摘要
背景:表皮生长因子作为一种辅助生长因子,但是对于骨髓间充质干细胞的生长是否必须尚无定论。目的:建立大鼠骨髓间充质干细胞分离提取以及鉴定的方法,同时探索表皮生长因子对其增殖能力以及迁移能力的影响,同时探索其潜在机制。方法:①采用全骨髓贴壁法,分离纯化大鼠骨髓间充质干细胞,培养至第3代,使用流式细胞仪检测细胞表面抗原CD29,CD45,CD90表达情况。通过成骨和成脂分化诱导,更进一步鉴定骨髓间充质干细胞;②同时通过CCK-8法和克隆形成实验测量表皮生长因子对骨髓间充质干细胞的第3代细胞的增殖能力的影响。同时通过Transwell小室验证表皮生长因子对骨髓间充质干细胞的第3代细胞迁移能力的影响。通过蛋白印迹实验检测PI3K/Akt和核因子κB信号通路相关蛋白表达水平,探索表皮生长因子的作用机制。结果与结论:①原代骨髓间充质干细胞呈多角形和梭形,随着培养时间增加,逐渐呈梭形为主。流式细胞术检测第3代细胞表面CD29,CD90表达呈阳性,CD45表达成阴性。并且对骨髓间充质干细胞的第3代细胞成功进行了成骨和成脂分化诱导;②表皮生长因子能够促进第3代细胞的增殖,且细胞中PI3K/Akt信号通路关键分子p-Akt表达水平的增加,同时其下游信号分子Bax表达水平降低而Bcl-2表达升高。表皮生长因子能够通过调节核因子κB信号通路分子p 65以及IκB的磷酸化水平,同时也增加了基质金属蛋白酶9的表达水平,从而促进大鼠骨髓间充质干细胞的迁移;③结果表明表皮生长因子促进骨髓间充质干细胞的增殖和迁移。
BACKGROUND:Epidermal growth factor is an auxiliary growth factor,but its effect on the growth of bone marrow mesenchymal stem cells(BMSCs)is uncertain.OBJECTIVE:To establish a mature method for isolation,extraction and identification of rat BMSCs,to investigate theeffects of epidermal growth factor(EGF)on the proliferation and migration ability of BMSCs and to explore its potentialmechanisms at the same time.METHODS:Rat BMSCs were isolated and purified using the improved whole bone marrow adherence method.After thecells were subcultured to the third generation,we detected the expression of cells surface antigens CD29,CD45andCD90by flow cytometry.BMSCs were further identified by osteogenic and adipogenic differentiation.Meanwhile,theeffect of EGF on the proliferation of passage3BMSCs was measured by cell counting kit-8and clonogenic assay.Andthe migration of P3cells was verified by Transwell chamber.In addition,we detected the expression of proteins related toPI3K/Akt and nuclear factor-κB signaling pathways by western blot assay.RESULTS AND CONCLUSION:The primary BMSCs were polygonal and spindle-shaped,and then gradually appearedto be spindle-shaped.The results of flow cytometry demonstrated that the passage3cells were positive for CD29andCD90,but negative for CD45.Furthermore,we successfully induced the osteogenic and adipogenic differentiation ofBMSCs in vitro.Additionally,our data demonstrated that EGF promoted the proliferation and migration of passage3BMSCs.The relative expression levels of p-Akt and Bcl-2of PI3K/Akt signaling pathway was up-regulated and theexpression of Bax was down-regulated.At the same time,the relative expression level of phosphorylated p65of nuclearfactor-κB signaling pathway was up-regulated and the expression of phosphorylated inhibitorκB was down-regulated.Moreover,the downstream protein of matrix metalloproteinase-9was up-regulated.Those proteins were related to themigaration of BMSCs.In summary,our results suggest that EGF could promote the proliferation and migration ofBMSCs.
作者
夏荣木
江其昌
杨龙
谢天宏
陆红玲
蒋正举
Xia Rong-mu;Jiang Qi-chang;Yang Long;Xie Tian-hong;Lu Hong-ling;Jiang Zheng-ju(Department of Biochemistry and Molecular Biology, Zunyi Medical College, Zunyi 563000, Guizhou Province, China;Department of Chest Surgery,Affiliated Hospital of Zunyi Medical College, Zunyi 563000, Guizhou Province, China;Ear-Nose-Throat Department, Affiliated Hospital of Zunyi Medical College, Zunyi 563000, Guizhou Province, China)
出处
《中国组织工程研究》
CAS
北大核心
2017年第29期4635-4641,共7页
Chinese Journal of Tissue Engineering Research
基金
贵州省科学技术基金(黔科合J字[2012]2360号)~~
关键词
表皮生长因子
间质干细胞
组织工程
Epidermal Growth Factor
Mesenchymal Stem Cells
Tissue Engineering
