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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-1β、TNF-α、IL-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。 Abstract:Objective To explore the protective effect of placenta-derived mesenchymal stem cells (P-MSCs) transplantation on intestinal injury in septic mice and its mechanism. Methods A total of 24 mice were randomly assigned to 3 groups, a sham operation group, a sepsis group that underwent cecal ligation and puncture (CLP) procedure, and a group that received CLP and P-MSCs treatment. Hereinafter, the three groups are referred to as the Sham group, the CLP group, and the CLP+P-MSCs group. For the mice in the Sham group, the abdomen was cut open and the cecum was exposed and then placed back in the abdomen. CLP was performed in the other two groups to establish the sepsis model. Mice in the Sham and the CLP groups received 0.1 mL of 0.9% NaCl injection in the tail vein 1 hour after operation, while mice in the CLP+P-MSCs group received 2×105 P-MSCs infusion 1 hour after operation. Intestinal and blood specimens were collected from the mice in each group 24 hours after P-MSCs transplantation. Hematoxylin and eosin (HE) staining of the intestinal tissue was performed for pathological evaluation. The serum concentrations of D-lactic acid, diamine oxidase (DAO), endotoxin, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and transforming growth factor (TGF)-β were determined by enzyme linked immunosorbent assay (ELISA). The gene expression of the relevant inflammatory factors in the small intestinal tissue was determined by real-time fluorescence polymerase chain reaction. The expression of zonula occludens protein-1 (ZO-1) and occludin protein in the intestine was determined by Western blot, the infiltration of intestinal macrophages was determined by immunohistochemical method, and the polarization of macrophages was determined by immunofluorescence. Results The exogenous transplantation of P-MSCs could form colonies in the injured intestines of septic mice. Compared with those of the CLP group, the intestinal injury of the CLP+P-MSCs group was significantly alleviated, the serum concentrations of D-lactic acid, DAO, endotoxin, IL-1β, IL-6, and TNF-α were significantly decreased (P<0.05), while the serum concentrations of IL-10 and TGF-β were significantly increased (P<0.05), the expression levels of IL-1β, TNF-α and IL-6 genes in the intestinal tissue were significantly decreased (P<0.05), while the expression levels of IL-10 and TGF-β genes were significantly increased (P<0.05), and the expression of ZO-1 and occludin proteins in the intestine was also significantly increased (P<0.05). In addition, the distribution of macrophages in the intestinal tissue of the CLP+P-MSCs group decreased significantly and the macrophages showed a tendency for M2 polarization. Conclusion Exogenous transplantation of P-MSCs can significantly reduce inflammatory injury and improve the intestinal barrier function in septic mice with intestinal injury. Reduction in the infiltration of macrophages and promotion of the polarization of macrophages from M1 to M2 may be the mechanisms underlying the reduction of inflammation. -
Key words:
- Sepsis /
- Mesenchymal stem cells /
- Intestinal injury /
- Immune imbalance /
- Macrophage polarization
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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
Gene Upstream primer sequence Downstream 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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