两种外泌体提取方法的比较以及外泌体对低氧肺动脉高压肺血管重构的作用  

Two extraction methods for exosomes and effect of exosomes on pulmonary arterial remodeling in hypoxia-induced pulmonary hypertension

作　　者：郭峰[1] 黄宁 胡长平 GUO Feng;HUANG Ning;HU Chang-ping(Department of Pharmacy,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052;Department of Pharmacology,Xiangya School of Pharmaceutical Sciences,Central South University,Changsha 410078)

机构地区：[1]郑州大学第一附属医院药学部,郑州450052 [2]中南大学湘雅药学院药理学系,长沙410078

出　　处：《中南药学》2022年第10期2286-2294,共9页Central South Pharmacy

基　　金：国家自然科学基金资助项目(No.82173817)。

摘　　要：目的 比较超高速差速离心法和试剂盒法提取肺动脉平滑肌细胞(PASMCs)源性外泌体的纯度,并评价超高速差速离心法提取外泌体的稳定性和适用性。探究低氧肺动脉高压(PH)大鼠血浆源性外泌体对PASMCs表型转化和肺动脉内皮细胞(PAECs)内皮间质转化的作用。方法 ① 收集PASMCs培养基上清液,分别用超高速差速离心法和试剂盒法提取外泌体,通过透射电子显微镜观察外泌体形态、纳米微粒示踪分析检测外泌体粒径分布、蛋白质免疫印迹法检测外泌体标志蛋白表达,评估外泌体纯度;② 收集PAECs培养基上清液和大鼠血浆,超高速差速离心法分别提取外泌体并评估外泌体纯度;③ 构建低氧PH大鼠模型,超高速差速离心法分别提取常氧和低氧大鼠血浆源性外泌体并作用于PASMCs和PAECs,免疫荧光检测各标志蛋白表达以评估外泌体对PASMCs表型转化和PAECs内皮间质转化的影响。结果 ① 超高速差速离心法提取的PASMCs、PAECs和血浆源性外泌体形态典型,纯度较高;而试剂盒法提取的PASMCs源性外泌体纯度较低。② 低氧PH大鼠血浆源性外泌体诱导PASMCs发生表型转化和PAECs发生内皮间质转化。结论 超高速差速离心法提取外泌体纯度优于试剂盒法,实验方法稳定且适用性强。低氧PH大鼠血浆源性外泌体可诱导PH肺血管重构。Objective To compare the purity of exosomes derived from pulmonary artery smooth muscle cells (PASMCs) by ultra-high speed differential centrifugation method and kit method,assess the stability and applicability of ultra-high speed differential centrifugation method for exosomes extraction,and determine the effect of plasma-derived exosomes extracted from hypoxic-induced pulmonary hypertension (PH) rats on the phenotypic transformation of PASMCs and the endothelial-mesenchymal transition (EndMT) of pulmonary artery endothelial cells (PAECs).Methods ① Ultra-high speed differential centrifugation method and kit method were used respectively to obtain the PASMCs-derived exosomes.Transmission electron microscope was used to observe the morphology of exosomes;nanoparticle tracing was used to observe the particle size distribution of exosomes;Western blot was used to detect the expression of exosomes biomarkers,and the purity of exosomes was ultimately evaluated.② Ultra-high speed differential centrifugation method was used to obtain the PAECs-derived and plasma-derived exosomes,and to evaluate the purity of exosomes.③ The hypoxia-induced PH model of rats was constructed.The plasma-derived exosomes from normoxic rats and hypoxic rats were obtained by ultra-high speed differential centrifugation,and were used to treat primary normoxic cultured PASMCs and PAECs derived from the pulmonary arteries of SD rats.The expression of various marker proteins was detected to assess the effect of exosomes on the phenotypic switching of PASMCs and PAECs-EndMT.Results ① The morphology of PASMCs,PAECs and plasma-derived exosomes extracted by ultra-high-speed differential centrifugation was typical,and the purity of exosomes was high.The purity of the PASMCs-derived exosomes extracted by the kit method was low.②The plasma-derived exosomes of hypoxic rats promoted the phenotypic switching of PASMCs and PAECs-EndMT.Conclusion The purity of exosomes extracted by ultra-high-speed differential centrifugation is higher than that by

关 键 词：外泌体 超高速差速离心法 试剂盒法 肺动脉内皮细胞 肺动脉平滑肌细胞 

分 类 号：R563[医药卫生—呼吸系统]