人胎盘蜕膜基层来源MSCs参与裸鼠皮肤创面修复的研究

发布时间：2018-04-05 11:34

本文选题：MSCs　切入点：胎盘　出处：《中国修复重建外科杂志》2016年05期

【摘要】：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200μL浓度为5×106个/m L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。
[Abstract]:Objective to investigate the placenta derived MSCs (placental decidua basalis base derived MSCs, PDB-MSCs) in the effect of full-thickness skin defects repair. Methods from voluntary donors of placental tissue by enzyme digestion combined with density gradient centrifugation method and PDBMSCs passage, take the fourth generation cells were used for experiments. Cell morphology was observed under inverted microscope. For osteogenic and adipogenic identification, detection of cell surface antigen expression to identify the cell. 42 4~5 week old female BALB/c nude mice were randomly divided into two groups (n=21); the 200 L concentration is 5 * 106 /m L fourth generation PDB-MSCs and volume PBS via intravenous injection in the experimental group and control group of nude mice; 3 d after injection of the experimental animal preparation on the back of a size of 1.5 cm * 1.5 cm full-thickness skin defect model. Postoperative 1,2,4,7,14,18,21,25,30 D gross observation of wound healing, the wound Start exuvial after measuring the rate of wound healing after surgery. 1,7,14,21,30 D were performed HE staining, observe the wound healing; 7,14,30 D experimental group by immunofluorescence staining. Results the cell positioning analysis decidua placenta identification of isolated cells was MSCs, multilineage differentiation ability and MSCs immunohistochemical phenotype. After two the mice survived to the end of the experiment. The general observation showed that the wound healing in experimental group compared with the control group, the experimental group after 12~14 D, the control group in 14~17 D began exuvial. Rates were significantly higher than those in control group 14,18,21 D wound healing after surgery in the experimental group (t=4.001, P=0.016; t=3.380, P=0.028; t=3.888, P=0.018 25,30 d); the two groups had no statistically significant difference (t=1.565, P=0.193; t=1.000, P=0.423). Histological observation showed that compared with the control group, the inflammatory reaction of skin wound tissue in experimental group is low, and the lapse of time and each layer of the skin tissue Gland regeneration. Immunofluorescence staining showed that, after each time point in the experimental group wound area can see mitochondrial antigen positive staining, suggesting the presence of human cells. Conclusion through enzyme digestion combined with density gradient centrifugation method can stably obtain PDB-MSCs, pre injected in nude mice, under the conditions of the skin injury to cells skin lesions and migration, promote healing of wound.

【作者单位】：四川大学华西医院生物治疗国家重点实验室·干细胞与组织工程研究室;四川大学华西医院公共技术实验中心;四川大学华西医院烧伤整形科;
【基金】：国家自然科学基金资助项目(31570970,31271049)~~
【分类号】：R641

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