Objective: To investigate the effectiveness of human placental decidua basalis derived mesenchymal stem cells (PDB-MSCs) in repairing full-thickness skin defect of nude mice.
Methods: Human placenta samples were obtained from healthy donor mothers with written informed consent. PDB-MSCs were isolated through enzymic digestion and density gradient centrifugation; the 4th passage cells were identified by cellular morphology, cell adipogenic and osteogenic differentiation, and phenotype evaluation. Forty-two 4-5-week-old BALB/c female nude mice were randomly divided into experimental group (n=21) and control group (n=21). The 4th passage PDB-MSCs solution (200 μL, 5×106/mL) was injected into the mice of experimental group via caudal vein; the mice of control group were given equal volume of PBS. The full-thickness skin defect model of 1.5 cm×1.5 cm in size was made after 3 days. The wound healing was observed generally at 1, 2, 4, 7, 14, 18, 21, 25, and 30 days after operation, and the wound healing rate was calculated after wound decrustation. HE staining was used to observe the wound repair at 1, 7, 14, 21, and 31 days; immunofluorescent staining was used for cellular localization at 7, 14, and 31 days after operation.
Results: Cells isolated from human placenta were MSCs which had multipotential differentiation ability and expressed MSCs phenotype. Animals survived to the end of the experiment. The general observation showed that the experimental group had a faster skin repairing speed than the control group; the time for decrustation was 12-14 days in experimental group and was 14-17 days after operation in the control group. The wound healing rate of experimental group was significantly higher than that of control group at 14, 18, and 21 days (t=4.001, P=0.016; t=3.380, P=0.028; t=3.888, P=0.018), but no significance was found at 25 and 30 days (t=1.565, P=0.193; t=1.000, P=0.423). HE staining showed lower inflammatory reaction, and better regeneration of the whole skin and glands with time in the experimental group. The immunofluorescent staining was positive in skin defect area of experimental group at different time points which displayed that human PDB-MSCs existed.
Conclusions: Through enzymic digestion and density gradient centrifugation, PDB-MSCs can be obtained. Pre-stored PDB-MSCs can mobilize to the defect area and participate in repair of nude mice skin.
目的: 探讨人胎盘蜕膜基层来源MSCs（placental decidua basalis derived MSCs，PDB-MSCs）参与裸鼠全层皮肤缺损修复的效果。.
方法: 取自愿捐赠人胎盘组织，采用酶消化法结合密度梯度离心法分离培养PDBMSCs并传代，取第4代细胞进行实验。倒置相差显微镜观察细胞形态，行成骨与成脂诱导鉴定，检测细胞表面特异性抗原表达以鉴定细胞。取42只4～5周龄雌性BALB/c裸鼠，随机分为两组（n=21）；分别将200 μL浓度为5×106个/mL的第4代PDB-MSCs及等体积PBS经尾静脉注射入实验组及对照组裸鼠体内；注射后3 d实验动物于背部制备大小为1.5 cm×1.5 cm全层皮肤缺损模型。术后1、2、4、7、14、18、21、25、30 d大体观察创面愈合情况，待创面开始脱痂后测量创面愈合率。术后1、7、14、21、30 d取材行HE染色，观察创面修复情况；7、14、30 d实验组采用免疫荧光染色法行细胞定位分析。.
结果: 经鉴定胎盘蜕膜组织所分离的细胞为MSCs，具有多向分化能力和MSCs免疫组织表型。术后两组裸鼠存活至实验完成。大体观察示，实验组创面愈合较对照组快，其中实验组于术后12～14 d、对照组于14～17 d开始脱痂。实验组术后14、18、21 d创面愈合率均显著高于对照组（t=4.001，P=0.016；t=3.380，P=0.028；t=3.888，P=0.018）；25、30 d两组比较差异无统计学意义（t=1.565，P=0.193；t=1.000，P=0.423）。组织学观察示，与对照组比较，实验组皮肤创面组织炎性反应低，并且随时间延长皮肤各层组织及腺体等再生良好。免疫荧光染色示，术后各时间点实验组皮肤创面区域均可见人线粒体抗原染色阳性，提示存在人源细胞。.
结论: 通过酶消化法结合密度梯度离心法可稳定获得PDB-MSCs，预先注射入裸鼠体内后，在皮肤损伤条件下细胞可向皮肤损伤部位迁移，并促进创面修复。.
Keywords: Mesenchymal stem cells; Nude mice; Placenta; Repair and reconstruction; Skin defect.