Gong Ming, Huang Sheng, Luo Jiaquan, Huang Baoding, Zhou Zhiyu, Dai Xuejun, Gao Manman, Li Liangping, Zou Xuenong
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2015 Jul;29(7):857-62.
To observe the genes expression of hypoxia inducible factor lα (HIF-1α) and HIF- 2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis.
High density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2% O2) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type II at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type II, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture.
Toluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group; extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type II was positive in cytoplasm; when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t = 8.345, P = 0.001; t = 7.683, P = 0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P < 0.05), but no significant difference was found at 3 weeks between the 2 groups (P > 0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P < 0.01). The mRNA expressions of collagen type II and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P < 0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P < 0.05).
HIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.
通过诱导人骨髓间充质干细胞（hBMSCs）向软骨细胞分化，观察缺氧诱导因子1α（HIF-1α）和HIF-2α的基因表达，为HIF参与软骨形成机制提供基础依据。
通过离心获得hBMSCs高密度细胞团，在缺氧（2% O₂）条件下，分别用含2%胎牛血清的H-DMEM培养基（对照组）和软骨诱导培养基（软骨诱导组）培养3周。培养3周后，采用免疫组织化学染色鉴定细胞外蛋白聚糖和Ⅱ型胶原。培养1周后，应用蛋白质免疫印迹法检测HIF-1α和HIF-2α蛋白水平。培养1、2、3周后，采用实时定量PCR检测HIF-1α、HIF-2α、Sox-9、Ⅱ型胶原、X型胶原和聚集蛋白聚糖的基因表达。
甲苯胺蓝染色显示，对照组细胞核稀疏，软骨诱导组细胞核密集；软骨诱导组细胞外基质染色比对照组深。Ⅱ型胶原免疫组织化学染色显示细胞质呈阳性；与软骨诱导组相比，对照组细胞核稀疏且颜色浅。培养1周后，软骨诱导组HIF-1α和HIF-2α蛋白表达水平显著低于对照组（t = 8.345，P = 0.001；t = 7.683，P = 0.002）。与对照组相比，软骨诱导组HIF-1α mRNA表达在1周时显著下调而在2周时显著上调（P < 0.05），但两组在3周时无显著差异（P > 0.05）。与对照组相比，软骨分化后HIF-2α mRNA表达显著下调，Sox-9 mRNA表达显著上调（P < 0.01）。软骨分化后2周和3周时，Ⅱ型胶原和X型胶原mRNA表达与对照组相比显著上调（P < 0.05）。聚集蛋白聚糖mRNA表达在软骨分化后的各时间点均显著上调（P < 0.05）。
HIF-1α可能参与缺氧条件下hBMSCs的软骨分化，而HIF-2α表达在整个过程中受到抑制，可能对分化有负面影响。
