中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (19): 4000-4010.doi: 10.12307/2025.047

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

高活性脐带间充质干细胞干预老年树鼩衰老脾脏的作用与机制

叶  丽1，2，田  川2，赵晓娟2，陈梦蝶2，叶倩倩1，2，李  强2，廖珠银2，李  晔2，朱向情2，阮光萍2，何志旭3，舒莉萍1，潘兴华2   

  1. 1贵州医科大学，基础医学院免疫学教研室，组织工程与干细胞实验中心，贵州省贵阳市   550004；2中国人民解放军联勤保障部队第九二〇医院干细胞与免疫细胞生物医药技术国家地方联合工程实验室，云南省细胞治疗技术转化医学重点实验室，昆明市干细胞与再生医学重点实验室，基础医学实验室，云南省昆明市   650032；3遵义医科大学附属医院儿科学教研室，贵州省遵义市   563100
  • 收稿日期:2024-01-08 接受日期:2024-03-16 出版日期:2025-07-08 发布日期:2024-09-12
  • 通讯作者: 潘兴华，博士，主任医师，中国人民解放军联勤保障部队第九二〇医院干细胞与免疫细胞生物医药技术国家地方联合工程实验室，云南省细胞治疗技术转化医学重点实验室，昆明市干细胞与再生医学重点实验室，基础医学实验室，云南省昆明市 650032； 共同通讯作者：舒莉萍，博士，教授，贵州医科大学，基础医学院免疫学教研室，组织工程与干细胞实验中心，贵州省贵阳市 550004
  • 作者简介:叶丽，女，1998年生，云南省楚雄州人，汉族，贵州医科大学在读硕士，主要从事脐带间充质干细胞抗衰老相关研究。
  • 基金资助:
    云南省科技计划项目重大科技专项(2018ZF007)，项目负责人：潘兴华；云南省基础研究专项面上项目(202301AT070302)，项目负责人：田川

Effect and mechanisms of highly active umbilical cord mesenchymal stem cells on aging spleen in elderly tree shrews

Ye Li1, 2, Tian Chuan2, Zhao Xiaojuan2, Chen Mengdie2, Ye Qianqian1, 2, Li Qiang2, Liao Zhuyin2, Li Ye2, Zhu Xiangqing2, Ruan Guangping2, He Zhixu3, Shu Liping1, Pan Xinghua2   

  1. 1Guizhou Medical University, Department of Immunology of Basic Medical College, Tissue Engineering and Stem Cell Experiment Center, Guiyang 550004, Guizhou Province, China; 2National and Local Joint Engineering Laboratory of Stem Cell and Immune Cell Biomedical Technology, 920 Hospital of China People’s Liberation Army Joint Logistics Support Force, Yunnan Provincial Key Laboratory of Cell Therapy Technology Translational Medicine, Kunming Key Laboratory of Stem Cell and Regenerative Medicine, Basic Medical Laboratory, Kunming 650032, Yunnan Province, China; 3Department of Pediatrics, Affiliated Hospital of Zunyi Medical University, Zunyi 563100, Guizhou Province, China
  • Received:2024-01-08 Accepted:2024-03-16 Online:2025-07-08 Published:2024-09-12
  • Contact: Pan Xinghua, PhD, Chief physician, National and Local Joint Engineering Laboratory of Stem Cell and Immune Cell Biomedical Technology, 920 Hospital of China People’s Liberation Army Joint Logistics Support Force, Yunnan Provincial Key Laboratory of Cell Therapy Technology Translational Medicine, Kunming Key Laboratory of Stem Cell and Regenerative Medicine, Basic Medical Laboratory, Kunming 650032, Yunnan Province, China; Co-corresponding author: Shu Liping, PhD, Professor, Guizhou Medical University, Department of Immunology of Basic Medical College, Tissue Engineering and Stem Cell Experiment Center, Guiyang 550004, Guizhou Province, China
  • About author:Ye Li, Master candidate, Guizhou Medical University, Department of Immunology of Basic Medical College, Tissue Engineering and Stem Cell Experiment Center, Guiyang 550004, Guizhou Province, China; National and Local Joint Engineering Laboratory of Stem Cell and Immune Cell Biomedical Technology, 920 Hospital of China People’s Liberation Army Joint Logistics Support Force, Yunnan Provincial Key Laboratory of Cell Therapy Technology Translational Medicine, Kunming Key Laboratory of Stem Cell and Regenerative Medicine, Basic Medical Laboratory, Kunming 650032, Yunnan Province, China
  • Supported by:
    Major Science and Technology Special Project of Yunnan Provincial Science and Technology Plan Project, No. 2018ZF007 (to PXH); Yunnan Province Basic Research Special General Project, No. 202301AT070302 (to TC)

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摘要：

文题释义：

脾脏：脾脏是人体最大的外周免疫器官，受多种因素干扰而极易发生衰老。衰老影响免疫系统，治疗脾脏衰老将从整体水平延缓衰老。
高活性脐带间充质干细胞：是课题组通过改良传统脐带间充质干细胞培养体系获得的新型脐带干细胞，其在猕猴卵巢衰老和胸腺衰老、SD大鼠骨质疏松症、树鼩多器官功能障碍综合征等疾病模型治疗中显示出较好的效果。

摘要
背景：脾脏具有储血、造血和免疫功能，随着年龄增长，脾脏结构退变、功能衰退引起免疫系统功能受损，进而加速机体衰老进程，而高活性脐带间充质干细胞治疗树鼩脾脏衰老尚未见报道。
目的：探讨高活性脐带间充质干细胞对树鼩脾脏衰老的干预作用及机制。
方法：从剖腹产的新生树鼩脐带组织中分离、培养和获得高活性脐带间充质干细胞，三系分化试剂盒检测成脂、成骨、成软骨分化能力，流式细胞术检测细胞周期和表面标志物。以感染复数值分别为100，120，140，160，180，200的吉凯基因绿色荧光蛋白转染第2代高活性脐带间充质干细胞，筛选最佳转染条件；转染后的第4代高活性脐带间充质干细胞尾静脉输注给老年治疗组树鼩，青年对照组和老年模型组不予特殊处理，治疗4个月时取脾脏组织，苏木精-伊红染色观察脾脏组织结构；β-半乳糖苷酶染色检测衰老相关半乳糖苷酶活性；免疫组织化学染色检测p21和p53蛋白表达水平；Ki67和PCNA免疫荧光染色检测细胞增殖活性；免疫荧光染色检测脾脏自噬蛋白分子Beclin1和APG5L/ATG5表达水平；活性氧荧光染色检测脾脏组织的活性氧含量；CD3免疫荧光染色检测总T淋巴细胞比例变化；酶联免疫吸附实验检测脾脏组织白细胞介素1β和转化生长因子β1分泌水平；DAPI复染细胞核观察绿色荧光蛋白标记的高活性脐带间充质干细胞在脾脏组织的分布情况。

结果与结论：①高活性脐带间充质干细胞呈核小短梭形、鱼群样生长，G0/G1期占比大，具有向成脂、成骨和成软骨分化潜能。②感染复数为140且转染72 h为吉凯基因绿色荧光蛋白标记树鼩高活性脐带间充质干细胞的最佳条件。③与老年模型组相比，老年治疗组树鼩脾脏组织细胞排列紧密，白髓面积增加(P < 0.01)，红白髓界线清晰，生发中心占比无显著差异(P > 0.05)，脾脏组织衰老相关半乳糖苷酶活性水平降低(P < 0.001)，衰老蛋白分子p21和p53表达下调(P < 0.001)，增殖相关分子Ki67和PCNA表达上调(P < 0.001，P < 0.05)，自噬相关分子Beclin1和APG5L/ATG5表达上调(P < 0.001)，活性氧含量降低(P < 0.001)，CD3+ T细胞比例增加(P < 0.05)，衰老相关分泌表型中白细胞介素1β分泌水平降低(P < 0.001)，转化生长因子β1分泌水平无显著差异(P > 0.05)。与青年对照组相比，以上检测指标在老年治疗组中均有显著差异(P < 0.05)。④冰冻组织切片观察可见老年治疗组树鼩脾脏组织中有绿色荧光蛋白标记的绿色荧光细胞。结果表明：静脉输注高活性脐带间充质干细胞可迁移至脾脏组织，抑制活性氧产生，下调衰老相关蛋白分子表达，诱导细胞自噬，促进细胞增殖，降低慢性炎症，进而改善脾脏组织结构和功能。

https://orcid.org/0009-0000-1366-6766 (叶丽) 


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

关键词: 高活性脐带间充质干细胞, 树鼩, 脾脏, 衰老, 免疫, 自噬, 炎症

Abstract: BACKGROUND: Spleen has the functions of blood storage, hematopoiesis, and immunity. With the increase of age, the structural degeneration and functional decline of spleen lead to the impairment of immune system function, thus accelerating the aging process of the body. The treatment of spleen aging in tree shrews with highly active umbilical cord mesenchymal stem cells has not been reported. 
OBJECTIVE: To explore the intervention effect and mechanism of highly active umbilical cord mesenchymal stem cells on spleen aging in tree shrews. 
METHODS: Highly active umbilical cord mesenchymal stem cells were isolated, cultured, and obtained from the umbilical cord tissue of newborn tree shrews by caesarean section. The differentiation abilities of adipogenesis, osteogenesis, and chondrogenesis were detected by three-line differentiation kit. Cell cycle and surface markers were detected by flow cytometry. The second generation of highly active umbilical cord mesenchymal stem cells were transfected with Genechem Green Fluorescent Protein with infection complex values of 100, 120, 140, 160, 180, and 200, respectively, to screen the best transfection conditions. After transfection, the fourth generation of highly active umbilical cord mesenchymal stem cells was injected into the tail vein of tree shrews in the elderly treatment group. The young control group and the aged model group were not given special treatment. After 4 months of treatment, the spleen tissue was taken and the structure of the spleen was observed by hematoxylin-eosin staining. β-Galactosidase staining was used to detect the activity of aging-related galactosidase. Immunohistochemical staining was used to detect the expression levels of p21 and p53 proteins. Ki67 and PCNA immunofluorescence staining was used to detect cell proliferation activity. Immunofluorescence staining was used to detect the expression levels of spleen autophagy protein molecules Beclin 1 and APG5L/ATG5. Reactive oxygen species fluorescence staining was used to detect the content of reactive oxygen species in spleen tissue. CD3 immunofluorescence staining was used to detect the change of the proportion of total T lymphocytes. The secretion levels of interleukin 1β and transforming growth factor β1 in spleen were detected by enzyme linked immunosorbent assay. The distribution of highly active umbilical cord mesenchymal stem cells labeled with green fluorescent protein in spleen tissue was observed by DAPI double staining of nucleus. 
RESULTS AND CONCLUSION: (1) Highly active umbilical cord mesenchymal stem cells grew in a short spindle shape with fish-like growth, with a large proportion of G0/G1 phase, and had the potential to differentiate into adipogenesis, osteogenesis, and chondrogenesis. (2) Multiplicity of infection=140 and transfection for 72 hours were the best conditions for labeling tree shrews highly active umbilical cord mesenchymal stem cells with Genechem Green Fluorescent Protein. (3) Compared with the aged model group, in the aged treatment group, the spleen tissue cells of tree shrews were arranged closely, and the area of white pulp was increased (P < 0.01); the boundary between red pulp and white pulp was clear; the proportion of germinal centers did not show statistically significant difference (P > 0.05). The activity level of galactosidase related to spleen tissue aging was decreased (P < 0.001), and the expression levels of aging protein molecules p21 and p53 were down-regulated (P < 0.001). The expression levels of proliferation-related molecules Ki67 and PCNA were up-regulated (P < 0.001, P < 0.05); expression levels of autophagy-related molecules Beclin 1 and APG5L/ATG5 were up-regulated (P < 0.001), and the content of reactive oxygen species decreased (P < 0.001), and the proportion of CD3+ T cells increased (P < 0.05). The secretion level of interleukin 1β in the aging-related secretion phenotype decreased (P < 0.001); no significant difference was found in transforming growth factor β1 level (P > 0.05). Compared with the young control group, the above indexes were significantly different in the elderly treatment group (P < 0.05). (4) Green fluorescent cells labeled with green fluorescent protein were observed in spleen tissue of tree shrews the elderly treatment group by frozen tissue section observation. The results show that intravenous infusion of highly active umbilical cord mesenchymal stem cells can migrate to spleen tissue, inhibit the production of reactive oxygen species, down-regulate the expression of aging-related proteins, induce autophagy, promote cell proliferation, reduce chronic inflammation, and then improve the structure and function of spleen tissue. 

Key words: highly active umbilical cord mesenchymal stem cell, tree shrew, spleen, aging, immune, autophagy, inflammation

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