含bFGF和维生素C的羊膜载体复合膜负载BMSCs修复兔创面的实验研究

AN EXPERIMENTAL STUDY OF RABBITS'WOUND REPAIR BY AMNIOTIC CARRIER COMPLEX MEMBRANE CONTAINING bFGF AND VITAMIN C AND LOADED WITH BMSCs

目的将含bFGF和维生素C(vitamin C,VitC)的羊膜载体复合膜负载BMSCs植入兔创面,探讨其对真皮新生和重建速度的影响以及促进表皮修复速度的最佳时期。方法取健康3月龄新西兰大耳白兔24只,体重1.0～1.5kg,雌雄不限,取骨髓5mL进行BMSCs体外分离、培养和传代,取第2代细胞调整至3.5×107个/mL备用。取新鲜牛羊膜制备大小为4.52cm2的羊膜载体复合膜。在每只兔背部脊柱两侧作3个创面,深度达深筋膜,面积约3.14cm2,随机分为A、B、C3组。A组创面植入含1mLBMSCs、10mLbFGF(0.2mg/L)和10mLVitC(0.02g/L)的羊膜载体复合膜,B组创面植入仅含有1mLBMSCs的羊膜载体复合膜,C组创面仅植入羊膜载体复合膜。术后行大体观察,术后7、14、21d取材行HE、Masson、Van Gieson、Ⅰ型胶原免疫组织化学染色观察,血管墨汁灌注法动态观察创面愈合情况。结果术后各组创面愈合良好。术后21d,A组创面与周边正常皮肤齐平;B组创面尚有凹陷;C组创面凹陷明显较B组大且深,周边组织分界明显较B组清楚。术后7d,各组创面无明显缩小。术后14d,各组创面逐渐缩小,形成新生表皮,A、B、C组创面平均愈合率分别为60%、41%和23%;术后21d,3组创面平均愈合率分别为99%、90%、81%;组间两两比较差异均有统计学意义(P<0.05)。术后7、14、21d,HE、Masson、Van Gieson染色示A组新生表皮层数及真皮成纤维细胞数、胶原纤维数量均多于B、C组,Ⅰ型胶原免疫组织化学染色示A组阳性细胞数多于B、C组,墨汁灌注法显示A组血管数量明显多于B、C组。结论添加BMSCs、bFGF和VitC的羊膜载体复合膜植入全层皮肤缺损后,能加速真皮的修复重建,并且在真皮修复全层皮肤过程中表皮有新生和重建的最佳时期。

Objective The amniotic carrier complex membrane, which contains bFGF and vitamin C （VitC） and is loaded with BMSCs, is planted into the deeply-partial wounds of rabbits. To explore its influence on the epidermis renascence and regenerating speed in the process of the dermis restore. Methods BMSCs were isolated from the marrows of 24 healthy 3-month-old New Zealand rabbits, male or female, weighing 1.0-1.5 kg. The BMSCs were cultured in vitro and purified, and then amniotic carrier complex membrane was prepared, whose size was 4.52 cm^2. Three deep-partial wounds, with the area of about 3.14 cm^2, were produced on the back of each rabbit. All the wounds were randomly divided into 3 groups： group A, group B and group C. Group A was the experimental group in which the amniotic carrier complex membrane was planted, including 1 ml BMSCs, 10 mL bFGF （0.2 mg/L） and 10 mL VitC （0.02 g/L）. In group B, the amniotic carrier complex membrane was planted, including only 1 mL BMSCs. In group C, the amniotic carrier complex membrane alone was planted. After the operation, general observation was conducted. At postoperative 7, 14 and 21 days, respectively, the observation by HE, Masson, Van Giesonr staining and immunohistochemical staining of collagen type I was performed. The ink perfusion method was performed to evaluate the velocity and the quality of the wound healing after the transplantation. Results All the wounds obtained good healing. At 14 days after the operation, the ratio of wound healing was 60%, 41% and 23% in groups A, B and C, respectively. At 21 days after the operation, the the ratio of wound healing was 99%, 90% and 81% in groups A, B and C, respectively. There were significant differences between any two groups （P 〈 0.05）. The depth of the newborn dermis, the number of the active collagen type I masculine cells and the number of the blood vessels in group A were better and more than in group B. And those in group B were better and more than in group C. At the exterior area of the newborn dermis, there was lots of regenerated epidermis from the peripheral normal skin, which in group A was better than in group B, and in group B was better than ingroup C. Conclusion The amniotic carrier complex membrane transplanted to deep-partial wounds, which is appended with BMSCs, b FGF and VitC, can accelerate repair and reconstruction of the dermis. There has an optimal time of the renascence and regeneration of the epidermis in the process of dermis repair.