TGF-β1Protects Human Intervertebral Disc Nucleus Pulpous Cells From Apoptosis

Objects:There is few research about the impact of TGF-β1on intervertebral disc nucleus pulpous cells apoptosis, even more no research on human nucleus pulpous cells apoptosis. This study establishs the human degenerated nucleus pulpous cell strain in vitro, using hydrogen peroxide to induce apoptosis, and try to explore the effect of TGF-β1on human nucleus pulpous cells apoptosis via immunofluorescence and flow cytometry to provide new theoretical foundation for the treatment of intervertebral disc degeneration.Methods:We extract nucleus pulpous tissue from ten young adults patients of lumber burst fracture proceeding pulpous excision surgery. After isolation and pancreatic enzyme and type II collagenase digestion, purified nucleus pulpous cells are cultured in DMEM/F12medium. While the cells grows well, we make passage and use the second generation of nucleus pulpous cells to experiment. The five total groups are divided into blank control group, TGF-β1treatment group, H2O2treatment group, H2O2+TGF-β1(10ng/ml) treatment group, H2O2+TGF-β1(100ng/ml) treatment group. In the last two groups, TGF-TGF-β1at specific concentration is preadministrated in cultures for5hours and remained in the medium during H2O2treatment. After48hours the cell morphology and apoptosis rate are observed by DAPI staining immunofluorescence and flow cytometry.Results:1. Plenty of nucleus pulpous cells apoptosis are founded after adding H2O2by DAPI staining, and flow cytometry indicates the apoptosis rate is28.5%, proving that H2O2induces human nucleus pulpous cells apoptosis successfully.2. Via flow cytometry,10ng/ml TGF-β1can make the nucleus pulposus cells apoptosis rate28.5%reduce to20.71%, and the difference is statistically significant(P<0.05);100ng/ml TGF-β1can reduce nucleus pulpous cells apoptosis rate to13.06%, compared to10ng/ml TGF-β1group, the difference is statistically significant(P<0.05).Conclusion:1.After induction of human nucleus pulpous cells apoptosis by H2O2, we find that TGF-β1can protect human nucleus pulpous cells from apoptosis through inspection of cell apoptosis morphology and apoptosis rate.And compared to10ng/ml TGF-β1group,100ng/ml TGF-β1inhibits nucleus pulpous cells apoptosis rate greater;2. TGF-β1cannot completely block human nucleus pulpous cells apoptosis.