Cheng Ao, He Pengjie, Zhang Junyu, Zheng Weiwei, Yang Min
Department of Orthopedics and Traumatology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu Anhui, 241001, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2020 Mar 15;34(3):318-322. doi: 10.7507/1002-1892.201908088.
To investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia .
The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO , 20%O ); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO , 1%O ); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.
HE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A ( <0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B ( <0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D ( >0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B ( <0.05).
Hypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
探讨缺氧条件下大鼠髓核细胞中缺氧诱导因子1α（HIF-1α）与自噬相关分子（Beclin1和LC3B）的表达及相关性。
从健康成年Sprague Dawley大鼠的髓核中提取髓核细胞并传代。第3代细胞经HE染色和Ⅱ型胶原酶免疫荧光染色鉴定后，随机分为4组。A组细胞在正常氧条件（37℃，5%CO₂，20%O₂）下培养8小时；B组细胞在缺氧条件（37℃，5%CO₂，1%O₂）下培养8小时；C组细胞转染HIF-1α小干扰RNA后在缺氧条件下培养8小时；D组细胞在缺氧条件下用自噬抑制剂3-MA培养8小时。采用蛋白质印迹法和实时荧光定量PCR（qRT-PCR）检测各组中HIF-1α及自噬相关分子（Beclin1和LC3B）的表达。
第3代髓核细胞HE染色显示细胞质呈淡粉红色，细胞核呈蓝黑色，Ⅱ型胶原酶免疫荧光染色呈阳性。蛋白质印迹法和qRT-PCR结果显示，B组中HIF-1α、Beclin1和LC3B蛋白及基因的相对表达量显著高于A组（P<0.05）；C组中HIF-1α、Beclin1和LC3B蛋白及基因的相对表达量显著低于B组（P<0.05）。B组和D组之间HIF-1α蛋白及基因的相对表达量差异无统计学意义（P>0.05）；而D组中Beclin1和LC3B蛋白及基因的相对表达量显著低于B组（P<0.05）。
缺氧可诱导大鼠髓核细胞中HIF-1α及自噬相关分子（Beclin1和LC3B）的表达，缺氧条件下大鼠髓核细胞中的HIF-1α与自噬相关分子的表达有关，即下调HIF-1α可显著降低自噬相关分子的表达，而缺氧条件下自噬水平的下调对HIF-1α的表达无或几乎无影响。
