Effects and mechanisms of human umbilical cord-derived mesenchymal stem cells on the thymus development in nude mice

doi:10.3969/j.issn.2095-4344.2014.32.009

Abstract

Abstract:

BACKGROUND: For autoimmune diseases, it is difficult to effectively solve the lack of immunological tolerance in patients by traditional treatments. Mesenchymal stem cells have the biological functions of tissue and organ regeneration and immune regulation.
OBJECTIVE: To explore the effects and mechanisms of human umbilical cord-derived mesenchymal stem cells on the development of thymus in nude mice.
METHODS: Human umbilical cord-derived mesenchymal stem cells were intraperitoneally injected into BABL/c nude mice at a dose of 2×106 per mouse. We analyzed the maturation and distribution of thymic epithelial cells in the thymus rudiment of nude mice and the thymopoiesis of this newly developed thymus rudiment; furthermore, we explored the therapeutic effects of human umbilical cord-derived mesenchymal stem cells.
RESULTS AND CONCLUSION: Our data showed well-organized cortex-medulla structure and an obvious improvement in the maturation of thymic epithelial cells in the rudiment. We further demonstrated the improved thymopoiesis and the enhanced export of mature T cells with the T regulatory cells increase in peripheral blood.
Furthermore, we found that human umbilical cord-derived mesenchymal stem cells could be engrafted in the thymus and express many cytokines, especially keratinocyte growth factor which is essential for the thymus development. These findings indicate that a new mechanism of human umbilical cord-derived mesenchymal stem cells can provide a proper microenvironment for the reconstitution and functional maturation of thymus in nude mice, and elicit another insight in terms of therapeutic efficacy of human umbilical cord-derived mesenchymal stem cells in many autoimmune diseases.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: umbilical cord, mesenchymal stem cells, thymus gland, T-lymphocytes, regulatory, fibroblast growth factor 7

CLC Number:

R394.2

Wang Li-ming, Wang Li-hua, Li Ming, Wang Qian-yun, Yang Jie, Liu Guang-yang, Cong Xiu-li,Liu Yong-jun. Effects and mechanisms of human umbilical cord-derived mesenchymal stem cells on the thymus development in nude mice[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5132-5139.

Figures/Tables 2

2.1 人脐带源间充质干细胞表型鉴定流式细胞仪检测结果显示，分离培养的间充质干细胞达到国际细胞治疗协会(ISCT)的鉴定标准[23](图1)。 2.2 人脐带源间充质干细胞能够促进裸鼠胸腺残基体积增大 Foxn1基因变异导致胸腺发育异常，但在裸鼠体内的胸腺部位，仍然存在着胸腺样器官，它由紧密的淋巴样组织组成，周围是一层薄薄的纤维囊和囊下界限不清的成熟的脂肪组织。为了确定人脐带源间充质干细胞是否能够促进胸腺发育，实验从大小和组织学两个方面来分析不同治疗时间点的治疗组和未治疗组小鼠胸腺的差别。从外观和质量来看，治疗组和未治疗组无显著性差异。但苏木精-伊红染色显示，治疗组小鼠比未治疗组小鼠的正常胸腺组织学结构区域显著性增大。而且，治疗组较未治疗组小鼠的胸腺组织出现了明显的皮髓质交界结构(图2)。这些数据表明，用人脐带源间充质干细胞治疗6周开始就可见胸腺结构的重塑，并且随着持续反复的治疗，胸腺结构不断向正常胸腺改善。 2.3 人脐带源间充质干细胞治疗促进裸鼠胸腺皮质和髓质结构重建以及胸腺上皮细胞的分化裸鼠的胸腺体积较小，且结构异常，没有明显的皮髓质区域之分，其中的大多数胸腺上皮细胞分化只停留在祖细胞阶段[24-29]。K8K5分别是成熟的皮质上皮细胞和髓质上皮细胞的标志，可用来评价胸腺上皮细胞的定位和组织特征。在正常胸腺中，髓质上皮细胞(medullary TECs，mTECs)组成K5+的髓质区域，周围被K8+的皮质所包绕。本实验中，人脐带源间充质干细胞治疗后6，8，10周的胸腺组织抗角蛋白免疫荧光染色显示，未治疗组裸鼠胸腺上皮细胞散在分布；相反，治疗组胸腺出现清晰的皮质(K5-K8+ TECs)和髓质(K5+K8- TECs)区域。此外，在治疗组胸腺的皮质和髓质区域均出现了紧密的上皮细胞网状结构，皮髓质交界区的结构接近正常胸腺(图3)。这表明人脐带源间充质干细胞能够促进胸腺上皮细胞的分化和增殖，重建裸鼠的胸腺结构。 2.4 人脐带源间充质干细胞增加胸腺生成能力，促进胸腺T细胞的输出研究发现人脐带源间充质干细胞能够明显改善裸鼠胸腺结构的基础上，实验进一步探讨胸腺T细胞的能力是否提高。在正常的胸腺组织，大多数的胸腺细胞是CD4+CD8+双阳的，而在裸鼠，由于胸腺上皮细胞和胸腺祖细胞缺乏相互作用，CD4+CD8+T细胞数量极少。应用人脐带源间充质干细胞后，CD4+CD8+胸腺细胞显著增加，并在髓质区域可见少量成熟的单阳性细胞(图4)。 2.5 人脐带源间充质干细胞增加裸鼠外周血中调节性T细胞数量 Tregs主要产生于胸腺，在成年健康小鼠外周血中，CD4+CD25+Foxp3+Tregs占CD4+T细胞的5%-10%[30]，通过抑制CD4+效应性T细胞的功能，在自身免疫耐受和免疫平衡中发挥着核心作用[31]。已有大量数据表明，缺乏CD4+CD25+Foxp3+Tregs与自身免疫性疾病的发生密切相关[32]。本研究发现，治疗后外周血中Tregs占CD4+T细胞的百分比较未治疗组显著增加(从1.95%平均增加到3.89%，图5)。 2.6 人脐带源间充质干细胞表达细胞因子和生长因子研究表明，脐带源间充质干细胞能够在表面黏附分子的介导下[1]，从血管中迁移到组织中[33]。作者用CM-Dil标记人脐带源间充质干细胞来观察腹腔注射后细胞在胸腺组织中的分布，结果表明，人脐带源间充质干细胞能够迁移并且定植到胸腺，主要存在于胸腺组织的边缘，在胸腺组织中心区域也有散在分布。此外，用real-time PCR检测到人脐带源间充质干细胞能够高表达多种与胸腺发育相关的细胞因子和生长因子，其中包括角质形成细胞生长因子、Ghrelin、白细胞介素15(图6)。"

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