[Proliferation effect of ligamentum flavum cells induced by transforming growth factor ß 1 and its effect on connective tissue growth factor].
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi
; 33(7): 883-888, 2019 Jul 15.
Article
en Zh
| MEDLINE
| ID: mdl-31298008
ABSTRACT
OBJECTIVE:
To investigate the effect of transforming growth factor ß 1 (TGF-ß 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-ß 1, 50 ng/mL CTGF, 3 ng/mL TGF-ß 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-ß 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 â , collagen type â ¢, and CTGF genes.RESULTS:
The morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type â and vimentin, and some cells expressed collagen type â ¢; 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 ( P<0.05), there was no significant difference in A value between the cells of each generation at the same time point ( P>0.05). 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 ( P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E ( P<0.05). There were also significant differences among groups A, C and groups B, D ( P<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 ( P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E ( P<0.05), and the difference between groups A, C and groups B, D was also significant ( P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type â , and collagen type â ¢ in group A was significantly higher than that in group E ( P<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 ( P<0.05). The mRNA relative expressions of collagen type â and collagen type â ¢ in group B was significantly higher than that in group E ( P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E ( P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type â and collagen type â ¢ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E ( P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E ( P>0.05).CONCLUSION:
TGF-ß 1 can promote CTGF, collagen typeâ , collagen type â ¢ gene level and protein expression in ligamentum flavum cells, and TGF-ß 1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.Palabras clave
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Ligamento Amarillo
/
Proliferación Celular
/
Factor de Crecimiento Transformador beta1
/
Factor de Crecimiento del Tejido Conjuntivo
Tipo de estudio:
Prognostic_studies
Idioma:
Zh
Revista:
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi
Año:
2019
Tipo del documento:
Article