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间充质干细胞移植对脓毒症小鼠肠道损伤的保护作用与机制的初步探讨

邓捷 黄尚卿 刘江涛 李薇 曾佳容 史惠卿 孙红玉

邓捷, 黄尚卿, 刘江涛, 等. 间充质干细胞移植对脓毒症小鼠肠道损伤的保护作用与机制的初步探讨[J]. 四川大学学报(医学版), 2023, 54(3): 565-573. doi: 10.12182/20230560508
引用本文: 邓捷, 黄尚卿, 刘江涛, 等. 间充质干细胞移植对脓毒症小鼠肠道损伤的保护作用与机制的初步探讨[J]. 四川大学学报(医学版), 2023, 54(3): 565-573. doi: 10.12182/20230560508
DENG Jie, HUANG Shang-qing, LIU Jiang-tao, et al. Protective Effect of Mesenchymal Stem Cell Transplantation on Intestinal Injury in Septic Mice and Its Mechanism[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCES), 2023, 54(3): 565-573. doi: 10.12182/20230560508
Citation: DENG Jie, HUANG Shang-qing, LIU Jiang-tao, et al. Protective Effect of Mesenchymal Stem Cell Transplantation on Intestinal Injury in Septic Mice and Its Mechanism[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCES), 2023, 54(3): 565-573. doi: 10.12182/20230560508

间充质干细胞移植对脓毒症小鼠肠道损伤的保护作用与机制的初步探讨

doi: 10.12182/20230560508
基金项目: 国家自然科学基金面上项目(No. 81772001)和西部战区总医院军事医学联合攻关项目(No. 2019LH04)资助
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    E-mail:shongyu2008@163.com

Protective Effect of Mesenchymal Stem Cell Transplantation on Intestinal Injury in Septic Mice and Its Mechanism

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  • 摘要:   目的  探讨胎盘间充质干细胞(placental-derived mesenchymal stem cells, P-MSCs)移植对脓毒症小鼠肠道损伤的保护作用与机制。  方法  将24只小鼠随机均分为3组:假手术(Sham)组、脓毒症(CLP)组与胎盘间充质干细胞治疗(CLP+P-MSCs)组。假手术组小鼠仅开腹游离盲肠后还纳,其余2组采用盲肠结扎穿孔法(CLP)构建脓毒症模型。Sham组和CLP组均于术后1 h经尾静脉注射0.9%NaCl 0.1 mL,CLP+P-MSCs组于术后1 h输注P-MSCs 2×105个。P-MSCs移植24 h后收集各组小鼠肠道和血液样本。肠道组织经HE染色后进行病理评估,酶联免疫吸附法对血清D-乳酸、二胺氧化酶(DAO)、内毒素、IL-1β、TNF-α、IL-6、IL-10、TGF-β浓度进行检测,实时荧光聚合酶链式反应法对小肠组织中相关炎性因子基因表达水平进行测定。蛋白免疫印迹法测定肠道中ZO-1与occludin蛋白的表达情况,免疫组化法对小肠巨噬细胞的浸润程度进行检测,免疫荧光法对巨噬细胞的极化情况进行检测。  结果  外源性移植的P-MSCs可在脓毒症小鼠损伤肠道中定植。相比CLP组,CLP+P-MSCs组肠道损伤明显减轻;血清D-乳酸、DAO、内毒素、IL-1β、IL-6、TNF-α浓度降低(P<0.05),IL-10、TGF-β升高(P<0.05);肠道组织中IL-1βTNF-αIL-6基因表达水平降低(P<0.05),IL-10、TGF-β升高(P<0.05);肠道中ZO-1与occludin蛋白表达量也升高(P<0.05)。同时,CLP+P-MSCs组肠道组织中巨噬细胞分布显著减少,巨噬细胞有向M2型极化的趋势。  结论  外源性P-MSCs移植可显著减轻脓毒症肠损伤小鼠的炎性损伤、改善肠屏障功能。减少巨噬细胞的浸润、促使巨噬细胞由M1型向M2型极化可能是其减轻炎症反应的机制之一。
  • 图  1  P-MSCs流式细胞仪检测结果

    Figure  1.  P-MSCs flow cytometry results

    A: CD90; B: CD73; C: CD14; D: CD34; E: CD19; F: CD45; G: CD105; H: HLA-DR.

    图  2  P-MSCs分化鉴定

    Figure  2.  P-MSCs differentiation identification

    A: before P-MSCs osteoblast differentiation and staining; B: P-MSCs osteoblast differentiation staining; C: before P-MSCs adipoblast differentiation and staining; D: P-MSCs adipoblast differentiation staining.

    图  3  各组小鼠小肠组织病理损伤

    Figure  3.  Pathological injury of the small intestine of the mice in each group

    * P<0.05, *** P<0.001; n=5.

    图  4  小鼠血清炎性指标

    Figure  4.  Findings for the serum inflammatory indices of the mice

    * P<0.05, ** P<0.01; n=5.

    图  5  小鼠小肠组织炎性因子基因表达

    Figure  5.  Gene expression of inflammatory factors in the small intestine of the mice

    * P<0.05, ** P<0.01; n=5.

    图  6  小鼠血清D-乳酸、DAO和ET检测

    Figure  6.  Determination of serum D-lactic acid, DAO, and ET in mice

    * P<0.05, ** P<0.01; n=5.

    图  7  小肠组织 ZO-1与occludin蛋白的Western blot检测

    Figure  7.  Examination of ZO-1 and occludin proteins in small intestine by Western blot

    * P<0.05, ** P<0.01, *** P<0.001; n=5.

    图  8  P-MSCs在小肠组织的定植

    Figure  8.  Colonization of P-MSCs in the small intestinal tissue

    A:distribution of CM-Dil labeled P-MSCs in small intestine (×400); B: statistical analysis of colonization (*** P<0.001; n=4).

    图  9  免疫组化检测小肠组织巨噬细胞浸润情况

    Figure  9.  Determination of infiltration of macrophages in the small intestinal tissues by immunohistochemical staining

    *** P<0.001; n=5.

    图  10  小肠组织中巨噬细胞的极化情况

    Figure  10.  Polarization of macrophages in the small intestinal tissues

    * P<0.05, ** P<0.01, *** P<0.001; n=5.

    表  1  qRT-PCR引物序列

    Table  1.   qRT-PCR primer sequence

    GeneUpstream primer sequenceDownstream primer sequence
    TNF-α 5′-AGAAGTTCCCAAATGGCCTC-3′ 5′-CCACTTGGTGGTTTGCTACG-3′
    IL-1β 5′-GCAACTGTTCCTGAACTCAACT-3′ 5′-ATCTTTTGGGGTCCGTCAACT-3′
    IL-6 5′-TAGTCCTTCCTACCCCAATTTCC-3′ 5′-TTGGTCCTTAGCCACTCCTTC-3′
    IL-10 5′-GTTACTTGGGTTGCCAAG-3′ 5′-TTGATCATCATGTATGCTTC-3′
    TGF-β 5′-AGAGCCCTGGATACCAACTATTG-3′ 5′-TGCGACCCACGTAGTAGACG-3′
    β-actin 5′-ACGGTCAGGTCATCACTATCG-3′ 5′-GGCATAGAGGTCTTTACGGATG-3′
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出版历程
  • 收稿日期:  2022-11-10
  • 修回日期:  2023-04-08
  • 网络出版日期:  2023-05-20
  • 刊出日期:  2023-05-20

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