碱性成纤维细胞生长因子复合骨髓间充质干细胞促进大鼠β射线皮肤损伤创面愈合作用的实验研究

发布时间：2018-12-20 12:35

【摘要】：目的：探讨碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF)复合骨髓间充质干细胞(bone mesenchymal stem cells，BMSCs)对β射线皮肤损伤创面愈合的影响及机制的研究。方法： 1．选用清洁级6-8周龄雄性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1×10~（6）/ml)细胞悬液。 2.选用清洁级3月龄雌性SD大鼠60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×30mm，建立急性深Ⅱ度β射线皮肤损伤动物模型。随机分为3组：bFGF+BMSCs复合治疗组(A组)、BMSCs治疗组（B组）、生理盐水对照组（C组）。A组，n=20，创面出现后将BMSCs细胞悬液(浓度为1×10~（6）/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bFGF。B组，n=20，创面出现后仅给予注射BMSCs细胞悬液，，方法同A组。C组，n=20，创面出现后给予注射生理盐水。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bFGF、VEGF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，6-8d后细胞生长进入指数生长期。传代细胞生长至第3代，用DiI标记BMSCs，在荧光显微镜下可观察到呈红色荧光的细胞。将细胞注入大鼠创面，24小时后，荧光显微镜下仍能观察创面组织切片中被DiI标记的BMSCs。 2.创面观察：大鼠照射后2周开始脱毛，3周局部皮肤出现红肿、水疱，4周出现创面，且逐渐增大，5周创面不再增大。①创面愈合时间：A组：30.40±1.52天，B组：38.81±2.80天，C组：45.62±3.95天。A治疗组创面愈合时间明显较其余两组快（P0.05），B治疗组较C组创面愈合时间快（P0.05）。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两组，B组多于C组。③免疫组化显示：治疗后第1、3、5周bFGF、VEGF阳性细胞光密度值A组明显高于B、C两组（P0.05），B组高于C组（P0.05）。结论： 1.应用直线加速器建立β射线皮肤损伤创面动物模型方法简便、剂量准确、可靠。 2. BMSCs体外培养生长稳定，传代后仍保持未分化状态。DiI标记BMSCs稳定、可靠，可以做BMSCs示踪研究。 3. bFGF复合BMSCs能促进β射线皮肤损伤创面的愈合，明显缩短创面愈合的时间。 4、bFGF复合BMSCs促进β射线皮肤损伤创面的愈合,其机制是：bFGF通过促进BMSCs向血管内皮细胞、表皮细胞、成纤维细胞等分化，增加创面局部组织中修复细胞数量, BMSCs又能促进VEGF、bFGF等生长因子的分泌，两者具有协同作用，加速β射线皮肤损伤创面的愈合。
[Abstract]:Aim: to investigate the effect and mechanism of basic fibroblast growth factor (basic fibroblast growth factor,bFGF) combined with bone marrow mesenchymal stem cell (bone mesenchymal stem cells,BMSCs) on the wound healing of 尾 -ray skin injury. Methods: 1. Three clean grade 6-8 week old male SD rats were killed by cervical dislocation. Bone marrow was collected from femur and tibia in aseptic condition. Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation and adherent culture. BMSCs (1 脳 10 ~ (6) / ml) cell suspension was prepared. 2. Sixty 3-month-old female SD rats of clean grade were used to establish the animal model of acute deep second-degree 尾 -ray skin injury by single irradiation of 尾 -ray (45Gy) produced by linear accelerator on the buttocks skin of rats with 40mm 脳 30mm. They were randomly divided into three groups: group A,), BMSCs (group B), group C (). A, n = 20), and control group (group C, n = 20). BMSCs cell suspensions (1 脳 10 ~ (6) / ml) 1ml were injected into the subcutaneous and dermis of rat wound after the wound appeared, and the bFGF.B group was sprayed regularly on the surface of the wound. After the wound appeared, only the BMSCs cell suspension was injected into the wound. Methods same as group A, group C, nonglutein 20, after the wound appeared, saline was injected into the wound. The wound healing was observed weekly, and the wound tissue 5mm 脳 5mm was removed under aseptic condition. The histopathological changes and bFGF, were observed by light microscopy and immunohistochemistry. The dynamic changes of VEGF expression. Results: 1. Culture and observation of Bone Marrow Mesenchymal Stem cells: after 24 hours of culture, a large number of cells were observed to grow on the wall, and after 6-8 days, the growth of the cells entered the exponential growth stage. After the passage of cells grew to the third generation, red fluorescent cells were observed under fluorescence microscope with DiI labeled BMSCs,. The cells were injected into the wound surface of rats. After 24 hours, the BMSCs. labeled by DiI could still be observed under the fluorescence microscope. 2. Wound observation: rats began to depilate 2 weeks after irradiation, local skin appeared redness and swelling at 3 weeks, blister appeared at 4 weeks, and gradually increased. 1 wound healing time at 5 weeks: group A: 30.40 卤1.52 days; Group B: 38.81 卤2.80 days, group C: 45.62 卤3.95 days. The healing time of wounds in group B was faster than that in group C (P0.05). 2 the number of epidermal cells, vascular endothelial cells and fibroblasts in wound tissues in group A was significantly higher than that in group B and C (P0.05). The light density of bFGF,VEGF positive cells in group A was significantly higher than that in group B and C at 5 weeks after treatment (P0.05 in), B group was higher than that in C group (P0.05). Conclusion: 1. The animal model of 尾-ray skin injury was established by linear accelerator. 2. BMSCs grew steadily in vitro and remained undifferentiated after passage. DiI labeled BMSCs was stable and reliable and could be used for BMSCs tracer study. 3. BFGF combined with BMSCs can promote the healing of 尾-ray skin injury wound and shorten the healing time. 4bFGF combined with BMSCs can promote the healing of 尾 -ray skin injury wound. The mechanism is that bFGF can promote the differentiation of BMSCs into vascular endothelial cells, epidermal cells, fibroblasts, and increase the number of repair cells in local tissue of wound, and BMSCs can promote VEGF,. The secretion of bFGF and other growth factors had synergistic effect on accelerating the wound healing of 尾-ray skin injury.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R818

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