【摘要】：目的:明確瘢痕愈合/再生修復(fù)與皮膚損傷深度之間的關(guān)系;研究骨髓間質(zhì)干細(xì)胞創(chuàng)面局部移植能否使更深層次的組織損傷再生修復(fù),以及相關(guān)的細(xì)胞、分子機(jī)制。方法:在SD大鼠背部建立連續(xù)性、漸進(jìn)性加深創(chuàng)面,取生長(zhǎng)狀態(tài)良好的第三代SD大鼠BM-MSCs/GFP,以PBS稀釋成1.0×10~6/300.0μL的細(xì)胞懸液,其中,200.0μL均勻注射于創(chuàng)緣皮內(nèi),100.0μL注射于創(chuàng)面淺層,對(duì)照組以相同方法注射同等劑量的PBS液,于移植后第1、3、5、7、14、21天照相后取材,行快速冰凍組織病理切片,觀察BM-MSCs/GFP移植后不同時(shí)間點(diǎn)在創(chuàng)面的數(shù)量、分布情況;行HE、Masson染色,確定創(chuàng)面再生的最深組織層次,并根據(jù)免疫組織化學(xué)染色,觀察Blimp1~+、Lgr6~+干細(xì)胞在創(chuàng)面的數(shù)量和VEGF的表達(dá)差異,初步探討相關(guān)的細(xì)胞分子機(jī)制。實(shí)驗(yàn)數(shù)據(jù)用x±s表示,各時(shí)間點(diǎn)創(chuàng)面收縮率、創(chuàng)面愈合率、創(chuàng)面BM-MSCs/GFP計(jì)數(shù)和免疫組化陽(yáng)性著色細(xì)胞比例用SPSS 13.0統(tǒng)計(jì)分析軟件兩因素方差分析;毛細(xì)血管計(jì)數(shù)用Dunnet t檢驗(yàn);創(chuàng)面再生組織層次用Χ~2檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)處理;P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1、對(duì)照組毛囊漏斗頂部以上平面的組織損傷再生修復(fù);BM-MSCs/GFP創(chuàng)面局部移植后組織再生層次加深至毛囊皮脂腺開口平面以上。2、BM-MSCs/GFP移植后隨時(shí)間延長(zhǎng)而減少,移植后21日,細(xì)胞數(shù)量約為移植后首日的1.0%。3、BM-MSCs/GFP創(chuàng)面局部移植后隨時(shí)間延長(zhǎng)向創(chuàng)面深層遷徙,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+干細(xì)胞在移植后第3、5日分別增加,BM-MSCs/GFP組顯著高于對(duì)照組(P0.05),至移植后21日兩組無明顯差異。5、VEGF表達(dá)量在BM-MSCs/GFP移植后第1日開始上升,第5日達(dá)到高峰,14日開始下降,BM-MSCs/GFP組表達(dá)量顯著高于對(duì)照組(P0.05)。6、BM-MSCs/GFP治療組每高倍視野毛細(xì)血管數(shù)為5.0±0.3,高于對(duì)照組的2.5±0.2(P0.05)。結(jié)論:1、BM-MSCs/GFP創(chuàng)面局部移植能使組織再生層次由對(duì)照組的毛囊漏斗頂部以上平面,加深到毛囊皮脂腺開口平面以上;2、盡管BM-MSCs/GFP移植后長(zhǎng)期存活率低,但移植早期的存活有利于自身Blimp1~+、Lgr6~+干細(xì)胞的活化和損傷后皮脂腺的修復(fù);3、BM-MSCs/GFP組再生深度的增加是移植干細(xì)胞和自身干細(xì)胞共同作用的結(jié)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 鹵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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R641

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