人脑胶质瘤细胞源性外泌体的分离与提纯研究

发布时间：2018-04-27 08:22

本文选题：胶质瘤 + exosome　；参考：《宁夏医科大学》2017年硕士论文

【摘要】：目的:体外培养U251细胞,分别利用差速离心法和试剂盒两种方法分离、提纯exosome,证明分离提纯的物质为exosome,并对两种方法进行优缺点比较。方法:体外培养U251细胞,观察细胞贴壁生长良好后,将培养基更换为不添加小牛血清的培养基,1周后将细胞培养上清液分两组进行收集,分别以差速离心法、试剂盒提纯exosome。然后在电子显微镜下通过液相载网法观察形态,并照相,确认exosome存在,对比两组的形态结构,记录直径大小,并对两种方法进行比较。结果:U251细胞可以分泌exosome,exosome小囊泡均能够在差速离心法和试剂盒两种方法下提取到。通过差速离心机进行差速离心提取的exosome与试剂盒提取的exosome结构相同,电镜结构为小囊泡状,表现为球体、椭球体,中央为低密度区,周围深染。两组测量直径均在35~160nm之间,差速离心法组测量直径均值为99.33±4.21nm,试剂盒组测量直径均值为98.08±4.13nm,经统计学分析两组方法提取的exosome直径随机抽样结果无显著性差异。结论:胶质瘤细胞可以分泌exosome。差速离心法、试剂盒均可以分离和提纯到形态结构相同的exosome。试剂盒具有操作相对简单,耗时短,样本需求量少等优点,但提取纯度低,科研成本高,临床难以普遍应用。差速离心法操作步骤繁多,耗时长,样本需求量大,但技术易掌握,科研成本低,提取纯度高,有助于exosome进一步研究以及向临床应用转化。
[Abstract]:Aim: to culture U251 cells in vitro and to isolate and purify U251 cells by differential centrifugation and kit respectively, and compare the advantages and disadvantages of the two methods. Methods: U251 cells were cultured in vitro. After the cells grew well, the culture medium was replaced with the medium without calf serum for 1 week. The supernatant of cell culture was collected into two groups, and the exosome was purified by differential centrifugation and kit. Then the morphology was observed by liquid phase netting method under electron microscope, and the existence of exosome was confirmed. The morphological structure and diameter of the two groups were compared, and the two methods were compared. Results the exosome-exosome vesicles could be obtained by differential centrifugation and kit. The structure of exosome extracted by differential centrifuge was the same as that of exosome extracted by reagent kit. The structure of electron microscope was vesicular, which was sphere, ellipsoid, low density area in the center and deep staining around it. The mean diameter of the two groups was 99.33 卤4.21 nm in the differential centrifugation group and 98.08 卤4.13 nm in the kit group. There was no significant difference in the random sampling results of the exosome diameter obtained by statistical analysis between the two groups. Conclusion: glioma cells can secrete exosome. Differential centrifugation and kit can be separated and purified to exosome with the same morphology and structure. The kit has the advantages of relatively simple operation, short time consuming and less sample demand, but the purity of extraction is low, the cost of scientific research is high, and it is difficult to be widely used in clinic. The differential centrifugation method has many steps, long time consuming, large sample demand, but the technology is easy to master, the cost of scientific research is low, and the purity of extraction is high, which is helpful for further study of exosome and its transformation to clinical application.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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