Proliferation effect of ligamentum flavum cells induced by transforming growth factor β 1 and its effect on connective tissue growth factor / 中国修复重建外科杂志

ABSTRACT

Objective: To investigate the effect of transforming growth factor β 1 (TGF-β 1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression. Methods: The ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-β 1, 50 ng/mL CTGF, 3 ng/mL TGF-β 1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-β 1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes. Results: The morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance ( A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different ( P0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E ( P0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E ( P0.05). Conclusion: TGF-β 1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β 1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.