes between other groups （P〉0.05）. Conclusion ADSCs can promote angiogenesis and regeneration of the expanded skin, have no effect on the fibrous capsule. Chitosan can inhibit the proliferation of fibrous capsule, so a combination of ADSCs and chitosan can inhibit the immediate retraction of the expanded skin.