缺氧和营养缺乏对软骨终板干细胞凋亡的影响及相关机制

发布时间：2018-08-11 19:35

【摘要】：目的 :观察缺氧和营养缺乏对软骨终板干细胞(cartilage endplate-derived stem cells,CESCs)凋亡的影响,探讨B细胞淋巴瘤/白血病-2/腺病毒E1B 19-k Da结合蛋白3(Bcl-2/adenovirus E1B 19-k Da-interacting protein 3,BNIP3)信号通路在其中的作用。方法 :从临床获取退变椎间盘软骨终板标本,分离培养软骨终板细胞,使用琼脂糖筛选获得CESCs并进行干细胞标志物鉴定,将第三代细胞分别在常氧/完全培养基(对照组)与缺氧和营养缺乏(实验组)条件下培养48h,通过流式细胞术检测细胞凋亡率,CCK-8法检测细胞活性,Western Blot检测BNIP3、Bcl-2关联x蛋白(Bax)、Bcl-2关联k蛋白(Bak)和低氧诱导因子1α(HIF-1α)蛋白表达水平。使用BNIP3小干扰RNA(siRNA)干扰CESCs中BNIP3基因,同时设置阴性干扰对照组(Scramble siRNA),同前分组处理后再次检测细胞凋亡率、细胞活性及BNIP3、Bax、Bak蛋白的表达水平。结果:对5例标本获得的CESCs进行了干细胞标志物鉴定,其中细胞表面粘附分子44(CD44)、CD73、CD90和CD105为阳性,CD34、CD45、CD11b、CD19和HLA-DR为阴性,提示CESCs具有干细胞特性。实验组细胞凋亡率为(29.12±0.65)%显著高于对照组的(14.87±2.03)%(P0.05);实验组的细胞增殖活性明显降低,为对照组的56.18%(P0.05)。与对照组相比,实验组BNIP3、Bax、Bak蛋白的表达均显著上调(P0.05)。使用BNIP3 siRNA干扰后,缺氧和营养缺乏引起的细胞凋亡增加和细胞增殖活力下降均被明显抑制;同时,缺氧和营养缺乏导致CESCs中BNIP3、Bax和Bak的蛋白表达升高也被显著逆转(P0.05)。结论:缺氧和营养缺乏能够诱导CESCs发生凋亡,此过程可能是通过上调BNIP3、Bax和Bak蛋白表达而发挥作用的。
[Abstract]:Aim: to investigate the effects of hypoxia and nutritional deficiency on apoptosis of cartilage endplate-derived stem cells in vitro, and to investigate the role of Bcl-2/adenovirus E1B 19-k Da-interacting protein 3 (BNIP3) signaling pathway in B-cell lymphoma / leukemia / adenovirus E1B 19-k Da binding protein 3 (Bcl-2/adenovirus E1B 19-k Da-interacting protein 3). Methods: the specimens of degenerative intervertebral disc cartilage endplate were obtained from clinic, the chondrocytes were isolated and cultured, CESCs was obtained by agarose screening, and stem cell markers were identified. The third generation cells were cultured in normoxic / complete medium (control group) and hypoxia and nutrition deficiency (experimental group) for 48h respectively. The apoptosis rate was detected by flow cytometry and the cell activity was detected by CCK-8 method. Western Blot was used to detect BNIP3Bcl 2 association. The expression levels of (Bak) and HIF-1 伪 protein associated with Bcl-2 in x protein (Bax) were detected. BNIP3 small interference RNA (siRNA) was used to interfere BNIP3 gene in CESCs, and the negative interference control group (Scramble siRNA),) was used to detect the apoptosis rate, cell activity and the expression level of BNIP3 Bax-Bak protein. Results: stem cell markers were identified in 5 cases of CESCs. The results showed that the cell surface adhesion molecule 44 (CD44) CD73 CD90 and CD105 were positive for CD34, CD45, CD11, CD19 and HLA-DR, indicating that CESCs had stem cell characteristics. The apoptosis rate of experimental group (29.12 卤0.65)% was significantly higher than that of control group (14.87 卤2.03)% (P0.05), and the proliferative activity of experimental group was significantly lower than that of control group (56.18%, P0.05). Compared with the control group, the expression of BNIP3 was significantly up-regulated in the experimental group (P0.05). The increase of apoptosis and the decrease of cell proliferation activity induced by hypoxia and nutrition deficiency were significantly inhibited after BNIP3 siRNA interference, and the increase of protein expression of BNIP3, Bax and Bak in CESCs was also significantly reversed by hypoxia and nutritional deficiency (P0.05). Conclusion: hypoxia and nutritional deficiency can induce apoptosis of CESCs, which may be mediated by up-regulating the expression of BNIP3, Bax and Bak proteins.
【作者单位】：第三军医大学附属新桥医院骨科;
【基金】：国家自然科学基金资助项目(编号:81272028)
【分类号】：R681.5

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