生长分化因子-11对高糖诱导的内皮祖细胞损伤的保护作用及机制  

Protective effect of growth differentiation factor-11 on endothelial progenitor cells cultured with high glucose and its possible mechanism

作　　者：张佳佳[1] 向光大[1] 李欢[1] 朱彪[1] 王力 郭孛 董靖[1] 刘敏[1] 向林[1] Zhang Jiajia;Xiang Guangda;Li Huan;Zhu Biao;Wang Li;Guo Bei;Dong Jing;Liu Min;Xiang Lin(Department of Endocrinology,Wuhan General Hospital of Guangzhou Military,Wuhan 430070,China)

机构地区：[1]广州军区武汉总医院内分泌科,430070

出　　处：《中华糖尿病杂志》2019年第4期270-275,共6页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(81370896、81570730).

摘　　要：目的探讨生长分化因子-11(GDF-11)对高糖诱导的大鼠骨髓来源内皮祖细胞(BM-EPC)增殖与凋亡的影响及可能机制。方法在体外不同葡萄糖浓度下培养BM-EPC,分别加入重组GDF-11蛋白(rGDF-11)、转化生长因子β(TGF-β)受体Ⅰ抑制剂SB431542等干预因素。根据实验目的及干预因素不同将细胞随机分组为:正常对照组、高糖组、高糖+rGDF-11组、高糖+rGDF-11+SB431542组。采用MTT测定增殖功能;膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶(Annexin Ⅴ-FITC/PI)双标记检测细胞凋亡率,Western blot检测细胞内Bcl-2、Bax,以及活性半胱天冬酶(Cleaved-caspase)3等凋亡蛋白表达水平。免疫荧光染色法检测各组细胞p-Smad2/3表达水平。多组间比较采用单因素方差分析,组间多重比较选用最小显著差异t检验。结果高糖可明显减弱增殖功能(吸光度值0.23±0.03比0.12±0.01,t=12.98,P<0.01),降低Bcl-2/Bax比例,升高凋亡关键酶Cleaved-caspase 3,细胞凋亡率增加[分别为0.45±0.09比1.21±0.23、0.28±0.02比0.07±0.00、(18.63±3.02)%比(2.82±0.46)%,t=3.04、-68.06、-7.60,均P<0.05]。而rGDF-11干预可上调Bcl-2/Bax比例,减少Cleaved-caspase 3表达,抑制细胞凋亡。SB431542与细胞共培养后GDF-11的抗凋亡作用均明显减弱(0.56±0.11比1.14±0.07,t=4.33,P<0.05)。高糖显著降低p-Smad2/3水平(荧光强度表示),GDF-11预处理可恢复细胞内p-Smad2/3水平,而SB431542明显抑制GDF-11诱导的细胞p-Smad2/3表达增加(0.89±0.12比1.21±0.12,t=-3.20,P<0.05)。结论 GDF-11可能通过激活TGF-β/Smad2/3通路减少高糖诱导的β细胞凋亡。Objective To investigate the effects of growth differentiation factor-11 (GDF-11) on bone marrow endothelial progenitor cells (BM-EPC) cultured with high glucose and its possible mechanism. Methods BM-EPC were cultured in vitro and divided into the following groups: normal glucose (NG) group, high glucose (HG) group, HG+recombinant GDF-11 (rGDF-11) group and HG+rGDF-11+TGF-β receptor Ⅰ inhibitor SB431542 group. Proliferation was assessed by MTT, and apoptosis of EPC were stained with Annexin Ⅴ-FITC and propidium iodide (PI), and assessed by flow cytometry. The expression of Bcl-2, Bax and Cleaved-caspase 3 were detected by western blot. The phosphorylation of Smad were assessed by immunofluorescence. Data among multiple groups were compared by using one-way ANOVA and the least significant difference t test. Results Compared with the vehicle control group, Bcl-2/Bax ratio decreased, the protein levels of Cleaved-caspase 3 increased and the percentage of apoptotic EPC elevated in those groups cultured with HG [0.45±0.09 vs 1.21±0.23, 0.28±0.02 vs 0.07±0.00,(18.63±3.02)% vs (2.82±0.46)%, t=3.04,-68.06,-7.60, all P<0.05]. However, the effects were blocked by pretreated with rGDF-11. Treatment of EPC with TGF-β receptor Ⅰ inhibitor SB431542 partially abolished the pro-proliferation and anti-apoptotic effects of GDF-11 (0.56±0.11 vs 1.14±0.07, t=4.33, P<0.05). Immunofluorescence analyses showed that Smad2/3 phosphorylation decreased in high glucose culturing. Pre-incubation with rGDF-11 restored p-Smad2/3 expression, however, GDF-11 mediated Smad2/3 activation was partially blocked by SB431542 (0.89±0.12 vs 1.21±0.12, t=-3.20, P<0.05). Conclusion GDF-11 may prevent high glucose-induced EPC apoptosis and improve proliferation through activation of TGF-β/Smad2/3 signaling pathway.

关 键 词：生长分化因子 细胞凋亡 细胞增殖 高糖 内皮祖细胞 

分 类 号：R711[医药卫生—妇产科学]