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Abstract：
BACKGROUND: Microvascular dysfunction is one of the most common pathological characteristics in Type 2 diabetes. Human mesenchymal stem cell-derived exosomes (hUCMSCs-Exo) have diverse functions in improving microcirculation; however, the molecular mechanism of hUCMSCs-Exo in regulating burn-induced inflammation is not well understood.
METHODS: hUCMSCs-Exo were extracted by hypervelocity centrifugation method, and exosome morphology was observed by transmission electron microscopy, exosome diameter distribution was detected by particle size analysis, and exosome specific proteins were identified by Western blot.2. DB/DB mice were randomly divided into exosomes group and PBS group. Exosomes and PBS were injected into the tail vein, respectively, and the calf muscle tissue was taken 28 days later. 0.5% Evans blue fluorescence assessment microvascular permeability. The expression of CD31 was detected by immunofluorescence.The morphology and function of microvessels in muscle tissue of lower limbs was evaluated by transmission electron microscopy.3. TMT proteomics was used to detect the changes of differential protein expression in lower limb muscle tissues of the PBS group and the exosome group, and data analysis was performed to screen key signal molecules and their involved biological pathways. Key signal molecules CD105 were verified by Western blot. The expression of TGF-β1 in exosomes were evaluated by Western blot.
RESULTS: Electron microscopy showed that hUCMSCs-Exo presented a uniform vesicle structure, and NTA showed that its diameter was about 160 nm. Western blot showed positive expression of specific proteins CD9, CD81 and TSG101 on exosomes.2. There is no significant change in blood glucose and body weight before and after the exosome treatment. The exosome group can significantly reduce the exudation of Evans blue. Compared with the PBS group. Meanwhile, CD31 immunofluorescence showed that the red fluorescence of exosome treatment was significantly increased, which was higher than that of PBS group. Transmission electron microscopy showed smooth capillary lumen and smooth and complete surface of endothelial cells in the exosome group, while narrow capillary lumen and fingerlike protrusion of endothelial cells in the PBS group.3.Quantitative analysis of TMT proteomics showed that there were 82 differential proteins, including 49 down-regulated proteins and 33 up-regulated proteins. Go enrichment analysis showed that the differential proteins were involved in molecular function, biological process, cell components,among which CD105 was one of the up-regulated proteins. Through literature search, CD105 was found to be related to endothelial cell proliferation. Therefore, this study verified the changes of CD105 in the exosome group, and it was used as the mechanism study of this study. 4. Western blot analysis showed that the expression of CD105 protein in lower limb muscle tissue of exosome group was significantly increased compared with that of PBS group. Based on the fact that CD105 is a component of the TGF-β1 receptor complex and exosomes are rich in growth factors and cytokines, this study further examined the expression of TGF-β1 in exosomes, and the results showed that exosomes had high expression of TGF-β1.
CONCLUSIONS: By improving the integrity of microvascular endothelial cells, hUCMSCs-Exo can improve the permeability of microvessels in diabetic lower muscle tissue, further promote the proliferation of lower limb muscle cells and inhibit the apoptosis of tissue cells. The mechanism may be associated with exosomes rich in TGF-β1, which is likely to promote endothelial cell proliferation and improve permeability through binding to the endothelial CD105/TβR-II receptor complex, while promoting angiogenesis and protecting skeletal muscle cells from apoptosis.
METHODS: hUCMSCs-Exo were extracted by hypervelocity centrifugation method, and exosome morphology was observed by transmission electron microscopy, exosome diameter distribution was detected by particle size analysis, and exosome specific proteins were identified by Western blot.2. DB/DB mice were randomly divided into exosomes group and PBS group. Exosomes and PBS were injected into the tail vein, respectively, and the calf muscle tissue was taken 28 days later. 0.5% Evans blue fluorescence assessment microvascular permeability. The expression of CD31 was detected by immunofluorescence.The morphology and function of microvessels in muscle tissue of lower limbs was evaluated by transmission electron microscopy.3. TMT proteomics was used to detect the changes of differential protein expression in lower limb muscle tissues of the PBS group and the exosome group, and data analysis was performed to screen key signal molecules and their involved biological pathways. Key signal molecules CD105 were verified by Western blot. The expression of TGF-β1 in exosomes were evaluated by Western blot.
RESULTS: Electron microscopy showed that hUCMSCs-Exo presented a uniform vesicle structure, and NTA showed that its diameter was about 160 nm. Western blot showed positive expression of specific proteins CD9, CD81 and TSG101 on exosomes.2. There is no significant change in blood glucose and body weight before and after the exosome treatment. The exosome group can significantly reduce the exudation of Evans blue. Compared with the PBS group. Meanwhile, CD31 immunofluorescence showed that the red fluorescence of exosome treatment was significantly increased, which was higher than that of PBS group. Transmission electron microscopy showed smooth capillary lumen and smooth and complete surface of endothelial cells in the exosome group, while narrow capillary lumen and fingerlike protrusion of endothelial cells in the PBS group.3.Quantitative analysis of TMT proteomics showed that there were 82 differential proteins, including 49 down-regulated proteins and 33 up-regulated proteins. Go enrichment analysis showed that the differential proteins were involved in molecular function, biological process, cell components,among which CD105 was one of the up-regulated proteins. Through literature search, CD105 was found to be related to endothelial cell proliferation. Therefore, this study verified the changes of CD105 in the exosome group, and it was used as the mechanism study of this study. 4. Western blot analysis showed that the expression of CD105 protein in lower limb muscle tissue of exosome group was significantly increased compared with that of PBS group. Based on the fact that CD105 is a component of the TGF-β1 receptor complex and exosomes are rich in growth factors and cytokines, this study further examined the expression of TGF-β1 in exosomes, and the results showed that exosomes had high expression of TGF-β1.
CONCLUSIONS: By improving the integrity of microvascular endothelial cells, hUCMSCs-Exo can improve the permeability of microvessels in diabetic lower muscle tissue, further promote the proliferation of lower limb muscle cells and inhibit the apoptosis of tissue cells. The mechanism may be associated with exosomes rich in TGF-β1, which is likely to promote endothelial cell proliferation and improve permeability through binding to the endothelial CD105/TβR-II receptor complex, while promoting angiogenesis and protecting skeletal muscle cells from apoptosis.
摘要：
背景：微血管功能障碍是2型糖尿病最常见的病理特征之一。人类间充质干细胞来源的外泌体（hUCMSCs-Exo）在改善微循环方面具有多种功能；然而，hUCMSCs-Exo调节烧伤诱导炎症的分子机制尚不清楚。
方法：采用超高速离心法提取hUCMSCs-Exo,并通过透射电子显微镜观察到外泌体形态，通过粒度分析检测外泌体直径分布，和外泌体特异性蛋白通过Westernblot鉴定。将DB/DB小鼠随机分为外泌体组和PBS组。尾静脉注射外泌体和PBS,分别,28天后取小腿肌肉组织。0.5%Evans蓝荧光考核微血管通透性。免疫荧光法检测CD31的表达。透射电镜观察下肢肌肉组织微血管的形态和功能。采用TMT蛋白质组学方法检测PBS组和外泌体组下肢肌肉组织差异蛋白表达的变化,并进行数据分析以筛选关键信号分子及其涉及的生物学途径。通过Western印迹验证关键信号分子CD105。Westernblot检测TGF-β1在外泌体中的表达。
结果：电子显微镜显示hUCMSCs-Exo呈现均匀的囊泡结构，和NTA显示其直径约为160nm。Westernblot显示外泌体上特异性蛋白CD9、CD81和TSG101阳性表达。外泌体治疗前后血糖和体重无显著变化。外泌体组能显著减少伊文思蓝的渗出。与PBS组比拟。同时,CD31免疫荧光显示外泌体处理的红色荧光显著增加，高于PBS组。透射电镜显示外泌体组毛细血管腔光滑，内皮细胞表面光滑完整，而PBS组中狭窄的毛细血管腔和内皮细胞的手指状突起3。TMT蛋白质组学定量分析结果显示,共有82个差异蛋白,包括49种下调蛋白和33种上调蛋白。Go富集分析表明，差异蛋白参与了分子功能，生物过程，细胞组件，其中CD105是上调蛋白之一。通过文献检索,发现CD105与内皮细胞增殖有关。因此,本研究验证了CD105在外泌体组中的变化，并作为本研究的机理研究。4.Westernblot分析显示,外泌体组下肢肌肉组织中CD105蛋白的表达量较PBS组显著增加。基于CD105是TGF-β1受体复合物的组成部分,外泌体富含生长因子和细胞因子,这项研究进一步检查了TGF-β1在外泌体中的表达，结果表明外泌体高表达TGF-β1。
结论：通过改善微血管内皮细胞的完整性，hUCMSCs-Exo可改善糖尿病下肌肉组织微血管通透性,进一步促进下肢肌细胞增殖，抑制组织细胞凋亡。其机制可能与富含TGF-β1的外泌体有关,可能通过与内皮CD105/TβR-II受体复合物结合,促进内皮细胞增殖,提高通透性,同时促进血管生成和保护骨骼肌细胞免受凋亡。
方法：采用超高速离心法提取hUCMSCs-Exo,并通过透射电子显微镜观察到外泌体形态，通过粒度分析检测外泌体直径分布，和外泌体特异性蛋白通过Westernblot鉴定。将DB/DB小鼠随机分为外泌体组和PBS组。尾静脉注射外泌体和PBS,分别,28天后取小腿肌肉组织。0.5%Evans蓝荧光考核微血管通透性。免疫荧光法检测CD31的表达。透射电镜观察下肢肌肉组织微血管的形态和功能。采用TMT蛋白质组学方法检测PBS组和外泌体组下肢肌肉组织差异蛋白表达的变化,并进行数据分析以筛选关键信号分子及其涉及的生物学途径。通过Western印迹验证关键信号分子CD105。Westernblot检测TGF-β1在外泌体中的表达。
结果：电子显微镜显示hUCMSCs-Exo呈现均匀的囊泡结构，和NTA显示其直径约为160nm。Westernblot显示外泌体上特异性蛋白CD9、CD81和TSG101阳性表达。外泌体治疗前后血糖和体重无显著变化。外泌体组能显著减少伊文思蓝的渗出。与PBS组比拟。同时,CD31免疫荧光显示外泌体处理的红色荧光显著增加，高于PBS组。透射电镜显示外泌体组毛细血管腔光滑，内皮细胞表面光滑完整，而PBS组中狭窄的毛细血管腔和内皮细胞的手指状突起3。TMT蛋白质组学定量分析结果显示,共有82个差异蛋白,包括49种下调蛋白和33种上调蛋白。Go富集分析表明，差异蛋白参与了分子功能，生物过程，细胞组件，其中CD105是上调蛋白之一。通过文献检索,发现CD105与内皮细胞增殖有关。因此,本研究验证了CD105在外泌体组中的变化，并作为本研究的机理研究。4.Westernblot分析显示,外泌体组下肢肌肉组织中CD105蛋白的表达量较PBS组显著增加。基于CD105是TGF-β1受体复合物的组成部分,外泌体富含生长因子和细胞因子,这项研究进一步检查了TGF-β1在外泌体中的表达，结果表明外泌体高表达TGF-β1。
结论：通过改善微血管内皮细胞的完整性，hUCMSCs-Exo可改善糖尿病下肌肉组织微血管通透性,进一步促进下肢肌细胞增殖，抑制组织细胞凋亡。其机制可能与富含TGF-β1的外泌体有关,可能通过与内皮CD105/TβR-II受体复合物结合,促进内皮细胞增殖,提高通透性,同时促进血管生成和保护骨骼肌细胞免受凋亡。
