【摘要】：目的：探討堿性成纖維細胞生長因子（basic fibroblast growth factor,bFGF)復合骨髓間充質(zhì)干細胞(bone mesenchymal stem cells，BMSCs)對β射線皮膚損傷創(chuàng)面愈合的影響及機制的研究。 方法： 1．選用清潔級6-8周齡雄性SD大鼠3只，頸椎脫臼法處死。在無菌條件下獲取股骨、脛骨，并收集骨髓，采用密度梯度離心法和貼壁培養(yǎng)法分離培養(yǎng)骨髓間充質(zhì)干細胞，傳代細胞生長至第3代，用DiI標記細胞，制備BMSCs(濃度為1×10~（6）/ml)細胞懸液。 2.選用清潔級3月齡雌性SD大鼠60只，應用直線加速器產(chǎn)生的β射線（45Gy）單次照射大鼠臀部皮膚40mm×30mm，建立急性深Ⅱ度β射線皮膚損傷動物模型。隨機分為3組：bFGF+BMSCs復合治療組(A組)、BMSCs治療組（B組）、生理鹽水對照組（C組）。A組，n=20，創(chuàng)面出現(xiàn)后將BMSCs細胞懸液(濃度為1×10~（6）/ml)1ml注入大鼠創(chuàng)面皮下及真皮層，單次注射，并定期向創(chuàng)面噴灑bFGF。B組，n=20，創(chuàng)面出現(xiàn)后僅給予注射BMSCs細胞懸液，，方法同A組。C組，n=20，創(chuàng)面出現(xiàn)后給予注射生理鹽水。每周觀察創(chuàng)面愈合情況，且在無菌條件下切取創(chuàng)面組織5mm×5mm，采用光鏡、免疫組化等方法檢測，分別于治療后第1、3、5周觀察各組大鼠創(chuàng)面組織病理學變化和bFGF、VEGF表達的動態(tài)變化。 結(jié)果： 1.骨髓間充質(zhì)干細胞的培養(yǎng)與觀察：原代細胞經(jīng)過24h培養(yǎng)后可見大量細胞貼壁生長，6-8d后細胞生長進入指數(shù)生長期。傳代細胞生長至第3代，用DiI標記BMSCs，在熒光顯微鏡下可觀察到呈紅色熒光的細胞。將細胞注入大鼠創(chuàng)面，24小時后，熒光顯微鏡下仍能觀察創(chuàng)面組織切片中被DiI標記的BMSCs。 2.創(chuàng)面觀察：大鼠照射后2周開始脫毛，3周局部皮膚出現(xiàn)紅腫、水皰，4周出現(xiàn)創(chuàng)面，且逐漸增大，5周創(chuàng)面不再增大。①創(chuàng)面愈合時間：A組：30.40±1.52天，B組：38.81±2.80天，C組：45.62±3.95天。A治療組創(chuàng)面愈合時間明顯較其余兩組快（P0.05），B治療組較C組創(chuàng)面愈合時間快（P0.05）。②光鏡觀察：觀察各組創(chuàng)面組織中的表皮細胞、血管內(nèi)皮細胞、成纖維細胞數(shù)量，A組明顯多于B、C兩組，B組多于C組。③免疫組化顯示：治療后第1、3、5周bFGF、VEGF陽性細胞光密度值A組明顯高于B、C兩組（P0.05），B組高于C組（P0.05）。 結(jié)論： 1.應用直線加速器建立β射線皮膚損傷創(chuàng)面動物模型方法簡便、劑量準確、可靠。 2. BMSCs體外培養(yǎng)生長穩(wěn)定，傳代后仍保持未分化狀態(tài)。DiI標記BMSCs穩(wěn)定、可靠，可以做BMSCs示蹤研究。 3. bFGF復合BMSCs能促進β射線皮膚損傷創(chuàng)面的愈合，明顯縮短創(chuàng)面愈合的時間。 4、bFGF復合BMSCs促進β射線皮膚損傷創(chuàng)面的愈合,其機制是：bFGF通過促進BMSCs向血管內(nèi)皮細胞、表皮細胞、成纖維細胞等分化，增加創(chuàng)面局部組織中修復細胞數(shù)量, BMSCs又能促進VEGF、bFGF等生長因子的分泌，兩者具有協(xié)同作用，加速β射線皮膚損傷創(chuàng)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 鹵1.52 days; Group B: 38.81 鹵2.80 days, group C: 45.62 鹵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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R818

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