The Relationship Between Integrin β1and Tumor Necrosis Factor-α In Osteoblast Apoptosis And Nitric Oxide Metabolizing

Objective: Tumor necrosis factor （TNF）-α is a proinflammatory cytokine. Integrinsβ1is atransmembrance signal molecule in cell membrane. Nitric oxide （NO） is a short-lived freeradical. TNF-α, Integrinsβ1and NO, they have different biological effect in cell action. Wewant to understand the role of TNF-α in the osteoblast activity, apoptosis, Integrinsβ1expression and NO metabolism. And to study the influence of TNF-α, Integrinsβ1inosteoblast apoptosis and NO metabolism.Methods：1. Higher activity of osteoblasts was obtained by using the method of osteoblastprimary culture of neonatal rat calvaria and subculture in vitro.2. TNF-α and α-MEM medium were made up with suspension liquid.Osteoblasts werecultured with0μg/L、10μg/L、50μg/L、100μg/L TNF-α concentration.According to the timeosteoblasts were divided into24h and48h group.3. According to the TNF-α concentration gradient and different time, we investigate theosteoblast activity, alkaline phosphatase activity.4. According to the TNF-α concentration gradient and different time, integrinβ1expression and osteoblast apoptosis were detected by the flow cytometry.According to GraceMethod, we detect NO₂^-concentration which is a product of nitric oxide. NO₂^-concentrationcan indicate NO concentration.5. We use integrin β1blocker to block integrin β1, and according to the TNF-αconcentration gradient to detect osteoblast apoptosis, then to investigate the interactionbetween TNF-α and integrin β1.Results：1.By using the method of osteoblast primary culture, the third passage culturedosteoblast were induced to format mineralized nodules, and Alizarin Bordeaux stainingmethod was used to detect the mineralized nodules. And the third passage cultured osteoblastactivity is able to conduct experiments.2. Along with TNF-α concentration increasing and the culture time extension, theosteoblast activity reduced and osteoblast apoptosis increased.3. When concentration of TNF-α is up to10μg/L, NO₂^-expression is maximum for48h.With the culture time extension, concentration of NO₂^-were be kept high concentration. Ithad significantly difference compared with the control group（P＜0.01）.4. Along with TNF-α concentration increasing and the culture time extension, integrin β1 expression reduced.5. After blocked integrinβ1, with TNF-α suspension liquid concentration increasing,osteoblast apoptosis increased. That had significantly difference compared with unblockedintegrinβ1group（P＜0.01）. And NO expression had no changed compared with unblockedintegrinβ1group.Conclusions：1. TNF-α can reduce the activity of osteoblast and alkaline phosphatase, the effect has timeand dose-dependent.2. TNF-α induce osteoblast apoptosis, the effect has time and dose-dependent.3. TNF-α can promote nitric oxide to metabolism and release, and for48h there occurs amaximum at10μg/L.Integrinβ1has no impaction on nitric oxide supersession in osteoblast.4.Osteoblasts in the cultivation of the metabolic process, integrin β1expression waspositive. High concentration of TNF-α can inhibit integrinβ1to express, this effect has timeand dose-dependent.TNF-α and integrinβ1have antagonism in osteoblast apoptosis.