Expression and analysis of hypoxia-inducible factor-1α in congenital microtia chondrocytes
10.3760/cma.j.cn114453-20230222-00038
- VernacularTitle:低氧诱导因子-1α在小耳畸形耳软骨细胞中的表达及差异分析
- Author:
Qiaoli SHANG
1
;
Leren HE
Author Information
1. 中国医学科学院北京协和医学院整形外科医院外耳整形再造一科，北京 100144
- Keywords:
Congenital microtia;
Hypoxia-inducible factor;
Oxygen concentration;
Birth defect
- From:
Chinese Journal of Plastic Surgery
2023;39(4):428-434
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the expression of hypoxia-inducible factor-1α (HIF-1α) in different types of congenital microtia chondrocytes and normal ear chondrocytes, and the relationship with oxygen concentrations.Methods:The experimental samples were from the patients with unilateral congenital microtia admitted to Plastic Surgery Hospital of Chinese Academy of Medical Sciences during September 1st, 2021 to October 31st, 2022, including 6 cases of unilateral lobular type congenital microtia (experimental group 1), 6 cases of conchal type (experimental group 2), and 6 cases of normal ear cartilage (experimental group 3). And auricle chondrocytes of each group were extracted respectively. The chondrocytes of each group were cultured in an incubator with an oxygen concentrations of 21%, 10%, 5%, and 1% respectively. After 12 h, total RNA was collected and the expression of HIF-1α at the mRNA level was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Immunofluorescence staining was used to detect the expression of HIF-1α protein in chondrocytes. Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA was used for comparison between groups, and SNK test was used for pairwise comparison. P<0.05 was considered statistically significant. Results:RT-qPCR results showed the relative expression levels of HIF-1α mRNA in ear chondrocytes under 21%, 10%, 5%, and 1% oxygen concentrations. In experimental group 1 the relative expression levels of HIF-1α mRNA were 0.031 8±0.006 0, 0.043 4±0.001 0, 0.049 5±0.000 9, 0.030 5±0.001 0 respectively; experimental group 2, 0.036 9±0.000 6, 0.047 4±0.000 8, 0.054 1±0.000 4, 0.034 1±0.001 8 respectively; experimental group 3, 0.041 6±0.000 4, 0.055 6±0.000 6, 0.070 1±0.001 9, 0.077 2±0.000 9 respectively; the difference of relative expression of HIF-1α mRNA in the three groups was statistically significant under the condition of 21% and 5% oxygen concentration ( P<0.01). Immunofluorescence staining showed that the HIF-1α protein in experimental groups 1 and 2 was partially located in the nucleus and partially located in the cytoplasm, while in the experimental group 3 it was located in the nucleus at 5% oxygen concentration. Conclusion:HIF-1α is expressed in both types of lobular and conchal congenital microtia and normal ear chondrocytes, and the expression level is negatively correlated with the severity of the deformity. When oxygen concentration varied from 21% to 1%, the changing trend of HIF-1α mRNA in type lobular and type conchal microtia is similar, and the expression level is negatively correlated with the severity of the abnormality, and the peak value appeared at 5% oxygen concentration, decreased at 1% oxygen concentration, but continued to increase in normal ear chondrocytes. At 5% oxygen concentration, the location of HIF-1α protein in the nucleus of chondrocytes with microtia is different from normal chondrocytes.
