Effects of rabbit adipose-derived mesenchymal stem cells on the healing of skin deep partial-thickness scald wound of rabbit
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摘要: 目的 探讨创面局部注射兔脂肪源性间充质干细胞(ADSC)对兔皮肤深Ⅱ度烫伤创面愈合的影响。 方法 取1只新西兰大白兔脂肪组织,分离ADSC,并进行传代培养,采用第3代细胞进行以下实验。将24只兔按照随机数字表法分为ADSC组12只和对照组12只,在兔背部近臀部两侧各造成1个直径约5 cm深Ⅱ度烫伤创面。于伤后第2天起ADSC组兔创面皮下注射2 mL经EdU标记的ADSC悬液(浓度为5×105个/mL),对照组兔创面同前注射2 mL无血清DMEM培养液,至创面愈合。每日观察2组兔创面愈合情况,记录其愈合时间。伤后第7、14、21、28天,2组各取3只兔,测量创面面积并计算愈合率;切取创面愈合组织,HE染色观察组织形态,Masson染色观察胶原纤维表达。倒置荧光显微镜下观察EdU标记的ADSC在伤后第28天创面愈合组织中的分布,ELISA法检测伤后第7、14、21天创面愈合组织中血管内皮生长因子(VEGF)及EGF表达。对数据行析因设计方差分析、配对样本
t 检验。 结果 (1)ADSC组兔创面愈合时间为伤后(19.5±1.1)d,明显短于对照组的(23.3±1.5)d,t =4.50,P <0.05。伤后第7天,2组兔创面均干燥,无明显渗出物,创周红肿逐渐消退;ADSC组兔创面愈合率为(15.1±2.4)%,与对照组的(13.7±3.1)%相近(t =1.20,P >0.05)。伤后第14天,ADSC组兔创面干燥、结痂明显,创面愈合率为(73.1±5.7)%;对照组兔创面有少量分泌物、少量结痂,创面愈合率为(52.9±5.1)%,明显低于ADSC组(t =8.06,P <0.01)。伤后第21天,ADSC组兔创面基本愈合,创面愈合率为(95.6±3.0)%;对照组兔仍有少量创面存在,创面愈合率为(78.6±3.7)%,明显低于ADSC组(t =9.73,P <0.01)。伤后第28天,2组兔创面均完全愈合,愈合率100%,ADSC组兔创面愈合皮肤组织质地及微血管反应优于对照组。(2)伤后第7天,2组兔创面愈合组织Fb增多,均有少量血管、胶原纤维增生,无明显差别。伤后第14天,ADSC组兔创面愈合组织Fb数量多于对照组,胶原纤维排列密集整齐;对照组兔创面愈合组织胶原纤维排列稀疏不规则。伤后第21天,2组兔创面愈合组织表皮层均有分化,ADSC组兔创面愈合组织胶原纤维仍较对照组密集。伤后第28天,ADSC组兔创面愈合组织新生表皮各个层次分化良好,优于对照组;2组兔创面愈合组织中均有大量粗大胶原纤维排列;ADSC组兔创面愈合组织中可见EdU标记的ADSC。(3)伤后第7天,ADSC组兔创面愈合组织VEGF及EGF表达与对照组相近(t 值分别为0.70和0.91,P 值均大于0.05)。伤后第14、21天,ADSC组兔创面愈合组织VEGF及EGF表达明显多于对照组(t 值为2.85~4.81,P 值均小于0.01)。 结论 ADSC移植对兔皮肤深Ⅱ度烫伤创面愈合有明显促进作用,能缩短创面愈合时间。-
关键词:
- 烧伤 /
- 伤口愈合 /
- 间质干细胞移植 /
- 脂肪源性间充质干细胞
Abstract: Objective To investigate the effects of local injection of rabbit adipose-derived mesenchymal stem cells (ADSCs) on the healing of skin deep partial-thickness scald wound of rabbit. Methods ADSCs were isolated from adipose tissue of one New Zealand rabbit and then sub-cultured. ADSCs of the third passage were used in the following experiments. Twenty-four rabbits were divided into ADSCs group (n =12) and control group (n =12) according to the random number table, and one deep partial-thickness scald wound with diameter of 5 cm on the two sides of the back near the buttocks was made. From post injury day (PID) 2, 2 mL suspension of EdU-labeled ADSCs with the number of 5×105 per mL was subcutaneously injected in wounds of rabbits in ADSCs group, while the rabbits in control group were given 2 mL serum-free DMEM until the wounds were healed. Wound healing processes of rabbits in two groups were observed every day, and the healing time was recorded. On PID 7, 14, 21, and 28, areas of wound of three rabbits in two groups were measured and the healing rates were calculated, respectively. The healed wound tissue was harvested to observe the morphology by HE staining, and the expression of collagen fiber was observed by Masson staining. The distribution of EdU-labeled ADSCs in healed wound tissue on PID 28 was observed by inverted fluorescence microscope. The expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) of healed wound tissue on PID 7, 14, and 21 were detected by enzyme-linked immunosorbent assay. Data were processed with analysis of variance of factorial design and paired samplest test. Results (1) The wound healing time of rabbits in ADSCs group was (19.5±1.1) d post injury, which was significantly shorter than that in control group [(23.3±1.5) d,t =4.50,P <0.05]. On PID 7, wounds of rabbits in two groups were dry with no obvious exudation, and redness and swelling around wounds disappeared gradually, the wound healing rate of rabbits in ADSCs group was (15.1±2.4)%, which was close to that in control group [(13.7±3.1)%,t =1.20,P >0.05]. On PID 14, wounds of rabbits in ADSCs group were dry and scabbed obviously, and the wound healing rate was (73.1±5.7)%, while wounds of rabbits in control group were little scabbed with little exudation, and the wound healing rate was significantly lower than that in ADSCs group [(52.9±5.1)%,t =8.06,P <0.01]. On PID 21, wounds of rabbits in ADSCs group were generally healed, and the wound healing rate was (95.6±3.0)%, while a few wounds still existed in rabbits of control group, and the wound healing rate was significantly lower than that in ADSCs group [(78.6±3.7)%,t =9.73,P <0.01]. On PID 28, wounds of rabbits in two groups were totally healed with the healing rate of 100%, and texture and microvascular responses of healed wound tissue in ADSCs group were better than those in control group. (2) On PID 7, fibroblasts in healed wound tissue of rabbits in two groups were all increased, and there were little vascular and collagen fiber proliferation with no obvious differences. On PID 14, the number of fibroblasts in healed wound tissue of rabbits in ADSCs group was more than that in control group, and the collagen fibers in healed wound tissue of rabbits in ADSCs group were arranged in dense and uniform, while those in control group were sparse and irregular. On PID 21, skin layers were differentiated in healed wound tissue of rabbits in two groups, and collagen fibers in healed wound tissue of rabbits in ADSCs group were still denser than that in control group. On PID 28, newborn skin was well differentiated in healed wound tissue of rabbits in ADSCs group, which was better than that in control group. There were a lot of thick collagen fibers in healed wound tissue of rabbits in two groups, and EdU-labeled ADSCs were involved in skin texture of rabbits in ADSCs group. (3) The expressions of VEGF and EGF in healed wound tissue of rabbits in two groups were similar on PID 7 (witht values respectively 0.70 and 0.91,P values above 0.05), which in ADSCs group were significantly higher than those in control group on PID 14 and 21 (witht values from 2.85 to 4.81,P values below 0.01). Conclusions The transplantation of ADSCs can promote the wound healing of skin deep partial-thickness scald wound of rabbit and shorten the wound healing time.
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