The effect of chondrocyte desdifferentiation on hypoxia and relationship between Collagen Prolyl 4- hydroxylase
10.3760/cma.j.cn121113-20200529-00358
- VernacularTitle:缺氧下软骨细胞失分化过程中胶原蛋白脯氨酰4-羟化酶的作用研究
- Author:
Chengming ZHANG
1
;
Jiangfeng FENG
;
Ziquan YANG
;
Peidong LIU
;
Wenjie XU
Author Information
1. 山西医科大学第二医院骨与软组织损伤修复省重点实验室，太原 030001
- From:
Chinese Journal of Orthopaedics
2020;40(12):778-789
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To observe the effect of hypoxia on the dedifferentiation process of chondrocytes in vitro and explore the role of collagen prolyl 4-hydroxylase (C-P4Hs).Methods:Chondrocytes were treated with COCl 2 for different concentrations, and selecting the optimal COCl 2 concentration for hypoxia inductionwas100 μmol/L, the mouse costal chondrocytes were divided into the normal oxygen group and the hypoxia group, and the indexes of the 3rd generation 0.5-72 h and the 1st, 3rd, 5th and 7th generation at 72 h were detected respectively. The proliferation rate was determined by CCK8 method and cell count, and the dynamic changes of HIF-1α, P4Hα1, P4Hα2 and Col II in each group were detected by RT-qPCR, IF and Western blot. Results:Costal chondrocytes were cultured under different concentration of COCl 2 for 48 h. When COCl 2>150 μmol/L, the proliferation rate ( P<0.05) was significantly decreased. Normal oxygen and hypoxia induced rib chondrocytes for 0-72 h, and RT-qPCR showed significant increases in P4Hα2 and Col II mRNA in hypoxia group ( P<0.05). IF showed that HIF-1α and P4Hα2 accumulated in the nucleus under hypoxia, and P4Hα2 gradually entered the cytoplasm from the nucleus. Westernblot analysis showed that HIF-1α and P4Hα2 protein expressions were significantly increased in hypoxia group ( P<0.05). The expression of Col II protein in hypoxia group ( P<0.05) increased at the induction stage. CCK8 and cell count results showed that the proliferation rate and cell number of each generation in the hypoxic group were significantly increased ( P<0.05), and there was still potential for proliferation when the cells were transferred to the 6-7 generation. RT-qPCR showed that hypoxia group each generation cells P4Hα2, Col II mRNΑ were significantly increased ( P<0.05). Westernblot results showed that HIF-1α, P4Hα2 and Col II protein expressions were increased in each generation of hypoxia group ( P<0.05). ConcIusion:Increased expression of P4Hα2 through hypoxia induced HIF-1α can accelerate post-translational modification of Col II in chondrocytes and increase synthesis and accumulation of Col II. P4Hα2 may be responsible for increased proliferation rate and delayed dedifferentiation of chondrocytes cultured in vitro under hypoxia condition.
