脓毒症大鼠来源的外泌体对WJ-MSC的免疫调控能力的影响

doi:10.3969/j.issn.1673-0364.2020.03.013

摘要/Abstract

摘要：

目的探究脓毒症大鼠外周血来源外泌体对人脐带华通胶来源间充质干细胞(WJ-MSC)免疫调控能力的影响。

方法采用盲肠穿孔结扎(CLP)方法建立脓毒症大鼠模型,分别提取对照组(假手术组)和实验组(CLP模型组)大鼠外周血的外泌体,鉴定其表面标志物及形态特征,并分别处理WJ-MSC,CCK8检测WJ-MSC的细胞活性,Annexin-V-FITC/PI检测凋亡细胞比例,qPCR检测细胞因子(IL-10、TNF-α)的mRNA水平;分别将两组外泌体处理后的WJ-MSC与LPS刺激后的人单核巨噬细胞(THP-1)共培养,qPCR检测THP-1细胞中细胞因子(IL-6、IL-10、TNF-α、IL-1β)的mRNA水平变化。

结果两组外泌体在表面标志物和形态特征上无明显差异;实验组外泌体不影响WJ-MSC的增殖活性,但可抑制其凋亡,同时能够上调IL-10的mRNA表达,下调TNF-α的mRNA表达;经过两组外泌体处理后的WJ-MSC,均能够降低LPS刺激下THP-1细胞中促炎性细胞因子(IL-1β,TNF-α)的表达,两组无明显差异。

结论脓毒症大鼠外周血来源的外泌体能够上调WJ-MSC中IL-10的表达,下调TNF-α的表达,可增强其免疫调控能力。

关键词: 脓毒症, 人脐带华通胶来源间充质干细胞, 外泌体, 免疫调控

Abstract:

Objective To explore the effect of peripheral blood exosomes from sepsis rats on the immune characteristics of human umbilical cord Wharton's jelly-derived mesenchymal stem cells (WJ-MSC).

Methods The sepsis rat model was established by means of cecal perforation ligation (CLP). Exosomes from peripheral blood of rats in the control group (sham operation group) and experimental group (CLP model group) were extracted to identify their surface markers and morphological characteristics, and pretreated to WJ-MSC. Cell activity of WJ-MSC was detected by CCK8, apoptosis of WJ-MSC was detected by Annexin V-FITC/PI, and qPCR was used to detect the mRNA levels of cytokines (IL-10, TNF-α). The pretreated WJ-MSC was co-cultured with human mononuclear macrophages cells (THP-1) stimulated by LPS, and the mRNA expression of cytokines (IL-6, lL-10, TNF-α, IL-1β) of THP-1 cells was detected by qPCR.

Results There was no significant difference in surface markers and morphological characteristics between the exosomes of experimental and control group. Exosomes of experimental group could not affect the proliferation of WJ-MSC, but inhibited its apoptosis, promoted the mRNA level of IL-10, and reduced the mRNA level of TNF-α. WJ-MSC pretreated by two kinds of exosomes could reduce the expression of pro-inflammatory cytokines (IL-1β,TNF-α) of LPS-stimulated THP-1 cells, but there was no significant difference between the two groups.

Conclusion Exosomes from peripheral blood of sepsis rats can up-regulate the expression of IL-10 in WJ-MSC and down-regulate the expression of TNF-α, which may enhance its immune regulation ability.

Key words: Sepsis, Mesenchymal stem cells derived from human umbilical cord Wharton's jelly, Exosomes, Immunoregulation

中图分类号:

Q813.2

方均燕, 宋阿会, 佟琰, 丁峰, 刘英莉. 脓毒症大鼠来源的外泌体对WJ-MSC的免疫调控能力的影响[J]. 组织工程与重建外科杂志, 2020, 16(3): 223-229.

Junyan FANG, Ahui SONG, Yan TONG, Feng DING, Yingli LIU. Effect of Exosomes Derived from Septic Rats on Immunoregulation of WJ-MSC[J]. Journal of Tissue Engineering and Reconstructive Surgery, 2020, 16(3): 223-229.

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基因 gene	引物序列 primer sequence
IL1-β	Forward:5’- ATGATGGCTTATTACAGTGGCAA -3’
	Reverse: 5’-GTCGGAGATTCGTAGCTGGA-3
TNF-α	Forward:5’-CCTCTCTCTAATCAGCCCTCTG-3’
	Reverse: 5’-GAGGACCTGGGAGTAGATGAG-3
IL-10	Forward:5’-ACGGCGCTGTCATCGATTT-3’
	Reverse: 5’-TAGAGTCGCCACCCTGATGT-3
IL-6	Forward:5’- ACTCACCTCTTCAGAACGAATTG-3’
	Reverse: 5’- CCATCTTTGGAAGGTTCAGGTTG-3