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

The effects of hypoxia and nutrition deprivation on cartilage endplate stem cells and the related mechanism

中文关键词:  软骨终板干细胞  缺氧和营养缺乏  凋亡  B细胞淋巴瘤/白血病-2（Bcl-2）腺病毒E1B 19-kDa结合蛋白  Bcl-2关联x蛋白  Bcl-2关联k蛋白

英文关键词:Cartilage endplate-derived stem cells  Hypoxia and nutrition deprivation  Apoptosis  BNIP3  Bax  Bak

中文摘要:

  【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-kDa结合蛋白3（Bcl-2/adenovirus E1B 19-kDa-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）%（P<0.05）；实验组的细胞增殖活性明显降低，为对照组的56.18%（P<0.05）。与对照组相比，实验组BNIP3、Bax、Bak蛋白的表达均显著上调（P<0.05）。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（P<0.05）。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。

英文摘要:

  【Abstract】 Objectives: To study the effects of hypoxia and nutrition deprivation on the apoptosis of cartilage endplate stem cells(CESCs) as well as the role of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3) in the process. Methods: The CESCs were separated from the clinically acquired human intervertebral disc endplate cartilage tissue, and the third generation cells were cultured under normoxia with full medium(control group) or hypoxia with nutrition deprivation condition(experimental group) for 48h. Then, the cell apoptosis rate was detected by flow cytometry, the cell viability was detected by CCK-8, and the protein expressions of BNIP3, Bax, Bak and hypoxia-inducible factor-1(HIF-1α) were detected by Western Blot. Furthermore, BNIP3 small interfering RNA(siRNA) was used to knock down the BNIP3 expression in CESCs, and the interference-negative control group was treated with control siRNA. Thereafter, cells were treated as described previously and the apoptosis rate, cell viability and the expressions of BNIP3, Bax and Bak were detected again. Results: The CESCs obtained from 5 clinical specimens were identified by stem cell markers. The CD44, CD73, CD90 and CD105 were positive while the CD34, CD45, CD11b, CD19 and HLA-DR were negative, suggesting that the CESCs had the characteristics of stem cells. The apoptosis rate of the experimental group was (29.12±0.65)%, which was significantly higher than that of the control group(14.87±2.03)%(P<0.05). The cell viabilty in the experimental group was notably lower than that in the control group, which was about 56.18% of the control group(P<0.05). Compared with the control group, the expressions of BNIP3, Bax and Bak in the experimental group were significantly up-regulated(P<0.05). Knockdown of BNIP3 by RNA interference attenuated the increase of cell apoptosis and decrease of cell viability in CESCs caused by hypoxia and nutrition deprivation. Additionally, hypoxia and nutrition deprivation-induced expressions of BNIP3, Bax and Bak were also inhibited. Conclusions: Hypoxia and nutrition deprivation may induce CESCs apopotosis via up-regulating the expressions of BNIP3, Bax and Bak.