【作者】 王静；

【导师】 蔡霞； 王志国；

【作者基本信息】 青岛大学 ， 外科学（烧伤）， 2019， 硕士

【摘要】 目的:1.提取人脂肪间充质干细胞,鉴定脂肪间充质干细胞,诱导脂肪间充质干细胞多向分化2.提取脂肪间充质干细胞来源的外泌体,鉴定脂肪间充质干细胞来源的外泌体3.观察脂肪间充质干细胞来源的外泌体对小鼠创面修复的影响。方法:1.收集正常成年女性行吸脂术后腹部浅层皮下脂肪,用酶消化法分离提取脂肪间充质干细胞进行原代培养,用细胞贴壁法纯化,胰酶消化传代。选取第3代细胞,进行流式细胞学鉴定;诱导第3代细胞向脂肪细胞分化,第14天油红O染色,观察分化情况;诱导第3代细胞向成骨细胞分化,第9天碱性磷酸酶染色,观察成骨化早期标志,第21天茜素红S染色,观察I型胶原钙化情况。2.收集第3-6代脂肪间充质干细胞的细胞培养液,差速离心法提取外泌体,BCA法测定蛋白浓度,透射电镜观察外泌体形态,粒度仪测外泌体直径分布,免疫磁珠法和流式细胞学相结合的方式检测外泌体及其来源细胞表面标志物。3.制备小鼠背部全层皮肤创面模型30只,随机分为EXO组、PBS组、空白组3组。EXO组经鼠尾静脉注射200uLEXO悬液,PBS组经鼠尾静脉注射200uLPBS,空白组不予处理。分别于第1、3、7、14天观察创面愈合情况,并统计分析各时间点残余创面面积,第7、14天处死小鼠,取组织行HE、Masson染色,观察纤维生成及胶原表达情况。结果:1.脂肪间充质干细胞经多次传代后,形态为长梭形,形似成纤维细胞,生长旺盛时呈旋涡状排列。流式细胞学检测显示CD29、CD44、CD90、CD105均为阳性表达,CD34、CD45为阴性表达。成骨诱导第9天时碱性磷酸酶染色呈蓝紫色,第21天时,细胞层次不清,排列紊乱,茜素红S染色可见红色矿化钙结节。成脂诱导14天后细胞胞浆内出现大量的透光性良好的大小不等的空泡,油红O染色呈橘红色。2.透射电镜显示外泌体形态为杯状,直径为81.225±22.226nm,有膜结构。蛋白浓度约为1.5mg/mL,直径分布集中于70-100nm,流式细胞学显示CD9、CD29、CD44、CD63、CD90、CD105为阳性表达,CD34、CD45为阴性表达。3.创面大体观察显示,第7天时,EXO组与另两组相比,残余创面面积稍小;第14天时,EXO与另两组相比,愈合速率明显加快,残余创面面积明显减少。HE染色显示,第7天,EXO组与另外两组相比,炎性细胞浸润稍少,纤维生成稍多,但均排列紊乱,上皮化不明显;第14天,3组炎性细胞浸润均明显减少,纤维组织排列,EXO组比PBS组、空白组更整齐,上皮化EXO组也更加明显。Masson染色显示,第7天,EXO组胶原表达比另两组稍高,虽均排列紊乱,但较另两组好;第14天,3组胶原表达都有所增高,EXO组增高尤为明显,排列也更加整齐。结论:1.脂肪间充质干细胞能通过较为简便的方法大量提取,并能通过流式细胞学与诱导多向分化成功鉴定。2.脂肪间充质干细胞来源的外泌体能通过超速离心法顺利提取,并通过透射电子显微镜、免疫磁珠法和流式细胞学方法相结合的方法成功鉴定。3.脂肪间充干细胞来源的外泌体经鼠尾静脉注射能够促进小鼠背部创面愈合。更多还原

【Abstract】 Objectives:1.Extracting human adipose-derived mesenchymal stem cells,identifying adipose-derived mesenchymal stem cells and inducing multidirectional differentiation of adipose-derived mesenchymal stem cells2.Extraction of adipose-derived mesenchymal stem cell-derived exosomes and identification of adipose-derived mesenchymal stem cell-derived exosomes3.To observe the effect of exosomes derived from adipose-derived mesenchymal stem cells on wound repair in mice.Methods:1.Collect the superficial subcutaneous fat of abdomen of normal adult women after liposuction and extract it by enzymatic digestion method.Adipose-derived mesenchymal stem cells were cultured in primary culture,purified by cell adherence method,and digested by trypsinase.The 3rd generation cells were identified by flow cytometry;the3rd generation cells were induced to differentiate into adipocytes,and the differentiation was observed by oil red O staining on the 14 th day;the 3rd generation cells were induced to differentiate into osteoblasts,and the early markers of osteogenesis were observed by alkaline phosphatase staining on the 9th day,and the calcification of collagen type I was observed by alizarin red S staining on the 21 st day.2.Cell culture medium of 3rd-6th generation adipose mesenchymal stem cells was collected,exosomes were extracted by differential centrifugation,protein concentration was determined by BCA,morphology of exosomes was observed by transmission electron microscopy,diameter distribution of exosomes was measured by particle size analyzer,and surface markers of exosomes and their source cells were detected by immunomagnetic beads and flow cytometry.3.Thirty full-thickness skin wound models were prepared and randomly divided into EXO group,PBS group and blank group.EXO group was injected with 200 u LEXO suspension via tail vein,PBS group was injected with 200 u LPBS via tail vein,blank group was not treated.The wound healing was observed on the 1st,3rd,7th and 14 th day,and the residual wound area was analyzed.The mice were killed on the 7th and 14 th day.The tissues were stained with HE and Masson to observe the fibrogenesis and collagen expression.Results:1.After several passages,adipose-derived mesenchymal stem cells are spindle-shaped and fibroblast-like in shape.They are arranged in a spiral pattern when they grow vigorously.Flow cytometry showed that CD29,CD44,CD90 and CD105 were positive,while CD34 and CD45 were negative.Alkaline phosphatase staining was blue-purple on the 9th day of osteogenesis induction.On the 21 st day,cell layers were unclear and disordered.Alizarin red S staining showed red mineralized calcium nodules.After 14 days of adipogenic induction,a large number of vacuoles with good light transmittance appeared in the cytoplasm.Oil red O staining was orange.2.Transmission electron microscopy showed that the exosome was cup-shaped with a diameter of 81.225+22.226 nm and a membrane structure.Flow cytometry showed that CD9,CD29,CD44,CD63,CD90 and CD105 were positive,while CD34 and CD45 were negative.3.Gross observation of wound surface showed that on the 7th day,the area of residual wound in EXO group was slightly smaller than that in the other two groups;on the 14 th day,the healing rate of EXO group was significantly faster than that in the other two groups,and the area of residual wound was significantly reduced.HE staining showed that on the 7th day,compared with the other two groups,inflammatory cell infiltration was slightly less and fibrogenesis was slightly more in EXO group,but the arrangement of inflammatory cells was disordered and epithelialization was not obvious.On the 14 th day,inflammatory cell infiltration was significantly reduced in all three groups,and the arrangement of fibrous tissue was more regular in EXO group than in PBS group and blank group,and the epithelialization of EXO group was more obvious.Masson staining showed that on the 7th day,the expression of collagen in EXO group was slightly higher than that in the other two groups,although the arrangement was disordered,but better than that in the other two groups.On the 14 th day,the expression of collagen in the three groups increased,especially in EXO group.Conclusions :1.Adipose-derived mesenchymal stem cells can be extracted in large quantities by relatively simple methods,and can be successfully identified by flow cytometry and induced multidirectional differentiation.2.The exosomes derived from adipose mesenchymal stem cells can be successfully extracted by ultracentrifugation and identified by transmission electron microscopy,immunomagnetic beads and flow cytometry.3.Intravenous injection of adipose-derived mesenchymal stem cell-derived exosomes via rat tail vein can promote wound healing in mice.更多还原