| Objective:Programmed cell death plays an important role in the intervertebral disc degeneration (IDD) but the role of autophagy and autophagic cell death in the pathogenic mechanisms and specific impact on IDD has not been documented. In this study, we builded a rat annulus fibrosus cells culture system in vitro, to explore the effects of pressure on the rat annulus fibrosus cells autophagy and mitochondrial damage stress, and analyze the possible mechanism and the role of autophagy and autophagic cell death in IVD degeneration.Methods:1. The rat annulus fibrosus cells were isolated, cultured and passaged; their morphological and biological characteristics were observed.The cell phenotypes were identified with the toluidine blue staining, under the Inverted phase contrast microscope.2. The first generations of well grown cells were taken into single cell suspension, and grew evenly in petri dishes or6-well plates. The cells were cultured for1day in Incubator, and then were divided into experimental groups and control groups. The experimental groups were placed in controllable pressure culture device under IMPa pressure and cultured for12h,24h,36h, and48h respectively. The control groups were cultured for48h in37℃,5%CO2incubator.3. Detected mitochondrial membrane potential depolarization level by JC-1fluorescence staining through Flow Cytometer, reflecting the degree of mitochondrial damage; the percentage of red fluorescence reduction was expressed as the reduction of mitochondrial membrane potential.4. MDA, SOD detection kit were used to measure the MDA content and SOD activity in mitochondria, which reflect the mitochondrial oxidative stress levels.5. Observed and compared the number of the dead cells between36h experimental group and the control group under inverted phase contrast microscope.6. Autophagy-related protein Beclin-1,LC3B-Ⅰ and LC3B-Ⅱ expression in cells were detected by Western Blot to reflect the level of autophagy of the cells.Results:1. Established the culture system of the rat annulus fibrosus cells in vitro successfully. The cell shapes were fusiform or polygonal mainly. Primary cells growth was relatively slow, the first generation and second generation cells grew fast, but the third generation cells and later slew down. The cells displayed intense toluidine blue metachromasia, cell nuclei showed vacuoles, typical nuclei showed blue and cytoplasm showed purple mitotic feature might be observed under Inverted phase contrast microscope.2. The percentage of red fluorescence reduction of JC-1fluorescence staining were greater as the pressurized culture time extended (0h-48h) in both the control groups and the experimental groups, which indicated that mitochondrial damage became worse due to the prolonged pressing time.3. With pressurized cultured time prolonging (0h-48h) in control and experimental groups, the MDA content became higher, SOD activity showed lower, suggesting that mitochondrial oxidative stress levels increased with increasing pressurized cultured time.4. The dead cell number of36h experimental groups increased significantly compared with the control group under the Inverted phase contrast microscope.5. Beclin-1expression levels were higher and the ratio LC3B-I/LC3B-II was smaller as pressurized cultured time was prolonged (0h-48h), which indicated the level of autophagy increased with increasing pressurized cultured time.Conclusion:The rat annulus fibrosus cells were cultured successfully in vitro; phenotypic identification of the cells was complied with annulus fibrosus cells characteristics. Pressure promotes rat annulus fibrosus cells mitochondrial damage stress, cell death ratio and cell autophagy levels. The mitochondrial damage stress may promote annulus fibrous autophagy and autophagic cell death, and involve in the formation of disc degeneration. |
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