| Osteogenesis imperfecta(OI) as known as brittle bone disease is one kind of inherited connective tissue disorder diseases. The typical clinical manifestations included multiple fractures, skeletal deformities, scoliosis, dentin dysplasia, blue sclera, flabby skin and joints laxity. The incidence of this rare disease is less than one in ten thousands and ninety percent pathogenic mutations occur in COL1A1 and COL1A2. Endoplasmic reticulum stress and cell autophagy triggered by the accumulation of mutant collagen in endoplasmic reticulum and these program enhanced degeneration of mutant collagen and promoted production of normal collagen.Research Objectives To verify the Endoplasmic reticulum stress and cell autophagy exist in OI's primary cells. To explore the effect of ER-phagy on collagen production in Osteogenesis imperfecta primary cells.Research method 1) Collect Normal Human and OI patients' skin, bone tissue samples and clinical information. 2) Culture osteoblast/fibroblast primary cells and extract RNA and protein from cells. 3) Verify Endoplasmic reticulum stress and cell autophagy by RT-q PCR. 4) Verify Endoplasmic reticulum stress and cell autophagy by western-blot. 5) Verify Endoplasmic reticulum stress and cell autophagy by morphologic observation. 6) Optimization method of collagen extraction from osteogenesis imperfecta-based primary cells. 7) Explore the degradation mechanism by pathway inhibitors and agonists. 8) Determine the influence of inhibitors by the analysis of collagen's production.Result1) Tissue processing and cell cultureAfter tissue processing, primary cells cultured in the EMDM with 10% fetal calf serum and 1% antibiotic and adhered within 24 hours.2) Verify of endoplasmic reticulum Compared with control group, ER stress associated genes Bip up-regulated 1.5-2.5 times, GRP94 up-regulated 2.1-3.3 times, ERp72 up-regulated 1.38-2.4 times, Hsp47 up-regulated 1.6-2.7 times in osteogenesis imperfecta cells. Compared with control group, ER stress associated proteins Bip?Grp94 and Erp72 up-regulated 7.56 times?7.34 times and 3.54 times, respectively3) Verify of cell autophagy Compared with control group, autophagy associated genes p62 up-regulated 2.2-2.4 times,LC3 up-regulated 1.7-3.2 times at the genetic level. The quantity of LC3I/LC3 II protein increased in osteogenesis imperfecta cells.4) Cell morphologic observation Obvious Autophagosomes were observed in osteogenesis imperfecta cells.5) Extraction of collagen Results For the collagen extracted from both primary osteoblast cells and its cellular medium, the optimal pepsin digestion concentration was 1mg/m L with the treatment of 5 hours. High amount of collagen extracted from cellular medium of primary osteoblast cells was produced with the combination treatment of 0.1~0.15 m M L-ascorbate and 0.1m M BAPN for 96 hour.6) The effect of autophagy inhibitors/ agonist on osteogenesis imperfecta cells Cell survival rate increased with the affection of autophagy inhibitors and autophagy activators accelerated cell death in osteogenesis imperfecta cells7) The effect of autophagy on procollagen production The experimental results showed that autophagy inhibitors would increase production of collagen and autophagy agonist had reverse effect in osteogenesis imperfecta cells.Colution There were obvious ER stress and cell autophagy in the osteogenesis imperfecta patients' cells with the improvement of modification associated molecular chaperone. Autophagy was activated by ER stress and degraded mutant protein and coped with stress. There are many different kinds of factors participating the regulation of autophagy. It can increase cell survival rate and improve the production of collagen by inhibited autophagy partly. This result can provided a new thought for osteogenesis imperfecta treatment. |
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