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人脐带间充质干细胞源性外泌体对低氧性肺动脉高压肺血管重构的影响

刘红^1,2,3, 张雨薇^1,4, 张晴晴^1,2,3, 王玉香^1,2,3, 格日力^1,2,3, 马兰^1,2,3,*

¹青海大学高原医学研究中心，西宁 810001；²高原医学教育部重点实验室，西宁 810001；³青海省高原医学应用基础重点实验室(青海-犹他高原医学联合重点实验室)，西宁 810001；⁴青海大学医学部公共卫生系，西宁 810001

摘要

本研究旨在探讨人脐带间充质干细胞源性外泌体(mesenchymal stem cells-derived exosomes, MSCs-Exo)对低氧诱导的肺动脉高压小鼠的作用。无菌条件下从人脐带分离培养间充质干细胞，收集上清，提取外泌体并鉴定。健康SPF级C57BL/6小鼠随机分为3组：常氧对照组、低氧组、低氧+MSCs-Exo组，每组7只。低氧组和低氧+MSCs-Exo组在氧浓度为10.08%、模拟海拔5 000 m的低压低氧舱连续饲养4周，建立低氧性肺动脉高压小鼠模型。低氧+MSCs-Exo组在低氧前及低氧第1、3、5、9天尾静脉注射MSCs-Exo，其他2组注射PBS。实验结束后，超声心动图检测各组小鼠肺动脉加速时间和射血时间比值(pulmonary arterial acceleration time/pulmonary arterial ejection time, PAAT/PET)、右心室游离壁厚度，计算右心室肥厚指数RV/(LV+S)；HE染色观察肺组织病理改变；EVG染色检测弹力纤维增生情况；免疫组织化学检测肺组织α-平滑肌肌动蛋白(α smooth muscle actin, α-SMA)表达；免疫荧光染色检测肺组织巨噬细胞浸润；qPCR检测肺组织中IL-1β、IL-33的表达水平、细胞因子微球检测技术检测IL-10的分泌；Western blotting检测各组肺组织M1型巨噬细胞标志物iNOS和M2型巨噬细胞标志物Arg-1及IL-33/ST2蛋白的改变。结果显示，与常氧对照组相比，低氧导致肺动脉压力增高和肺血管重构，巨噬细胞浸润增加，IL-1β、IL-33细胞因子表达增加(P < 0.05)及IL-33/ST2通路上调(P < 0.05)。MSCs-Exo干预后，PAAT/PET增加(P < 0.05)，右心室游离壁变薄(P < 0.05)，右心室肥厚指数RV/(LV+S)降低(P < 0.05)，肺小血管α-SMA表达降低(P < 0.05)，肺组织中炎症因子IL-1β、IL-33表达降低，IL-10分泌升高(P < 0.05)。此外，MSCs-Exo上调Arg-1，下调iNOS及IL-33/ST2 (P < 0.05)。以上结果提示，人脐带MSCs-Exo可能通过免疫调节作用来缓解低氧性肺动脉高压。

关键词： 低氧性肺动脉高压; 肺血管重塑; 间充质干细胞及外泌体; 免疫调节

Effects of human umbilical cord mesenchymal stem cells (MSCs)-derived exosomes on pulmonary vascular remodeling in hypoxic pulmonary hypertension

LIU Hong^1,2,3, ZHANG Yu-Wei^1,4, ZHANG Qing-Qing^1,2,3, WANG Yu-Xiang^1,2,3, GE Ri-Li^1,2,3, MA Lan^1,2,3,*

¹Research Center for High Altitude Medicine, Qinghai University, Xining 810001, China；²Key Laboratory of High Altitude Medicine of Ministry of Education, Xining 810001, China；³Qinghai Key Laboratory of High Altitude Medicine Application Foundation (Qinghai-Utah Joint Key laboratory of High Altitude Medicine), Xining 810001, China；⁴Department of Public Health, Faculty of Medicine, Qinghai University, Xining 810001, China

Abstract

The present study aimed to investigate the effect of human umbilical cord mesenchymal stem cells (MSCs)-derived exosomes (MSCs-Exo) on mice with hypoxic pulmonary hypertension (HPH). MSCs were isolated and cultured from human umbilical cords under aseptic conditions, and exosomes were extracted from the supernatants and identified. Healthy SPF C57BL/6 mice were randomly divided into three groups: normoxic group, hypoxic group, and hypoxic+MSCs-Exo group. Mice in the hypoxic group and the hypoxic+MSCs-Exo group were maintained for 28 d at an equivalent altitude of 5 000 m in a hypobaric chamber to establish HPH mouse model. The mice in the hypoxic+MSCs-Exo group were injected with MSCs-Exo via tail vein before hypoxia and on days 1, 3, 5 and 9 of hypoxia, and the mice in the other two groups were injected with PBS. At the end of the experiment, echocardiography was performed to detect pulmonary arterial acceleration time/pulmonary arterial ejection time ratio (PAAT/PET), right ventricular free wall thickness, and right ventricular hypertrophy index RV/(LV+S). HE staining was performed to observe the lung tissue morphology. EVG staining was performed to observe elastic fiber hyperplasia. Immunohistochemistry was performed to detect α smooth muscle actin (α-SMA) expression in lung tissue. Immunofluorescence staining was used to detect macrophage infiltration in lung tissue. qPCR was performed to detect IL-1β and IL-33 in lung tissue, and cytometric bead array was performed to detect IL-10 secretion. Western blotting was used to detect the M1 macrophage marker iNOS, M2 macrophage marker Arg-1 and IL-33/ST2 pathway proteins in lung tissues. The results showed that hypoxia increased pulmonary artery pressure and pulmonary vascular remodeling, increased macrophage infiltration, IL-1β and IL-33 expression (P < 0.05) and upregulated the IL-33/ST2 pathway (P < 0.05). Compared with the hypoxic group, MSCs-Exo treatment increased PAAT/PET (P < 0.05), decreased right ventricular free wall thickness (P < 0.05), right ventricular hypertrophy index RV/(LV+S) (P < 0.05), α-SMA expression in small pulmonary vessels (P < 0.05), and inflammatory factors including IL-1β and IL-33 expression in lung tissue, however increased IL-10 secretion (P < 0.05). In addition, MSCs-Exo treatment upregulated Arg-1 and downregulated iNOS and IL-33/ST2 (P < 0.05). The results suggest that MSC-Exo may alleviate HPH through their immunomodulatory effects.

Key words: hypoxic pulmonary hypertension; pulmonary vascular remodelling; mesenchymal stem cells and exosomes; immunomodulation

收稿日期：　　录用日期：

通讯作者：马兰　　E-mail: judyml-325@163.com

DOI: 10.13294/j.aps.2024.0006

引用本文：

刘红, 张雨薇, 张晴晴, 王玉香, 格日力, 马兰. 人脐带间充质干细胞源性外泌体对低氧性肺动脉高压肺血管重构的影响[J]. 生理学报 2024; 76 (1): 33-44.

LIU Hong, ZHANG Yu-Wei, ZHANG Qing-Qing, WANG Yu-Xiang, GE Ri-Li, MA Lan. Effects of human umbilical cord mesenchymal stem cells (MSCs)-derived exosomes on pulmonary vascular remodeling in hypoxic pulmonary hypertension. Acta Physiol Sin 2024; 76 (1): 33-44 (in Chinese with English abstract).