The expression and function of fibrillin 1 in engineered cartilage tissue constructed with pig auricular chondrocytes
10.3760/cma.j.cn114453-20200201-00023
- VernacularTitle:原纤维蛋白1在猪耳软骨细胞工程化软骨组织中的表达及作用
- Author:
Kexin DONG
1
;
Ning KANG
;
Kexin SUN
;
Xia LIU
;
Haiyue JIANG
Author Information
1. 中国医学科学院北京协和医学院整形外科医院研究中心 100144
- Keywords:
Tissue engineering;
Cartilage;
Fibrillin 1;
Chondrocytes
- From:
Chinese Journal of Plastic Surgery
2020;36(6):664-671
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the expression of fibrillin 1 (FBN1) in engineered cartilage tissue which is constructed with pig auricular chondrocytes, and the effect of overexpression of FBN1 on chondrocyte phenotype and extracellular matrix expression.Methods:Take three Chang Feng hybrid pigs’ unilateral auricle cartilage, extract chondrocytes and expand to the passage 2, then inoculate at a density of 6.0×10 7/ml (200 μl) onto PGA-PLA scaffold materials whose diameter is 10 mm and thickness is 2 mm, then culture in vitro for 5 weeks to construct 9 pieces of chondrocyes and materials mixture tissues; implant 6 pieces into 3 nude mice, 2 pieces each, culture for 10 weeks, construct 6 pieces of engineered cartilage tissue. HE staining was used to detect the morphology of 3 pieces of chondrocyes and materials mixture tissues cultured in vitro for 5 weeks and tissue engineered cartilage after 10 weeks culture in nude mice; take 3 pieces of pig ear cartilage tissue of 2 cm×1 cm size and 3 pieces of engineered cartilage tissues of 1.0 cm×0.5 cm size, extract RNA, and detect the difference of FBN1 mRNA expression by qPCR; immunohistochemical staining was used to detect the difference in protein expression and distribution of FBN1 between 3 cases of the other side pig ear cartilage tissue and engineered cartilage tissue with size of 0.5 cm×0.5 cm; transfected empty vector plasmids and FBN1 overexpression plasmids into the passage 2 generation of pig auricular chondrocytes, and used qPCR to detect the expression changes of genes related to cartilage extracellular matrix, the experiment was repeated three times. The obtained data was analyzed with Graph Pad Prism 6.0 for two-tailed unpaired t test, P<0.05 means the difference has statistically significant. Results:HE staining result showed that a large number of chondrocytes and small cartilage lacuna were seen in chondrocyes and materials mixture tissues after 5 weeks of in vitro culture. After 10 weeks of in vivo cultivation, obvious cartilage lacuna were formed in the engineered cartilage tissues; The relative expression level of FBN1 mRNA in engineered cartilage tissue (0.001 41±0.000 28) was significantly lower than that in normal pig auricular cartilage tissues (0.003 58±0.000 34), and the difference has statistically significant ( t=4.913, P=0.008); FBN1 in normal tissues were distributed in cartilage lacuna and cartilage membrane, while the engineered cartilage tissues were mostly distributed in the periosteum, and the closer to the center of the cartilage tissue, the less. The relative expression of FBN1 mRNA in the FBN1 overexpression groups was 0.018 03±0.000 64, while the empty vector control groups was 0.006 13±0.001 03, the difference have statistically significant ( P<0.001, t=9.708). After overexpressed FBN1, the relative expression levels of cartilage-related genes: SOX9, COL2A1, ACAN in chondrocytes were increased to 1.273±0.071, 1.315±0.104, 1.928±0.280 times, and the difference have statistically significant( t=3.874, 3.311, 3.044; P=0.018, 0.001, 0.038). Conclusions:FBN1 mRNA expression and protein expression in engineered cartilage tissue constructed with pig auricular chondrocytes was significantly lower than normal auricular cartilage tissue; in vitro experiments show that FBN1 promotes the expression of cartilage-related genes such as COL2A1, ACAN in chondrocytes.
