目的探讨BMP-2联合低氧环境诱导BMSCs向软骨表型分化的可行性,并进一步研究其生物学机制。方法取4周龄健康清洁级雌性SD大鼠骨髓采用贴壁法体外培养BMSCs,取第2代细胞根据培养条件不同分为4组:常氧对照组(A组)、常氧加BMP-2诱导液组(B组)、低氧(O2浓度3%)对照组(C组)和低氧加BMP-2诱导液组(D组)。倒置相差显微镜下观察细胞形态变化,培养7、14、21 d阿利新蓝染色检测各组软骨基质糖胺聚糖(glycosaminoglycans,GAG)分泌水平,21 d时Western blot检测细胞内Ⅱ型胶原和低氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)蛋白表达水平,RT-PCR检测成软骨、成骨以及低氧相关基因表达水平。结果诱导培养21 d,D组细胞变为类圆形,细胞密度降低,细胞周边呈陷窝样,基质包裹细胞;A、B、C组均未见上述典型变化。D组阿利新蓝染色明显较其他组深,并随诱导时间延长蓝染加深,21 d时出现成片深染蓝色,其他组各时间点仅见散在少量的淡染蓝色。Western blot检测D组细胞内Ⅱ型胶原蛋白表达水平较其他组显著增高,C、D组HIF-1α蛋白表达水平较A、B组显著增高,差异均有统计学意义(P<0.05)。RT-PCR检测D组成软骨分化相关指标Ⅱ型胶原α1(collagenⅡα1,COL2α1)、聚集蛋白聚糖表达最高,而B组成骨分化相关指标COL1α1、ALP、Runt相关转录因子2表达水平最高,C、D组低氧相关指标HIF-1α较A、B组显著增强,差异均有统计学意义(P<0.05)。结论 BMP-2联合低氧(O2浓度3%)环境可以诱导大鼠BMSCs向软骨分化,并抑制其成骨分化,HIF-1α可能是参与促软骨生成过程中的一个重要信号分子。 OBJECTIVE: To investigate the feasibility of BMP-2 combined with hypoxia to induce the differentiation of BMSCs to cartilage phenotype and to further investigate its biological mechanism. Methods The bone marrow of 4-week-old healthy female Sprague-Dawley rats was cultured in vitro by adherent method. The second passage cells were divided into 4 groups according to the culture conditions: normoxic control group (A group), normoxia plus BMP-2 (Group B), hypoxia (3% O2) control group (group C) and hypoxia plus BMP-2 induction group (group D). The morphological changes of the cells were observed under an inverted phase contrast microscope. The levels of glycosaminoglycans (GAG) in cartilage were detected by Alcian blue staining on day 7, 14 and 21, respectively. Western blot was used to detect the type Ⅱ collagen and low The expression of hypoxia-inducible factor 1α (HIF-1α) protein was detected by RT-PCR. The expression of HIF-1α was detected by RT-PCR. Results After cultured for 21 days, the cells in group D became roundish, the cell density decreased, the periphery of the cells became dimpled, and the matrix wrapped cells. The typical changes were not seen in groups A, B and C. The staining of Aliskiren in group D was deeper than that of other groups, and the blue staining deepened with the prolongation of induction time. The deep blue staining appeared on day 21 and the other groups were scattered with a small amount of lightly stained blue. Western blot showed that the expression of type Ⅱ collagen in group D was significantly higher than that in other groups. The protein expression of HIF-1α in group C and D was significantly higher than that in group A and B (P <0.05). The expression of collagenⅡα1 (COL2α1), a marker of cartilage differentiation, was the highest in D group by RT-PCR, while COL1α1, ALP and Runt-related transcription factor 2 were the highest in group B, C and D The hypoxia-related index HIF-1α in group A and B were significantly increased (P <0.05). Conclusion BMP-2 combined with hypoxia (3% O2 ​​concentration) can induce rat BMSCs to differentiate into cartilage and inhibit osteogenic differentiation. HIF-1α may be an important signaling molecule in the process of promoting cartilage formation.