Chinese Journal of Tissue Engineering Research ›› 2017, Vol. 21 ›› Issue (13): 1992-1997.doi: 10.3969/j.issn.2095-4344.2017.13.005
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Li Jiang-feng, Ding Shi-chao, Qi Ya-wei, Li Jin, Zeng Guo-fang, Lai Qiao, Liu Li, Zhang Pei-hua
Revised:
2017-02-12
Online:
2017-05-08
Published:
2017-06-09
Contact:
Zhang Pei-hua, M.D., Chief physician, Master’s supervisor, Institute of Plastic Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong Province, China;Liu Li, M.D., Assistant researcher, Institute of Plastic Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong Province, China
About author:
Li Jiang-feng, Studying for master’s degree, Institute of Plastic Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong Province, China
Supported by:
the National Natural Science Foundation of China in 2015, No. 81570260; the Natural Science Foundation of Guangdong Province in 2014, No. 2014A030313535; the Science and Technology Plan of Guangdong Province in 2016, No. 2016A020214018; the Special Financial Funds for Science and Technology in Zhanjiang in 2014, No. 2014C01022; the Doctor Fund of Guangdong Medical University in 2015, No. BJ201510; the Doctoral Initial Fund of the Affiliated Hospital of Guangdong Medical University in 2015, No. BJ201505
CLC Number:
Li Jiang-feng, Ding Shi-chao, Qi Ya-wei, Li Jin, Zeng Guo-fang, Lai Qiao, Liu Li, Zhang Pei-hua. Vitamin C treatment promotes cell proliferation of human adipose-derived stem cells under high glucose conditions[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 1992-1997.
2.1 人脂肪干细胞的形态学特征 第3代人脂肪干细胞形态均一,呈纺锤形,增殖迅速,胞质均匀透明,饱体饱满,折光度良好,胞核成圆形或卵圆形(图1A);高糖环境中培养4周后,人脂肪干细胞形态大小不一,细胞宽大,增殖缓慢,胞质粗糙,折光性较差(图1B)。 2.2 CCK-8检测细胞活力确定维生素C最佳剂量 在图2中,维生素C对于高糖处理后的人脂肪干细胞增殖有促进作用,并呈剂量依赖性,以200 μmol/L时促增殖作用最强,与250 μmol/L比较差异有显著性意义(P < 0.05)。 2.3 CCK-8检测维生素C作用在时间上的变化 从图3中可见,培养24 h时,3组间人脂肪干细胞活力比较差异无显著性意义;培养48 h时,对照组与200 μmol/L维生素C处理组细胞活力比较差异无显著性意义,但两者明显高于0 μmol/L维生素C处理组(P < 0.05);培养72 h时,0 μmol/L维生素C处理组细胞有增殖趋势,但明显低于对照组和200 μmol/L维生素C处理组(P < 0.01),对照组低于200 μmol/L维生素C处理组(P < 0.05)。 2.4 Western Blot检测信号通路相关蛋白表达水平 各组间p-AKT表达量比较差异无显著性意义;200 μmol/L维生素C处理组p-ERK表达明显高于对照组、0 μmol/L维生素C处理组(P < 0.01),见图4。这提示维生素C促进高糖环境下人脂肪干细胞增殖与ERK/MAPK信号通路相关。 2.5 EdU检测细胞增殖 由图5可见,空白对照组EdU阳性率低于对照组(P < 0.01),维生素C组EdU阳性率高于空白对照组(P < 0.01);在2个通路抑制剂组中,加入ERK/MAPK通路抑制剂U0126后,EdU阳性率较维生素C组降低(P < 0.01);加入PI3K/AKT通路抑制剂LY294002后,EdU阳性率与维生素C组无差异,进一步明确了维生素C通过ERK/MAPK信号通路促进高糖环境下人脂肪干细胞的增殖。"
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