[Proliferation effect of ligamentum flavum cells induced by transforming growth factor β and its effect on connective tissue growth factor].

Objective: To investigate the effect of transforming growth factor β (TGF-β ) 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 , 50 ng/mL CTGF, 3 ng/mL TGF-β +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 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 ( ) value of each generation of cells increased gradually, and the value of the same generation of cells at each time point was significantly different ( <0.05), there was no significant difference in value between the cells of each generation at the same time point ( 0.05). After cultured for 24 hours, MTT assay showed that the value of cells in groups A and B was significantly higher than that of group E ( <0.05). After adding CTGF neutralizing antibody, the value of cells in groups C and D decreased, but it was still higher than that of group E ( <0.05). There were also significant differences among groups A, C and groups B, D ( <0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E ( <0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E ( <0.05), and the difference between groups A, C and groups B, D was also significant ( <0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E ( <0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E ( <0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E ( <0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E ( >0.05); 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 ( <0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E ( >0.05).

Conclusion: TGF-β can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β can synergistically promote proliferation of ligamentum flavum cells through CTGF.