Article(id=1203033501986681461, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203033494428541350, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2023.05.0545, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1650816000000, receivedDateStr=2022-04-25, revisedDate=null, revisedDateStr=null, acceptedDate=1656345600000, acceptedDateStr=2022-06-28, onlineDate=1764755138154, onlineDateStr=2025-12-03, pubDate=1685203200000, pubDateStr=2023-05-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764755138154, onlineIssueDateStr=2025-12-03, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764755138154, creator=13701087609, updateTime=1764755138154, updator=13701087609, issue=Issue{id=1203033494428541350, tenantId=1146029695717560320, journalId=1189873630562394117, year='2023', volume='48', issue='5', pageStart='489', pageEnd='626', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1764755136353, creator=13701087609, updateTime=1764756085669, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1203037476202967229, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203033494428541350, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1203037476202967230, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203033494428541350, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=545, endPage=551, ext={EN=ArticleExt(id=1203033502334808717, articleId=1203033501986681461, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Effect of mesenchymal stem cells on inflammatory factors and T-lymphocyte subsets in naturally aged sepsis rats, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the effects of mesenchymal stem cells (MSCs) on inflammatory factors and T-lymphocyte subsets in naturally aged sepsis rats. Methods Ninety naturally aged SD rats (21 months old) were divided into 3 groups according to the random number table method: 36 rats in the model group, 36 rats in the treatment group, and 18 rats in the sham group. In each group, animals were then randomly divided equally into three sub-groups. The rats were severed and executed after 6-h, 24-h, and 72-h anesthesia for modeling or treatment, respectively. The sepsis model was established by the cecum ligation perforation (CLP) method. The sham group was operated as the model group, except that the cecum ligation and perforation method was not used. After 1-hour of modeling, the rats in the treatment group were injected with umbilical cord MSCs through the tail vein, and the rats in the model group and the sham group were injected with the same volume of saline through the tail vein. The 72-hour survival rate of rats in each group was observed, and the levels of interleukin (IL)-1β, IL-6, IL-10, IL-17A, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β were measured by Aimplex method. The spleens of rats in each group were stained with TUNEL and the apoptosis of spleen lymphocytes was observed by laser confocal microscopy; the ratio of CD4+/CD8+ T cells in each group was analyzed by flow cytometry. Results The rats in the sham group showed no abnormalities during the observation period, and the survival rate at 72-hour after surgery was 100.00%. The rats in the model group showed symptoms such as vertical hair, loss of appetite, and depression after surgery, while these symptoms were improved in the treated rats, and the survival rate at 72-hour after CLP surgery was significantly higher in the treatment group than in the model group (66.67% vs.41.67%, P<0.01). Compared with the sham group, the model group showed increased levels of IL-1β, IL-6, and IL-17A, blurred spleen structure, increased proportion of apoptotic lymphocytes, and decreased proportion of CD4+/CD8+ T lymphocytes in peripheral blood and spleen, all with statistically significant differences (P<0.05). Compared with the model group, the treatment group showed reduced levels of IL-1β, IL-6 and IL-17A, increased levels of IL-10, clear spleen structure, significantly reduced lymphocyte apoptosis, and increased proportions of CD4+/CD8+ T lymphocytes in peripheral blood and spleen, all with statistically significant differences (P<0.05). Conclusions MSCs were able to significantly improve the survival rate of naturally aged sepsis rats, suppress excessive inflammatory responses, reduce lymphocyte apoptosis, and improve the immune function of the body, thus exerting a protective effect on naturally aged sepsis rats.

, correspAuthors=Hong-Jun Kang, authorNote=null, correspAuthorsNote=
* E-mail:
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目的 探讨间充质干细胞(MSCs)对自然衰老脓毒症大鼠炎性因子及T淋巴细胞亚群的影响。方法 将90只自然衰老的SD大鼠(21月龄)按随机数字表法分为3组,其中模型组36只,治疗组36只,假手术组18只,各组大鼠再随机平均分为3组,分别在造模或治疗后6、24和72 h麻醉断头处死取材。采用盲肠结扎穿孔法(CLP)建立脓毒症模型。假手术组除不行盲肠结扎穿孔法外,其他操作同模型组。治疗组大鼠在造模1 h后,经尾静脉注射脐带MSCs,模型组和假手术组在造模1 h后经尾静脉注射等量的生理盐水。观察各组大鼠72 h生存率,通过Aimplex法检测白细胞介素(IL)-1β、IL-6、IL-10、IL-17A、肿瘤坏死因子(TNF)-α及转化生长因子(TGF)-β的水平。对各组大鼠脾组织行TUNEL染色,激光共聚焦显微镜观察脾淋巴细胞凋亡情况;流式细胞术分析各组大鼠CD4+/CD8+ T细胞比值。结果 假手术组大鼠观察期内无异常,术后72 h存活率为100.00%。模型组大鼠术后出现竖毛、食欲不振及精神萎靡等症状,而这些症状在治疗组大鼠中有所缓解,且治疗组CLP术后72 h存活率明显高于模型组(66.67% vs. 41.67%,P<0.01)。与假手术组相比,模型组IL-1β、IL-6和IL-17A等促炎因子水平升高,脾结构模糊、淋巴细胞凋亡比例增加,外周血和脾中CD4+/CD8+ T淋巴细胞比值降低,差异均有统计学意义(P<0.05);与模型组相比,治疗组IL-1β、IL-6和IL-17A等促炎因子水平降低,抗炎因子IL-10水平升高,脾结构清晰、淋巴细胞凋亡明显减少,外周血和脾中CD4+/CD8+ T淋巴细胞比值升高,差异均有统计学意义(P<0.05)。结论 MSCs能够明显提高自然衰老脓毒症大鼠的生存率,抑制过度的炎症反应,减少淋巴细胞凋亡,改善机体免疫功能,从而发挥保护作用。

, correspAuthors=康红军, authorNote=null, correspAuthorsNote=
康红军,E-mail:
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王陆,博士研究生,主要从事脓毒症及热射病等方面的基础研究

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A. 生存曲线;B. 24、72 h生存率;**P<0.01

, figureFileSmall=yNMxDa/M6eGH35LuC7VefA==, figureFileBig=kk4qPMt+LjXv7WrGgipZFg==, tableContent=null), ArticleFig(id=1203033509729366049, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203033501986681461, language=EN, label=Fig. 2, caption=Comparison of inflammatory factor levels in peripheral blood cells in each group of rats ($\bar{x}±s$, n=5), figureFileSmall=tPbbdmda2X57jKQY5CM0aQ==, figureFileBig=mkaUxCSJ6x+Plr68l/3dWg==, tableContent=null), ArticleFig(id=1203033509830029352, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203033501986681461, language=CN, label=图2, caption=各组大鼠外周血细胞炎性因子水平比较($\bar{x}±s$, n=5)

IL-1β. 白细胞介素-1β;IL-6. 白细胞介素-6;IL-17A. 白细胞介素-17A;TNF-α. 肿瘤坏死因子-α;IL-10. 白细胞介素-10;TGF-β.转化生长因子-β;***P<0.001

, figureFileSmall=tPbbdmda2X57jKQY5CM0aQ==, figureFileBig=mkaUxCSJ6x+Plr68l/3dWg==, tableContent=null), ArticleFig(id=1203033509930692653, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203033501986681461, language=EN, label=Fig. 3, caption=TUNEL staining and HE staining of rat spleen in each group, figureFileSmall=FRHaf/Akom/X5LUW+tmS4Q==, figureFileBig=rpv3FEg2uQeYA3JhyqSQBw==, tableContent=null), ArticleFig(id=1203033510039744565, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203033501986681461, language=CN, label=图3, caption=各组大鼠脾组织TUNEL染色及HE染色

A. 脾TUNEL染色 (bar=100 μm);B. 脾20倍视野下凋亡细胞数(n=3);C. 24 h脾HE染色 (bar=100 μm);***P<0.001

, figureFileSmall=FRHaf/Akom/X5LUW+tmS4Q==, figureFileBig=rpv3FEg2uQeYA3JhyqSQBw==, tableContent=null), ArticleFig(id=1203033510152990777, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203033501986681461, language=EN, label=Fig. 4, caption=Comparison of T cell subsets in peripheral blood and spleen of rats in each group (n=5), figureFileSmall=e/66GnO7xyZyteiyE+IQgw==, figureFileBig=c3lVbiG50YGm0xzWpp9sUQ==, tableContent=null), ArticleFig(id=1203033510257848382, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1203033501986681461, language=CN, label=图4, caption=各组大鼠外周血及脾T细胞亚群水平比较(n=5)

A. 外周血与脾T淋巴细胞亚群圈门图;B. 外周血与脾T淋巴细胞亚群比值比较;**P<0.01,***P<0.001

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间充质干细胞对自然衰老脓毒症大鼠炎性因子及T淋巴细胞亚群的影响
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王陆 1, 2 , 邓子辉 3 , 赵妍 2 , 李云 1, 2 , 刘育妍 1, 2 , 杨萌萌 2 , 周飞虎 2 , 康红军 2, *
解放军医学杂志 | 基础研究 2023,48(5): 545-551
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解放军医学杂志 | 基础研究 2023, 48(5): 545-551
间充质干细胞对自然衰老脓毒症大鼠炎性因子及T淋巴细胞亚群的影响
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王陆1, 2, 邓子辉3, 赵妍2, 李云1, 2, 刘育妍1, 2, 杨萌萌2, 周飞虎2, 康红军2, *
作者信息
  • 1解放军医学院,北京 100853
  • 2解放军总医院第一医学中心重症医学科,北京 100853
  • 3解放军总医院研究生院基础医学教研室,北京 100853
  • 王陆,博士研究生,主要从事脓毒症及热射病等方面的基础研究

通讯作者:

康红军,E-mail:
Effect of mesenchymal stem cells on inflammatory factors and T-lymphocyte subsets in naturally aged sepsis rats
Lu Wang1, 2, Zi-Hui Deng3, Yan Zhao2, Yun Li1, 2, Yu-Yan Liu1, 2, Meng-Meng Yang2, Fei-Hu Zhou2, Hong-Jun Kang2, *
Affiliations
  • 1Medical School of Chinese PLA, Beijing 100853, China
  • 2Department of Critical Care Medicine, the First Medical Center of Chinese PLA General Hospital, Beijing 100853, China
  • 3Department of Basic Medicine, Graduate School, Chinese PLA General Hospital, Beijing 100853, China
出版时间: 2023-05-28 doi: 10.11855/j.issn.0577-7402.2023.05.0545
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目的 探讨间充质干细胞(MSCs)对自然衰老脓毒症大鼠炎性因子及T淋巴细胞亚群的影响。方法 将90只自然衰老的SD大鼠(21月龄)按随机数字表法分为3组,其中模型组36只,治疗组36只,假手术组18只,各组大鼠再随机平均分为3组,分别在造模或治疗后6、24和72 h麻醉断头处死取材。采用盲肠结扎穿孔法(CLP)建立脓毒症模型。假手术组除不行盲肠结扎穿孔法外,其他操作同模型组。治疗组大鼠在造模1 h后,经尾静脉注射脐带MSCs,模型组和假手术组在造模1 h后经尾静脉注射等量的生理盐水。观察各组大鼠72 h生存率,通过Aimplex法检测白细胞介素(IL)-1β、IL-6、IL-10、IL-17A、肿瘤坏死因子(TNF)-α及转化生长因子(TGF)-β的水平。对各组大鼠脾组织行TUNEL染色,激光共聚焦显微镜观察脾淋巴细胞凋亡情况;流式细胞术分析各组大鼠CD4+/CD8+ T细胞比值。结果 假手术组大鼠观察期内无异常,术后72 h存活率为100.00%。模型组大鼠术后出现竖毛、食欲不振及精神萎靡等症状,而这些症状在治疗组大鼠中有所缓解,且治疗组CLP术后72 h存活率明显高于模型组(66.67% vs. 41.67%,P<0.01)。与假手术组相比,模型组IL-1β、IL-6和IL-17A等促炎因子水平升高,脾结构模糊、淋巴细胞凋亡比例增加,外周血和脾中CD4+/CD8+ T淋巴细胞比值降低,差异均有统计学意义(P<0.05);与模型组相比,治疗组IL-1β、IL-6和IL-17A等促炎因子水平降低,抗炎因子IL-10水平升高,脾结构清晰、淋巴细胞凋亡明显减少,外周血和脾中CD4+/CD8+ T淋巴细胞比值升高,差异均有统计学意义(P<0.05)。结论 MSCs能够明显提高自然衰老脓毒症大鼠的生存率,抑制过度的炎症反应,减少淋巴细胞凋亡,改善机体免疫功能,从而发挥保护作用。

脓毒症  /  间充质干细胞  /  T淋巴细胞亚群  /  炎症  /  自然衰老大鼠

Objective To investigate the effects of mesenchymal stem cells (MSCs) on inflammatory factors and T-lymphocyte subsets in naturally aged sepsis rats. Methods Ninety naturally aged SD rats (21 months old) were divided into 3 groups according to the random number table method: 36 rats in the model group, 36 rats in the treatment group, and 18 rats in the sham group. In each group, animals were then randomly divided equally into three sub-groups. The rats were severed and executed after 6-h, 24-h, and 72-h anesthesia for modeling or treatment, respectively. The sepsis model was established by the cecum ligation perforation (CLP) method. The sham group was operated as the model group, except that the cecum ligation and perforation method was not used. After 1-hour of modeling, the rats in the treatment group were injected with umbilical cord MSCs through the tail vein, and the rats in the model group and the sham group were injected with the same volume of saline through the tail vein. The 72-hour survival rate of rats in each group was observed, and the levels of interleukin (IL)-1β, IL-6, IL-10, IL-17A, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β were measured by Aimplex method. The spleens of rats in each group were stained with TUNEL and the apoptosis of spleen lymphocytes was observed by laser confocal microscopy; the ratio of CD4+/CD8+ T cells in each group was analyzed by flow cytometry. Results The rats in the sham group showed no abnormalities during the observation period, and the survival rate at 72-hour after surgery was 100.00%. The rats in the model group showed symptoms such as vertical hair, loss of appetite, and depression after surgery, while these symptoms were improved in the treated rats, and the survival rate at 72-hour after CLP surgery was significantly higher in the treatment group than in the model group (66.67% vs.41.67%, P<0.01). Compared with the sham group, the model group showed increased levels of IL-1β, IL-6, and IL-17A, blurred spleen structure, increased proportion of apoptotic lymphocytes, and decreased proportion of CD4+/CD8+ T lymphocytes in peripheral blood and spleen, all with statistically significant differences (P<0.05). Compared with the model group, the treatment group showed reduced levels of IL-1β, IL-6 and IL-17A, increased levels of IL-10, clear spleen structure, significantly reduced lymphocyte apoptosis, and increased proportions of CD4+/CD8+ T lymphocytes in peripheral blood and spleen, all with statistically significant differences (P<0.05). Conclusions MSCs were able to significantly improve the survival rate of naturally aged sepsis rats, suppress excessive inflammatory responses, reduce lymphocyte apoptosis, and improve the immune function of the body, thus exerting a protective effect on naturally aged sepsis rats.

sepsis  /  mesenchymal stem cells  /  T-lymphocyte subsets  /  inflammation  /  naturally aged rats
王陆, 邓子辉, 赵妍, 李云, 刘育妍, 杨萌萌, 周飞虎, 康红军. 间充质干细胞对自然衰老脓毒症大鼠炎性因子及T淋巴细胞亚群的影响. 解放军医学杂志, 2023 , 48 (5) : 545 -551 . DOI: 10.11855/j.issn.0577-7402.2023.05.0545
Lu Wang, Zi-Hui Deng, Yan Zhao, Yun Li, Yu-Yan Liu, Meng-Meng Yang, Fei-Hu Zhou, Hong-Jun Kang. Effect of mesenchymal stem cells on inflammatory factors and T-lymphocyte subsets in naturally aged sepsis rats[J]. Medical Journal of Chinese People’s Liberation Army, 2023 , 48 (5) : 545 -551 . DOI: 10.11855/j.issn.0577-7402.2023.05.0545
脓毒症是由宿主对炎症反应失调引起的危及生命的器官功能障碍,是重症监护室患者死亡的首要原因[1-2]。全球每年有3 000万例脓毒症患者,病死率达30%~40%[3-4],其中60%以上是大于65岁的老年患者[5]。最新研究发现,高龄是脓毒症患者死亡的独立危险因素,其病死率随着年龄增加而增高[6]。近年来,以抗感染、抗休克及支持治疗为主的综合治疗手段降低了脓毒症的病死率,但病情严重程度却逐年加重[7]。还有研究发现,不管是脓毒症早期的炎性因子风暴,还是后期的免疫功能抑制阶段,免疫系统都发挥着重要作用[8-9]。随着人口老龄化的日益严重,脓毒症的发病率也将持续增高[10]。有研究发现,高龄老年患者(85岁以上)的脓毒症发病率是成年患者(18~64岁)的31倍,是老年患者(65~84岁)的3倍[11]。因此,研究高龄脓毒症的免疫机制,对于早期控制炎性因子风暴,调节机体免疫状态,进而改善患者的生存率及长期预后,具有重要的科学价值及临床意义。
间充质干细胞(mesenchymal stem cells,MSCs)可与多种类型的免疫细胞相互作用,调节后者的代谢、迁移、细胞毒性及炎症反应等功能[12]。目前已有研究将MSCs用于治疗脓毒症,发现其可减轻全身炎症反应,降低器官损伤水平[13],提高脓毒症动物的生存率[14],但其机制相对复杂,仍有很多问题尚未解决。目前全世界已注册10余项利用MSCs治疗脓毒症的临床试验,其中有3项Ⅰ期临床试验结果已公布,均证实了一定剂量MSCs治疗脓毒症患者的安全性[15-17]。目前研究纳入的实验动物均是年轻的动物,少有将MSCs应用于自然衰老脓毒症动物的研究报道,这与脓毒症患者大多数为老年患者这一临床现状不符。本研究采用自然衰老的SD大鼠建立脓毒症模型,观察MSCs改善老龄脓毒症大鼠生存率及免疫功能的效果,明确MSCs对老龄脓毒症大鼠早期炎性因子风暴的抑制作用及对T细胞的保护作用,以期为其临床应用奠定基础。
21月龄雄性SD大鼠90只,体重(801±79) g,购自斯贝福(北京)生物技术有限公司[实验动物生产许可证号:SCXK(京)2019-0010]。大鼠饲养于SPF级屏障环境中,湿度55%,温度20~26 ℃,12 h/12 h光/暗循环。本实验流程通过解放军总医院实验动物伦理委员会批准(批准号:2017-X13-10)。按随机数字表法将90只SD大鼠分为3组,其中模型组36只,治疗组36只,假手术组18只,各组大鼠再随机平均分为3组,分别在造模或治疗后6 h、24 h及72 h麻醉断头处死取材。
采用盲肠结扎穿孔术(cecum ligation and puncture,CLP)建立脓毒症模型,腹腔注射戊巴比妥钠(50 mg/kg)麻醉,开腹后使用无菌4-0线在盲肠1/2处结扎,选取22G针头在结扎处远端贯穿盲肠1次,挤出少许肠内容物,将盲肠还纳腹腔。假手术组大鼠行开腹手术后,仅翻动盲肠,不进行盲肠结扎及针刺穿孔。治疗组大鼠在造模1 h后,经尾静脉注射5×106个脐带MSCs,模型组及假手术组在造模1 h后经尾静脉注射等量生理盐水。
人脐带来源于解放军总医院第一医学中心健康的顺产产妇,提供者无慢性传染性疾病、血液系统疾病及其他病史。人脐带分离、培养、扩增MSCs至生长良好的第3代,制备成1×106个细胞/ml的制剂,经鉴定后符合国际标准。均与孕妇签署知情同意书,且已获得解放军总医院伦理委员会批准(批准号:2013-107-01)。
造模或治疗完成后,分别于6 h、12 h、18 h、24 h、36 h、48 h及72 h观察各组大鼠生存情况,计算生存率。
从每组中随机选取5只大鼠,进行如下研究。手术后6 h、24 h及72 h分别处死各组大鼠,取血,通过Aimplex检测白细胞介素(interleukin, IL)-1β、IL-6、IL-10、IL-17A、肿瘤坏死因子(tumor necrosis factor,TNF)-α及转化生长因子(transforming growth factor,TGF)-β的水平。
将脾组织置于4%多聚甲醛中固定24 h,冲洗后脱水,清洗组织后置于包埋盒中,将组织块修整后切片(5 μm)。PBST洗3次后,每张玻片上加125 μl TUNEL工作液,使其全部覆盖样本,37 ℃避光孵育1 h。PBST洗3次,DAPI染核并滴加抗荧光淬灭剂,使用激光共聚焦显微镜观察。
将各组大鼠脾脏研磨,制备脾脏组织悬液。取外周血和脾脏组织悬液各1 ml,分别加入CD3-APC、CD4-FITC、CD8-PE、CD45-PreCP各10 μl,避光孵育15 min后,加入500 μl红细胞裂解液,避光放置15 min,离心后上机测定CD4+、CD8+ T细胞比例及CD4+/CD8+ T细胞比值。
采用SPSS 22.0软件进行统计分析。计量资料以$\bar{x}±s$表示,行方差齐性检验。若方差齐,多组间比较采用单因素方差分析,组间进一步两两比较采用LSD-t检验;若方差不齐,采用Welch近似F检验,组间进一步两两比较采用Dunnett's T3法。应用GraphPad Prism软件8.0版进行统计图绘制。采用Kaplan-Meier法分析生存率,统计方法为Log-rank(Mantel-Cox)检验。P<0.05为差异有统计学意义。
在观察时间内,假手术组大鼠未出现死亡,生存率为100.00%。模型组大鼠在造模完成后6 h内即出现死亡,24 h生存率为58.33%(14/24),72 h生存率为41.67%(5/12)。治疗组大鼠出现死亡的时间整体较模型组延后,其24 h生存率为79.17%(19/24),72 h生存率为66.67%(8/12)。模型组与治疗组术后72 h生存率比较,差异有统计学意义(Log-rank χ2=9.872,P<0.01,图1)。
与假手术组比较,模型组术后6、24及72 h的IL-1β、IL-6、IL-17A及TNF-α水平均明显升高(P<0.001),TGF-β水平降低(P<0.05),但IL-10水平比较差异无统计学意义(P>0.05);与模型组比较,治疗组术后6、24及72 h的IL-1β、IL-6、IL-17A及TNF-α水平均明显降低(P<0.001),IL-10、TGF-β水平升高(P<0.001)。本研究还观察了各组大鼠6、24及72 h外周血炎性因子水平的变化,其中假手术组所有炎性因子水平在各时间点的差异均无统计学意义(P>0.05);模型组IL-1β、IL-6及IL-17A在24 h达高峰,72 h有所降低,TNF-α水平在72 h内持续升高,IL-10及TGF-β的水平比较差异均无统计学意义(P>0.05);治疗组IL-1β及TGF-β水平持续降低,IL-6、TNF-α及IL-10在72 h内持续升高,IL-17A水平在24 h达到最低点(图2)。
与假手术组比较,模型组大鼠脾中凋亡细胞数明显增加,从发病早期即开始出现细胞大量凋亡,并持续存在。治疗组大鼠脾中凋亡细胞数明显低于模型组,与假手术组接近,未出现明显的细胞凋亡现象(图3A、B)。在24 h对各组大鼠脾组织行HE染色,发现假手术组大鼠脾组织结构清晰,白髓与红髓分布规则整齐;模型组大鼠脾结构模糊,红髓充血明显,出现淋巴细胞聚集和炎性浸润;治疗组大鼠结构轮廓较为清晰,红髓充血及淋巴细胞浸润明显改善(图3C)。
流式细胞检测结果显示,与假手术组比较,模型组外周血CD4+/CD8+ T细胞比值在24 h明显降低(P<0.001),脾脏CD4+/CD8+ T细胞比值在6 h、24 h及72 h均明显升高(P<0.01);与模型组比较,治疗组外周血CD4+/CD8+ T细胞比值在6 h及24 h均明显升高(P<0.01),脾脏CD4+/CD8+ T细胞比值在6 h及72 h明显升高(P<0.01,图4)。
脓毒症是由宿主对炎症反应失调引起的危及生命的器官功能障碍[1],发病早期即出现“炎性因子风暴”,剧烈的炎症反应是其最早也是最重要的病理生理特点。早期强烈的炎症反应会造成免疫细胞与免疫因子的大量消耗,这也是脓毒症后期免疫功能抑制的重要原因[18]。目前,衰老相关研究大多采用D-半乳糖诱导动物早衰模型,该模型鼠虽然某些指标接近自然衰老鼠,但在免疫学、行为学等方面与自然衰老鼠存在较大差异[19]。因此,本研究采用自然衰老的SD大鼠,通过CLP建立脓毒症动物模型,从生存率、炎性因子水平、脾淋巴细胞凋亡程度及T细胞亚群等方面评价MSCs对老龄脓毒症动物的治疗效果,旨在为下一步研究提供良好的模型。
很多研究证实,应用MSCs治疗脓毒症不仅能够提高实验动物的生存率,还能改善重要器官的功能。临床上脓毒症患者病死率高,同样CLP脓毒症动物的7 d病死率达到70%[20],28 d病死率达到80%[12],而经过MSCs治疗后,生存率均有明显提高,肺脏、肝脏及脾脏等重要脏器功能也有明显改善,器官病理损伤明显减轻[21]。本研究发现,MSCs可明显提高老龄脓毒症大鼠的生存率,改善其一般状况,与上述研究的结论一致。
细胞因子风暴的本质是由各种刺激引起的过度免疫反应,是脓毒症早期最重要的病理生理特点,也是其造成多器官功能障碍及长期免疫抑制的主要原因[22]。正常情况下,机体通过分泌抗炎因子来调节炎症反应的程度,清除有害物质,维持细胞内稳态[23-24]。但是,当这个平衡被破坏时,早期反应性细胞因子进一步触发细胞因子的级联瀑布反应,造成包括IL-6、IL-12及巨噬细胞炎性蛋白-1α在内的大量炎性因子激活并释放,形成失控的全身性炎症反应[25-26]。在成年脓毒症动物研究中已经发现,MSCs可降低血清中的炎性因子水平[27-28]。本研究也发现,MSCs降低了老龄脓毒症大鼠外周血IL-1β、IL-6及TNF-α等促炎细胞因子的水平,升高了IL-10、TGF-β等抗炎细胞因子的水平,从而在一定程度上避免了细胞因子风暴的发生及发展。
脾作为最大的外周免疫器官,聚集了大量的淋巴细胞,是机体发生特异性免疫应答的重要部位。脓毒症导致B淋巴细胞及效应性T细胞凋亡增加,造成了机体对后续感染的免疫麻痹[29]。目前研究证实,脓毒症会造成脾中大量淋巴细胞凋亡,且脾细胞及淋巴细胞的凋亡数量与炎症的严重程度及患者预后密切相关[30]。有研究发现在脓毒症患者中,28 d死亡组患者的淋巴细胞凋亡率明显高于28 d存活组[31]。本研究通过TUNEL染色发现,老龄脓毒症大鼠脾中淋巴细胞凋亡比例明显升高,经过MSCs治疗后,淋巴细胞凋亡数量明显减少,表明MSCs可抑制脓毒症造成的淋巴细胞凋亡,也说明MSCs对于改善免疫功能紊乱及长期的免疫抑制有积极作用。为进一步研究免疫功能的变化,本研究通过流式细胞术检测外周血与脾中T细胞亚群的变化情况,发现治疗组大鼠外周血与脾中CD4+/CD8+T细胞比值均高于模型组,一方面可能是脓毒症引起急性炎症反应激活大量的细胞毒性CD8+ T细胞,继而造成该细胞相对百分比升高,另一方面也可能与脓毒症引起T淋巴细胞凋亡有关。CD4+/CD8+ T细胞比值通常用来提示机体免疫功能,经过MSCs治疗后,这一比值明显升高,表明MSCs能够改善老龄脓毒症大鼠的免疫功能。
综上所述,本研究结果表明,MSCs可明显提高老龄脓毒症大鼠的生存率,抑制全身炎症反应,减少脓毒症引起的脾脏淋巴细胞凋亡,通过调节T细胞亚群改善机体免疫功能,证实了MSCs对老龄脓毒症大鼠的保护作用,为后续研究提供了重要基础。
  • 国家自然科学基金(81671966)
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2023年第48卷第5期
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doi: 10.11855/j.issn.0577-7402.2023.05.0545
  • 接收时间:2022-04-25
  • 首发时间:2025-12-03
  • 出版时间:2023-05-28
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  • 收稿日期:2022-04-25
  • 录用日期:2022-06-28
基金
National Natural Science Foundation of China(81671966)
国家自然科学基金(81671966)
作者信息
    1解放军医学院,北京 100853
    2解放军总医院第一医学中心重症医学科,北京 100853
    3解放军总医院研究生院基础医学教研室,北京 100853

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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