骨髓间质干细胞创面移植促进深层皮肤组织损伤再生

发布时间：2019-04-09 20:13

【摘要】：目的:明确瘢痕愈合/再生修复与皮肤损伤深度之间的关系;研究骨髓间质干细胞创面局部移植能否使更深层次的组织损伤再生修复,以及相关的细胞、分子机制。方法:在SD大鼠背部建立连续性、渐进性加深创面,取生长状态良好的第三代SD大鼠BM-MSCs/GFP,以PBS稀释成1.0×10~6/300.0μL的细胞悬液,其中,200.0μL均匀注射于创缘皮内,100.0μL注射于创面浅层,对照组以相同方法注射同等剂量的PBS液,于移植后第1、3、5、7、14、21天照相后取材,行快速冰冻组织病理切片,观察BM-MSCs/GFP移植后不同时间点在创面的数量、分布情况;行HE、Masson染色,确定创面再生的最深组织层次,并根据免疫组织化学染色,观察Blimp1~+、Lgr6~+干细胞在创面的数量和VEGF的表达差异,初步探讨相关的细胞分子机制。实验数据用x±s表示,各时间点创面收缩率、创面愈合率、创面BM-MSCs/GFP计数和免疫组化阳性着色细胞比例用SPSS 13.0统计分析软件两因素方差分析;毛细血管计数用Dunnet t检验;创面再生组织层次用Χ~2检验进行统计学处理;P0.05为差异有统计学意义。结果:1、对照组毛囊漏斗顶部以上平面的组织损伤再生修复;BM-MSCs/GFP创面局部移植后组织再生层次加深至毛囊皮脂腺开口平面以上。2、BM-MSCs/GFP移植后随时间延长而减少,移植后21日,细胞数量约为移植后首日的1.0%。3、BM-MSCs/GFP创面局部移植后随时间延长向创面深层迁徙,皮脂腺、血管壁等部位早期有散在分布,21日后消失。4、Blimp1~+、Lgr~6+干细胞在移植后第3、5日分别增加,BM-MSCs/GFP组显著高于对照组(P0.05),至移植后21日两组无明显差异。5、VEGF表达量在BM-MSCs/GFP移植后第1日开始上升,第5日达到高峰,14日开始下降,BM-MSCs/GFP组表达量显著高于对照组(P0.05)。6、BM-MSCs/GFP治疗组每高倍视野毛细血管数为5.0±0.3,高于对照组的2.5±0.2(P0.05)。结论:1、BM-MSCs/GFP创面局部移植能使组织再生层次由对照组的毛囊漏斗顶部以上平面,加深到毛囊皮脂腺开口平面以上;2、尽管BM-MSCs/GFP移植后长期存活率低,但移植早期的存活有利于自身Blimp1~+、Lgr6~+干细胞的活化和损伤后皮脂腺的修复;3、BM-MSCs/GFP组再生深度的增加是移植干细胞和自身干细胞共同作用的结果。
[Abstract]:Aim: to investigate the relationship between scar healing / regeneration and the depth of skin injury, and to investigate whether the local transplantation of bone marrow mesenchymal stem cells (BMSCs) can make deeper tissue regeneration and repair, as well as the related cellular and molecular mechanisms. Methods: a continuous and progressive wound was established in the back of SD rats. The BM-MSCs/GFP, of the third generation SD rats in good growth condition was diluted into 1.0 脳 10 ~ 6 / 300.0 渭 L cell suspension by PBS, in which 2000.0 渭 L was injected uniformly into the wound margin. 100.0渭 L was injected into the superficial layer of the wound. In the control group, the same dose of PBS solution was injected in the same way. After 1,3,5,7,14,21 days after transplantation, the specimens were taken and the specimens were taken for quick frozen histopathology. The number and distribution of wounds at different time points after BM-MSCs/GFP transplantation were observed. HE,Masson staining was performed to determine the deepest tissue level of wound regeneration. According to immunohistochemical staining, the number of Blimp1~ and Lgr6~ stem cells in the wound and the difference of VEGF expression were observed, and the related cellular and molecular mechanisms were preliminarily discussed. The experimental data were expressed by x-table s. The wound contraction rate, wound healing rate, BM-MSCs/GFP count and the proportion of positive staining cells were analyzed by SPSS-13.0 statistical analysis software. Capillary count was analyzed by Dunnet-t test; wound regeneration tissue level was statistically treated by X ~ 2 test; P0.05 was statistically significant. Results: 1Regeneration and repair of tissue injury above the top of the funnel of hair follicles was observed in the control group; After local transplantation of BM-MSCs/GFP, the level of tissue regeneration was deepened above the orifice level of hair follicle sebaceous gland. 2. The number of cells on 21 days after transplantation was about 1.0% of that of the first day after transplantation, and it decreased with the prolongation of the time after transplantation, and the number of cells on 21 days after transplantation was about 1.0% of that of the first day after transplantation. After local transplantation of BM-MSCs/GFP wound, it migrated to the deep layer of the wound over time, the sebaceous gland and vascular wall were scattered in the early stage and disappeared 21 days later. 4. Blimp1 ~, Lgr~6 stem cells increased on the 3rd and 5th day after transplantation, respectively. The expression of BM-MSCs/GFP was significantly higher in BM-MSCs/GFP group than in control group (P0.05), and there was no significant difference between the two groups on the 21st day after transplantation. 5. The expression level of VEGF increased on the 1st day after transplantation, reached the peak on the 5th day, and decreased on the 14th day after transplantation. The expression level in BM-MSCs/GFP group was significantly higher than that in control group (P0.05). The number of capillaries per high visual field in the treatment group was 5.0 卤0.3, which was higher than that in the control group (P 0.05). Conclusion: 1. Local transplantation of BM / MSCs / GFP can make the tissue regeneration level deepen from the top of hair follicle funnel to the orifice level of sebaceous gland of hair follicle in the control group. 2. Although the long-term survival rate after BM-MSCs/GFP transplantation is low, the early survival is beneficial to the activation of Blimp1~, Lgr6~ stem cells and the repair of sebaceous gland after injury. 3. The increase of regeneration depth in BM / MSCs / GFP group was the result of the co-action of transplanted stem cells and their own stem cells.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641

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