超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件（英文）  

Optimization of repeated freeze-thaw and ultrasonication for collection of lysate of adipose-derived stem cells

作　　者：王俊怡[1] 金银鹏[2] 李洪超[1] 孟凌玉[2] 李莉[2] 王晓今[2] 周荣[2] 陈成伟[2] 傅青春[2] 程明亮[3] Wang Jun-yi Jin Yin-peng Li Hong-chao Meng Ling-yu Li Li Wang Xiao-jin Zhou Rong Chen Cheng-wei Fu Qing-chun Cheng Ming-liang(Clinidal Medical College of Guizhou Medical University, Gujyang 550004, Guizhou Province, China Shanghai Liver Diseases Research Center, the 85th Hospital of PLA, Shanghai 200235, China Department of Infection, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China)

机构地区：[1]贵州医科大学临床医学院,贵州省贵阳市550004 [2]解放军第八五医院上海肝病研究中心,上海市200235 [3]贵州医科大学附属医院感染科,贵州省贵阳市550004

出　　处：《中国组织工程研究》2017年第17期2631-2637,共7页Chinese Journal of Tissue Engineering Research

基　　金：the Medical Innovation Project of the Nanjing Military Region,No.14ZX01;China Hepatitis Prevention and Treatment Foundation-Tian Qing Liver Research Fund Project,No.TQGB20150104~~

摘　　要：背景:旁分泌在间充质干细胞治疗方面起主要作用。提取间充质干细胞裂解液,可以避免直接间充质干细胞本身所致栓塞、肿瘤以及免疫排斥反应等问题,然而目前提取间充质干细胞裂解液的方法存在差异。目的:比较超声破碎及反复冻融方法获得间充质干细胞裂解液的差异。方法:(1)从健康的人腹部皮下脂肪中分离出脂肪间充质干细胞,经贴壁筛选法纯化细胞,胎牛血清培养基体外培养扩增,并采用流式细胞检测仪鉴定其常用表面标记;(2)采用反复冻融及超声细胞破碎2种破壁方法,将1×10~9 L^-1,2×10~9 L^-1,4×10~9 L^-1 3种不同密度的细胞分别置于生理盐水与双蒸水中进行破碎,比较细胞破碎率及裂解液蛋白含量的差异。结果与结论:(1)通过体外分离人体脂肪组织,可获得脂肪间充质干细胞;(2)细胞裂解时,细胞浓度越高,破壁所需时间越长,而破碎时间越长,则细胞破碎率越大;(3)生理盐水中超声细胞破碎法得到的蛋白含量较其他条件高;(4)结果提示在生理盐水环境下使用超声方法提取脂肪间充质干细胞裂解液尤为有效,且推荐细胞浓度为4×10~9 L^-1以内。BACKGROUND： It has been believed mesenchymal stem cells （MSCs） play a role in treatment through paracrine mechanism. Various side effects such as embolism, tumorigenesis and immunological reaction caused by direct injection of MSCs can be avoided by extracting MSC lysate. However, there is a larger difference in current collection methods and standards of MSC lysate. OBJECTIVE： To compare repeated freeze-thaw and ultrasonication for the collection of lysate of MSCs. METHODS： Adipose-derived mesenchymal stem cells （ADMSCs） were isolated from the abdominal subcutaneous fat of healthy individuals, and purified with adherence screening method, followed by in vitro amplification using fetal bovine serum medium. The common surface makers of these cells were tested by flow cytometry （1×109, 2×109, 4×109/L）. Repeated freeze-thaw and ultrasonication were employed for cell cytoclasis at three different densities respectively in saline and double distilled water, and a comprehensive comparison was performed on cytoclasis rate and the content of protein in cell lysate between the two methods. RESULTS AND CONCLUSION： （1） ADMSCs obtained from in vitro isolated human adipose tissue grew in a swirl or radial pattern with a homogenous size and neat arrangement. CD44, CD90, CD105 and other commonly used surface markers were highly expressed. （2） The study for optimization of lysate collection revealed that the higher cell density implicated a longer time for cell wall disruption and cytoclasis, as well as significantly increased cytoclasis rate. （3） BCA protein assay showed that the highest content of protein was obtained in saline solvent using ultrasonication method. Comprehensive analysis on the results leads to a conclusion that ultrasonication method with saline as the solvent is the optimized method for extraction of ADMSCs lysate, and the cell concentration of less than 4×109/L is recommended.

关 键 词：干细胞 脂肪干细胞 间充质干细胞 脂肪间充质干细胞 超声 反复冻融 细胞裂解液 细胞治疗 细胞外膜泡 

分 类 号：R394.2[医药卫生—医学遗传学]