血管内皮生长因子联合碱性成纤维细胞生长因子改善骨髓间充质干细胞复制性衰老  

Vascular endothelial growth factor combined with basic fibroblast growth factor improves replicative senescence of bone marrow mesenchymal stem cells

作　　者：施伟丽 刘珊珊[1,2] 常红波 高海霞[2] 王新洲 秦楠 吴鸿 Shi Weili;Liu Shanshan;Chang Hongbo;Gao Haixia;Wang Xinzhou;Qin Nan;Wu Hong(Second Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou 450002,Henan Province,China;Central Laboratory of Henan Province Hospital of TCM,Zhengzhou 450002,Henan Province,China)

机构地区：[1]河南中医药大学第二临床医学院,河南省郑州市450002 [2]河南省中医院中心实验室,河南省郑州市450002

出　　处：《中国组织工程研究》2024年第31期4958-4963,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年项目(81803771),项目负责人:施伟丽;河南省中医药科学研究专项(2021JDZX2018),项目负责人:施伟丽。

摘　　要：背景:间充质干细胞在体外扩增过程中易衰老,极大地阻碍了其体内外应用,如何改善间充质干细胞的复制性衰老,是组织工程学亟待解决的问题。目的:探讨血管内皮生长因子联合碱性成纤维细胞生长因子能否改善复制性传代导致的骨髓间充质干细胞衰老。方法:全骨髓贴壁法提取大鼠骨髓间充质干细胞,选取第2代细胞为正常对照组,第7代及以后代数细胞为衰老模型组,给予血管内皮生长因子(50μg/L)、碱性成纤维细胞生长因子(10μg/L)及二者联合干预。CCK-8法检测细胞增殖情况,β-半乳糖苷酶活性染色观察细胞衰老情况,鬼笔环肽染色和吉姆萨染色分别观察细胞骨架大小及集落形成能力,qRT-PCR检测衰老相关基因P16、P21和P53表达水平。结果与结论:①血管内皮生长因子联合碱性成纤维细胞生长因子可促进衰老骨髓间充质干细胞的增殖,第9天开始进入平台期,且第9天联合干预组吸光度值较模型组明显升高(P<0.05);②联合干预组细胞表型标记物未见改变,细胞形态由宽大变细长;③与模型组比较,联合干预组β-半乳糖苷酶阳性率显著降低(P<0.01);细胞核个数增加(P<0.001);细胞骨架总面积增加(P<0.01);集落形成能力增强(P<0.05);P16表达水平降低(P<0.01)。以上结果提示:血管内皮生长因子联合碱性成纤维细胞生长因子可改善复制性传代导致的骨髓间充质干细胞衰老,且不改变细胞表型。BACKGROUND:Mesenchymal stem cells are susceptible to senescence during in vitro expansion,which greatly hinders their application in vivo and in vitro.How to improve the replicative senescence of mesenchymal stem cells is an urgent problem to be solved in tissue engineering.OBJECTIVE:To determine whether vascular endothelial growth factor combined with basic fibroblast growth factor can improve the aging of bone marrow mesenchymal stem cells caused by replicative passage.METHODS:Rat bone marrow mesenchymal stem cells were extracted by whole bone marrow adhesion method.Passage 2 cells were selected as normal control group.Passage 7 and later algebraic cells were selected as aging model group.Vascular endothelial growth factor(50μg/L),basic fibroblast growth factor(10μg/L),and their combination were administered.Cell proliferation was detected by CCK-8 assay.Cell senescence was observed byβ-galactosidase activity staining.Cytoskeleton size and colony formation ability were observed by phalloidine staining and Giemsa staining,respectively,and the levels of senescence-related genes P16,P21,and P53 were detected by qRT-PCR.Gene expression levels of P16,P21,and P53 were tested by qRT-PCR.RESULTS AND CONCLUSION:(1)Vascular endothelial growth factor combined with basic fibroblast growth factor could promote the proliferation of aged bone marrow mesenchymal stem cells,which began to enter the plateau stage on day 9,and the absorbance value of the combined intervention group was significantly higher than that of the model group on day 9(P<0.05).(2)The phenotypic markers of the cells in the combined intervention group did not change,and the cell morphology changed from broad to slender.(3)Compared with the model group,the positive rate ofβ-galactosidase was significantly decreased(P<0.01);the number of nuclei increased(P<0.001);the total area of cytoskeleton increased(P<0.01);colony formation ability was enhanced(P<0.05);expression level of P16 was decreased(P<0.01)in the combined intervention group.These results indica

关 键 词：血管内皮生长因子 碱性成纤维细胞生长因子 骨髓间充质干细胞 复制性衰老 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R394.2