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  

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

作　　者：叶丽 田川 赵晓娟 陈梦蝶 叶倩倩 李强 廖珠银 李晔 朱向情 阮光萍 何志旭 舒莉萍 潘兴华 Ye Li;Tian Chuan;Zhao Xiaojuan;Chen Mengdie;Ye Qianqian;Li Qiang;Liao Zhuyin;Li Ye;Zhu Xiangqing;Ruan Guangping;He Zhixu;Shu Liping;Pan Xinghua(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;Department of Pediatrics,Affiliated Hospital of Zunyi Medical University,Zunyi 563100,Guizhou Province,China)

机构地区：[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

出　　处：《中国组织工程研究》2025年第19期4000-4010,共11页Chinese Journal of Tissue Engineering Research

基　　金：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。

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,G_(0)/G_(1)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�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 t

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 

分 类 号：R459.9[医药卫生—治疗学] R392.9[医药卫生—临床医学] R392.31