Biological effects of GV115-Caspase3 siRNA on human intervertebral disc nucleus pulpous cells

English Keywords:Nucleus pulposus cell  Lentivirus  Caspase-3  RNAi  Gene therapy

English Abstract:

  【Abstract】 Objectives: To explore the biological effects of GV115-Caspase3 siRNA on human intervertebral disc nucleus pulposus(hIDNP) cells. Methods: The hIDNP cells were collected from 12 patients with traumatic vertebral burst fracture(22-36 years old). The hIDNP cells were isolated and subcultured by using tissue culture technique. The cells of second generation were grouped into GV115-Caspase3 siRNA group, GV115 group and control group. The hIDNP cells were transfected by GV115-Caspase3 siRNA and detected by the immunofluence method. The viability of hIDNP cells was detected by MTT method. The synthesis of collagen type Ⅱ and glycosaminoglycan were detected by Western-Blot and Antonopulos methods. Results: The hIDNP cells were successfully isolated and cultured. After one week, the hIDNP cells reached 80% confluence, which were subcultured and transfected by GV115-Caspase3 siRNA, and the percentage of positive cells was (85.6±1.3)％ under a fluorescence microscope. The Caspase-3 expression in GV115-Caspase3 siRNA group[(19.4±3.2)%] was less than that in the GV115 group[(84.3±9.2)%] or the control group[(83.9±8.7)%], the difference was statistically significant(P＜0.05). The OD value of GV115-Caspase3 siRNA group(1.56±0.21) was higher than that of GV115 group(0.91±0.15) or control group(0.92±0.17), the difference was statistically significant(P＜0.05). The collagen type Ⅱ immunoblotting staining intensity of GV115-Caspase3 siRNA group(1.32±0.09) was higher than that of GV115 group(0.81±0.05) or control group(0.79±0.04). The glycosaminoglycan content of GV115-Caspase3 siRNA group(0.56±0.09) was higher than that of GV115 group(0.35±0.06) or control group(0.34±0.05), the difference was statistically significant. Conclusions: GV115-Caspase3 siRNA can efficiently transfect the hIDNP cells, enhance the cells′ viability and promote the synthesis of extracellular matrix.