Objective: To investigate the effects of human adipose-derived mesenchymal stem cells (ADSCs) and platelet-rich plasma (PRP) on healing of wounds with full-thickness skin defects in mice. Methods: ADSCs were isolated from the lumbar and abdominal fat donated voluntarily by a healthy woman undergoing liposuction in the Department of Plastic Surgery of Guangzhou General Hospital of Guangzhou Military Area Command, and the cells were cultured and identified. ADSCs of the second passage were used in the following experiments. The venous blood of the volunteer was taken, and PRP was obtained by secondary centrifugation. Thirty-six C57BL/6 mice were divided into simple injury group (n=12), simple ADSCs treatment group (n=12), and ADSCs+ PRP treatment group (n=12) according to the random number table. Each mouse was inflicted with a 1 cm×1 cm wound with full-thickness skin defect on the back. Immediately after injury, the wounds of mice in simple injury group were subcutaneously injected with 1 mL normal saline, the wounds of mice in simple ADSCs treatment group were subcutaneously injected with 1 mL phosphate buffer solution-blended ADSCs suspension (with concentration of 5×10(5) /mL, the same below), and the wounds of mice in ADSCs+ PRP treatment group were subcutaneously injected with 1 mL mixture of PRP and ADSCs (1∶2 volume ratio). Three mice in each group were taken on post injury day (PID) 3, 5, 7, and 14 to observe the gross condition of wound, and the wound healing rate was calculated. On PID 3, 5, and 7, the non-healing wound tissue and 0.5 cm normal skin tissue around the wound margin were taken after gross observation. The inflammation, re-epithelialization, and angiogenesis of tissue were observed by hematoxylin and eosin staining, and the re-epithelialization rate was calculated. The collagen synthesis of tissue was observed by masson staining. Immunohistochemistry was used to observe the expression of macrophages of tissue samples collected on PID 3 and 5. Data were processed with analysis of variance of factorial design and Least-Significant Difference test. Results: (1) On PID 3, the wounds of mice in ADSCs+ PRP treatment group were with granulation tissue regeneration, redness, and swelling, and the wounds of mice in the other two groups were ruddy and with effusion. On PID 5, the wounds of mice in ADSCs+ PRP treatment group had less redness and swelling, which were dry with obvious scab, and wounds of mice in the other two groups were obviously red and swollen. On PID 7, scab formed basically on wounds of mice in the three groups. On PID 14, the wounds of mice in the three groups basically healed, and their crusts were off. On PID 3, 5, 7, and 14, the wound healing rates of mice in ADSCs+ PRP treatment group were obviously higher than those of the other two groups (P<0.05 or P<0.01). On PID 5 and 7, the wound healing rates of mice in simple ADSCs treatment group were obviously higher than those of simple injury group (P<0.01). (2) On PID 3, granulation tissue regeneration of wounds in ADSCs+ PRP treatment group was more than that in the other two groups. On PID 5, inflammatory reaction of wounds of mice was mild in ADSCs+ PRP treatment group, which was severe in the other two groups. On PID 7, the re-epithelialization process of wounds of mice was almost completed in ADSCs+ PRP treatment group, and the number of new vessels was more in ADSCs+ PRP treatment group than in the other two groups. The migration distance of regenerated epithelia around the wound edge in simple injury group and simple ADSCs treatment group was short. On PID 3, 5, and 7, the re-epithelialization rates of wounds of mice in ADSCs+ PRP treatment group were (37.6±4.5)%, (59.1±1.3)%, and (89.2±4.3)%, respectively, significantly higher than (25.7±1.5)%, (34.5±4.4)%, and (50.8±2.7)% in simple injury group and (29.1±0.8)%, (42.6±2.9)%, and (72.9±3.0)% in simple ADSCs treatment group (P<0.01). On PID 5 and 7, the re-epithelialization rates of wounds of mice in simple ADSCs treatment group were significantly higher than those in simple injury group (P<0.05 or P<0.01). (3) On PID 3 and 5, a quite large number of new collagen fibers appeared in granulation tissue of wounds of ADSCs+ PRP treatment group, while the collagen fibers in the other two groups were less. On PID 7, the granulation tissue of mice in ADSCs+ PRP treatment group decreased, and a large number of new collagen fibers appeared. The collagen fibers in wounds tissue of mice in simple ADSCs treatment group increased, while the collagen fibers deposited in wounds tissue of mice in simple injury group was still less. (4) On PID 3 and 5, the numbers of macrophages in wounds tissue of mice in simple ADSCs treatment group were 4.7±0.6 and 5.3±0.6 respectively, obviously lower than 6.3±0.6 and 7.7±0.6 in injury group (P<0.05 or P<0.01); the numbers of macrophages in wounds tissue of mice in ADSCs+ PRP treatment group were 3.0±1.1 and 2.7±0.5, significantly lower than those in the other two groups (P<0.05 or P<0.01). Conclusions: Human PRP and ADSCs are involved in the early inflammation, metaphase of tissue proliferation, and re-epithelialization and shaping process of late stage of wounds with full-thickness skin defects in mice. The combination of ADSCs and PRP may be a comparatively good combination to improve the speed and quality of wound healing.
目的： 探讨人脂肪源性间充质干细胞(ADSC)和富血小板血浆(PRP)对小鼠全层皮肤缺损创面愈合的影响。 方法： 取1名2018年2月在广州军区广州总医院整形外科接受吸脂手术的健康女性自愿捐赠的腰腹部脂肪，分离培养ADSC并进行鉴定，采用第2代细胞进行以下实验。抽取该志愿者静脉血，通过二次离心法得到PRP。将36只C57BL/6小鼠按随机数字表法分为单纯损伤组12只、单纯ADSC治疗组12只、ADSC联合PRP治疗组12只，在每只小鼠背部制备1个1 cm×1 cm全层皮肤缺损创面。伤后即刻，单纯损伤组小鼠创面皮下注射1 mL生理盐水，单纯ADSC治疗组小鼠创面皮下注射PBS混匀的ADSC悬液(浓度为5×10(5)个/mL，下同)1 mL，ADSC联合PRP治疗组小鼠创面皮下注射PRP与ADSC按1∶2体积比混合的液体1 mL。伤后3、5、7、14 d，每组各取3只小鼠，观察创面大体情况并计算创面愈合率；伤后3、5、7 d，大体观察后切取未愈合创面组织及创缘周围0.5 cm正常皮肤组织，行苏木素－伊红染色观察组织炎症反应、再上皮化、血管新生情况并计算创面再上皮化率，行Masson染色观察组织胶原新生情况，另对伤后3、5 d标本采用免疫组织化学法观测巨噬细胞表达。对数据行析因设计方差分析、LSD检验。 结果： (1)伤后3 d，ADSC联合PRP治疗组小鼠创面可见肉芽组织新生、红肿，其余2组小鼠创面红润、有渗液。伤后5 d，ADSC联合PRP治疗组小鼠创面红肿减轻、干燥、结痂明显，其余2组小鼠创面红肿明显。伤后7 d，3组小鼠创面基本结痂。伤后14 d，3组小鼠创面痂皮脱落、基本愈合。伤后3、5、7、14 d，ADSC联合PRP治疗组小鼠创面愈合率明显高于其余2组(P<0.05或P<0.01)；伤后5、7 d，单纯ADSC治疗组小鼠创面愈合率明显高于单纯损伤组(P<0.01)。(2)伤后3 d，ADSC联合PRP治疗组小鼠创面肉芽组织新生较其余2组多。伤后5 d，ADSC联合PRP治疗组小鼠创面炎症反应较轻，其余2组小鼠创面炎症反应重。伤后7 d，ADSC联合PRP治疗组小鼠创面几乎完成再上皮化过程，新生血管数多于其余2组；其余2组小鼠创缘再生上皮迁移距离较近。伤后3、5、7 d，ADSC联合PRP治疗组小鼠创面再上皮化率分别为(37.6±4.5)%、(59.1±1.3)%、(89.2±4.3)%，显著高于单纯损伤组的(25.7±1.5)%、(34.5±4.4)%、(50.8±2.7)%和单纯ADSC治疗组的(29.1±0.8)%、(42.6±2.9)%、(72.9±3.0)%(P<0.01)；伤后5、7 d，单纯ADSC治疗组小鼠创面再上皮化率显著高于单纯损伤组(P<0.05或P<0.01)。(3)伤后3、5 d，ADSC联合PRP治疗组小鼠创面肉芽组织中有较多新生胶原纤维，其余2组小鼠创面组织中胶原纤维较少。伤后7 d，ADSC联合PRP治疗组小鼠创面肉芽组织减少，出现大量新生胶原纤维；单纯ADSC治疗组小鼠创面组织中胶原纤维增多；单纯损伤组创面组织中胶原纤维沉积仍较少。(4)伤后3、5 d，单纯ADSC治疗组小鼠创面组织中巨噬细胞数分别为(4.7±0.6)、(5.3±0.6)个，明显少于单纯损伤组的(6.3±0.6)、(7.7±0.6)个(P<0.05或P<0.01)；ADSC联合PRP治疗组小鼠创面组织中巨噬细胞数分别为(3.0±1.1)、(2.7±0.5)个，明显少于其余2组(P<0.05或P<0.01)。 结论： 人PRP和ADSC参与小鼠全层皮肤缺损创面早期炎症和中期组织增殖以及后期再上皮化与塑形过程，ADSC和PRP联合治疗可能是提高创面愈合速度与质量的较佳组合方案。.
Keywords: Adipose-derived mesenchymal stem cell; Mesenchymal stem cell transplantation; Platelet-rich plasma; Wounds and injuries.