A Preliminary Study IL-1β Human Intervertebral Disc Nucleus Pulposus Cells To Apoptosis

Backgroud. Intervertebral disc(IVD)is the key structure in the function unit of spinal columnmotion. Degenerative disc disease(DDD)has become a common disease, which significantly impact on the quality of life nowadays. Intervertebral disc degeneration is a continuous, complex, multifactorial process include abnormal stress, trauma, nutritional disturbance, inflammatory reaction, growth factor alteration, protease activation and genetic factor et al. However,the real mechanism of disc degeneration is not fully understood. In recent years, the detection of IVD cell apoptosis in degenerative disc has provided a new sight into the pathology of IVD degeneration. IVD cell apoptosis may play an important role in the process of intervertebral disc degeneration. Some researchers have optimistically predicted that inhibition of IVD cell apoptosis in degenerative disc should be a way to prevent intervertebral disc from degeneration. Inflammatory factors such as IL-1β, IL16, IL6, IL8, IL-1a, IL12, and TNF-a et al may play great roles in the apoptosis of IVD cell. It was reported that IL-1β may induce the apoptosis of IVD cells. The research of apoptosis of NP cells induced by IL-1β may help with the treatment of Degenerative disc disease.Objective. To investigate the effect of IL-1β、IL-1a、IL-6on NP cells from patients of Intervertebral disc degeneration, especially the effect of IL-1β on the Apoptosis of NP cells which various in concentration and time. And Confirm that human nucleus pulposus cells in apoptosis induced by IL-1β.Materials and methods.IVDs were obtained from patients with disc herniation undergoing discectomy. The discs were divided into AF and NP tissues, and NP cells were cultured invitro.(1) Phenotype of NP cells were verified.(2) Use CCK-8to observe the effect of IL-1β on the nucleus pulposus cells in vitro, in the concentration of Ong/mL,25ng/mL,50ng/mL,100ng/mL at the age of culture of Oh,24h,48h,72h, and compare it with IL-1a, IL-6under similar conditions.(3) Observe the apoptosis of NP cells induced by IL-1β with Annexin V-FITC Apoptosis Detection Kit and Mitochondrial membrane potential assay kit with JC-1, and.calculate the apoptosis index with the flow cytometry. Confirm the expression of cleaved caspase3with western blot.Results.(1) NP cells grew in monolayer with a polygonal structure. Collagen-Ⅱ expression was positive.(2)Without FBS,50ng/ml of IL-1β already had great toxicity to the NP cells at48h. And10%FBS can inhibit the apopsis induced by IL-1β(3) The apoptosis of NP cells induced by IL-1β was observed, and confirmed by cleaved caspase3.Conclusions.(1) NP cells grew in monolayer with a polygonal structure. Collagen-Ⅱ expression was positive.(2) IL-1β had great toxicity to the NP cells with no FBS in Certain concentration at48h, which IL-la, IL-6might not. And10%FBS can inhibit the apopsis induced by IL-1β.(3) The apoptosis of NP cells induced by IL-1β was observed, and confirmed by cleaved capase3.