奶牛乳源外泌体提取方法的优化及效果评价  

Optimization and Evaluation of Extraction Methods of Milk Exosomes from Dairy Cows

作　　者：赵莹 王靖雷 王萌[1] 王立斌[1,2] 张倩 李志杰[1] 马鑫 余四九 潘阳阳[1,2] ZHAO Ying;WANG Jinglei;WANG Meng;WANG Libin;ZHANG Qian;LI Zhijie;MA Xin;YU Sijiu;PAN Yangyang(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Gansu Embryo Engineering Research Center of Cattle and Sheep,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]甘肃省牛羊胚胎工程技术研究中心,兰州730070

出　　处：《动物营养学报》2024年第2期1265-1276,共12页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：甘肃农业大学青年导师基金(GAU-QDFC-2022-02);甘肃省重点人才项目(2022RCXM016);国家自然科学基金地区项目(32160859);甘肃省杰出青年基金(20JR10RA561)。

摘　　要：乳源外泌体在疾病治疗及载体效应等领域应用颇为广泛,但目前乳源外泌体的提取方法仍步骤繁琐,耗时长,难以推广应用。为建立省时、高效和稳定的乳源外泌体提取方法,本试验在前期已建立的差速超速离心法、凝乳酶法、乙酸法及蔗糖密度梯度离心法的基础上,通过增加离心速度、改变乙酸或凝乳酶用量、调整蔗糖密度梯度、减少离心步骤进行逐一优化。通过透射电子显微镜、纳米粒子跟踪分析、蛋白浓度检测、免疫印迹鉴定比较乳源外泌体形态结构、粒径大小、蛋白浓度、粒子浓度、特异性标记蛋白的差异,筛选出最优提取方法。结果显示:4种优化后提取方法均可分离获得具有典型“茶托”形状,介于30~200 nm的细胞外囊泡,但优化后的乙酸法提取乳源外泌体最多,视野密度高达80%以上,粒径大小为(151.0±47.2)nm,粒子浓度为6.2×10^(12)个/mL,均高于其他优化方法;优化后的乙酸法提取乳源外泌体的特异性标记蛋白[肿瘤易感基因101(TSG101)、热休克蛋白70(HSP70)、CD81和CD9]均表达量较高,其他3种优化方法提取到的乳源外泌体特异性标记蛋白发生不同程度降解,且提取乳源外泌体所得样品蛋白浓度均显著高于优化的乙酸法(P<0.05)。结果表明,优化后奶牛乳源外泌体乙酸提取法具有方法简单、耗时短、纯度高、特异性标记蛋白稳定等优点,该方法为外泌体需求量大的试验、医疗、工业生产等奠定一定基础。Milk exosomes have been widely used in the treatment of diseases and carrier effects,but the current extraction methods of milk exosomes are still complicated and time-consuming,and it is difficult to promote their application.In order to establish a time-saving,efficient and stable extraction method for exosomes from milk sources,this experiment was optimized one by one by increasing the centrifugation speed,changing the amount of acetic acid or rennet and the density gradient of sucrose on the basis of the previously established differential ultra-centrifugal method,rennet method,acetic acid method and sucrose density gradient,and reducing the centrifugation steps.Through transmission electron microscopy,nanoparticle tracking analysis,protein concentration detection and western blot identification,the morphological structure,particle size,protein concentration,particle concentration and specific labeled protein of milk exosomes were compared,and the optimal extraction method was selected.The results showed that:extracellular vesicles with typical“saucer”shape ranging from 30 to 200 nm could be isolated by all the four optimized extraction methods,but the optimized acetic acid method extracted the most milk exosomes,with a field density of more than 80%,a particle size of(151.0±47.2)nm,and a particle concentration of 6.2×10^(12) pcs/mL were higher than those in other optimized methods.The specific marker proteins of milk exosomes extracted by the optimized acetic acid method[tumor susceptibility gene 101(TSG101),heat shock protein 70(HSP70),CD81 and CD9]all had high expression levels,while the milk exosome specific marker proteins extracted by the other three optimized methods were degraded to different degrees.The protein concentration of extractive milk exosomes was significantly higher than that of the optimized acetic acid extraction group(P<0.05).The results show that the optimized acetic acid extraction method of milk exosomes from dairy cows have the advantages of simple method,short time consuming,

关 键 词：乳源外泌体 差速超速离心法 乙酸 凝乳酶 提取鉴定 

分 类 号：S811.3[农业科学—畜牧学]