Objective: To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor a (TNF-alpha).
Methods: The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/ F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-alpha, and DMEM/F12 medium containing 10% PBS, 100 ng/mL TNF-alpha, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-alpha group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry.
Results: Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-alpha group had no expression. The cell apoptosis rates of TNF-alpha group, Ad-hIGF-1 group, and control group were 34.24% +/- 4.60%, 6.59% +/- 1.03%, and 0.40% +/- 0.15%, respectively. The early apoptosis rates of TNF-alpha group, Ad-hIGF-1 group, and control group were 22.16% +/- 2.69%, 5.03% +/- 0.96%, and 0.49% +/- 0.05%, respectively; the late cell apoptosis rates were 13.96% +/- 4.86%, 10.68% +/- 3.42%, and 0.29% +/- 0.06%, respectively. Compared with TNF-alpha group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P < 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P < 0.05).
Conclusion: Ad-hIGF-1 could ingibit the apoptosis of nucleus pulposus cells induced by TNF-alpha.