【作者】 张毓姣；

【导师】 沈国良；

【作者基本信息】 苏州大学 ， 烧伤外科学， 2013， 硕士

【摘要】 目的：应用表皮细胞生长因子（epidermal growth factor，EGF）复合骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）异体移植治疗β射线皮肤损伤，从病理学、生物化学和分子生物学等方面多角度探讨表皮细胞生长因子复合骨髓间充质干细胞异体移植对β射线皮肤损伤创面促愈作用的机制，为β射线皮肤损伤的治疗提供理论依据及新思路。方法：1.选用清洁级6-8周龄SD大鼠3只，颈椎脱臼法处死。在无菌条件下获取股骨、胫骨。并收集骨髓，采用密度梯度离心法和贴壁培养法分离培养骨髓间充质干细胞，传代细胞生长至第3代，用DiI标记细胞，制备BMSCs（浓度为1×106/ml）细胞悬液。2.选用清洁级3月龄雌性SD大鼠80只，应用直线加速器产生的β射线（45Gy）单次照射大鼠臀部皮肤40mm×40mm，建立急性深Ⅱ度β射线皮肤损伤动物模型。随机分为4组：EGF＋BMSCs复合治疗组（A组）、EGF治疗组（B组）、BMSCs异体移植治疗组（C组）、生理盐水对照组（D组）。A组：n=20，创面出现后将BMSCs细胞悬液（浓度为1×106/ml）1ml注入大鼠创面皮下及真皮层，单次注射，并定期向创面喷洒EGF。B组：n=20，创面出现后仅定期向创面喷洒EGF，方法同A组。C组：n=20，创面出现后仅给予注射BMSCs细胞悬液，方法同A组。D组：n=20，创面出现后仅给予注射生理盐水。每周观察创面愈合情况，且在无菌条件下切取创面组织5mm×5mm，采用光镜、免疫组化等方法检测，分别于治疗后第1、3、5周观察各组大鼠创面组织病理学变化和EGF、转化生长因子β（transforming growth factor beta,TGF-β）、血小板衍生生长因子（platelet derived growth factor，PDGF）、神经生长因子（nerve growth factor，NGF）表达的动态变化。结果：1.骨髓间充质干细胞的培养与观察：原代细胞经过24h培养后可见大量细胞贴壁生长，6-8后细胞生长进入对数生长期。传代细胞生长至第3代，用DiI标记BMSCs，在荧光显微镜下观察到呈红色荧光的细胞。将细胞注入大鼠创面，24h后，荧光显微镜下仍能观察到创面组织切片中被DiI标记的BMSCs。2.创面观察：大鼠照射2w后开始脱毛，3w局部皮肤出现红肿、水疱，4w出现创面，且逐渐增大，5w创面不再增大。①创面愈合时间：A组：30.41±1.49，B组：38.79±2.91，C组：38.83±2.95，D组：45.81±3.89。A治疗组创面愈合时间明显较其余组快（P＜0.05），B、C治疗组较D组创面愈合时间快（P＜0.05）；②光镜观察：观察各组创面组织中的表皮细胞、血管内皮细胞、成纤维细胞数量，A组明显多于B、C、D组，B、C组多于D组。③免疫组化显示：治疗后第3、5周EGF、TGF-β、PDGF、NGF阳性细胞光密度值A组明显高于B、C、D组（P＜0.05），B、C组高于D组（P＜0.05）。结论：1.应用直线加速器产生的β射线，剂量为45Gy可建立深II度β射线皮肤损伤创面动物模型，方法简便、剂量准确、可靠。2. EGF复合BMSCs能有效促进β射线皮肤损伤创面的愈合，明显缩短创面愈合的时间。3.应用表皮细胞生长因子(EGF)复合骨髓间充质干细胞(BMSCs)促进β射线皮肤损伤创面的愈合，其机制是：EGF可促进BMSCs向上皮细胞、成纤维细胞、血管内皮细胞的分化及增殖，促进伤口再上皮化，增加基质形成和结缔组织收缩的作用，BMSCs又可分泌大量的EGF、TGF-β、PDGF、NGF等生长因子，两者具有协同作用，可加速β射线皮肤损伤创面的愈合。更多还原

【Abstract】 Objectives: To investigate the effect and mechanism of the role of epidermal growthfactor combined with bone marrow mesenchymal stem cells in promoting healing ofβ-ray irradiated skin inyury.Methods:1.3SD rats(6-8weeks old),clean grade, were killed using cervical dislocation method.We obtained the femur and tibia bone marrow under sterile conditions, isolated andcultured bone mesenchemal stem cells(BMSCs) by DiI when the passage grow intopassage3,and then we prepared the BMSCs suspension(1×106/ml in concentration).2.60female SD rats (age:3-months old),clean grade. We applied single dosage(45Gy)of β-ray irradiation by linear accelerator on buttock skin (40mm×40mm) in rats, andestablished acute deep II β-irradiated skin injury model of rats. Those60SD female ratswere randomly divided into4groups: EGF+BMSCs (Group A), EGF((Group B),BMSCs((Group C),normal saline control(Group D).In Group A,N=20,1ml BMSCssuspension(1×106/ml in concentration) were injected subcutaneously after the woundappeared, and EGF were sprayed to the wound regularly; In Group B, n=20, EGF wasmerely sprayed to the wound regularly, with the same method as the Group A; In GroupC,n=20, BMSCs was merely injected when the wound appeared, with the same methodas the Group A; In Group D, n=20,the control rats,normal saline was injected when thewound appeared with the same method as the Group A. We observed the wound healingevery week, and excised the wound tissue by5mm×5mm in size under sterile conditions inthe1st,3,and5th week after the treatment. The samples were examined with lightmicroscopy, and immunohistochemistry stained with EGF,TGF,PDGF,NGF antibodies. Results:1.Growth and observation of BMSCs: After24hours of primary cultures, there weremany cells adhering in the wall of the culture dishes, and the culture entered into theexponential growth phase6-8days later. After3times of subculture, we labeled the BMSCscells with DiI and checked them with fluorescence microscope, red fluorescence was found.After injecting these cells into the wound, red fluorescence still could be detected withfluorescence microscope.2. Wound observation: hair began to fall in2weeks after irradiation, redness appearedlocally on the skin, and blisters were formed in3weeks after irradiation, the woundappeared and then gradually increased in4weeks after irradiation, and the wound becamestable5weeks after irradiation.①Wound healing time: in group A. in group B. in group C.in group D.②Light microscope: the epidermal cells, vascular endothelial cells, andfibroblasts in group A were significantly more than the cells in group B、C and D. Thesecells in group B、C were significantly morethan in group D.③Immunohistochemistryshowed that: EGF,TGF,PDGF,NGF-optical density value of the group A in the1st,3rd,and5th week after BMSCs suspension injection combined with EGF was significantly morethan that of group B、C and D(P＜0,05)，these factors of group B、C were significantlymore than that of group D(P＜0,05).Conclusions:1. Application of β-ray linear accelerator in establishing animal model of skin damageis simple. The dose is accurate and reliable.2.EGF combined with BMSCs can promote the healing of β-ray skin injury irradiated,and reduce wound healing time.3.EGF combined with BMSCs can promote the healing of β-ray irradiated skininjury. The mechanism is that EGF can induce the differentiation of BMSCs into theepithelial cells, fibroblasts, vascular endothelial cells, and can enhance the viability ofepithelial cells and other tissue repair cells, increase the number of repair cells in localwound，regulate collagen metabolism and related enzyme，regulate the informationpathways of cell cycles and apoptosis, synergistic effect between EGF and BMSCs, andcan enhance the healing of β-ray irradiated skin injury.更多还原