Real-time quantitative PCR analysis of UPR genes in human osteoarthritic chondrocytes specimens treated with tunicamycin compared to the untreated (control) chondrocytes. Homo sapiens

Human osteoarthritic (OA) chondrocytes were obtained from the cartilage of femoral condyles and tibial plateaus from three patients undergoing total knee arthroplasty. The chondrocytes were released from the cartilage, seeded at high density, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic mixture (100 units/ml penicillin base and 100 μg/ml streptomycin base) at 37°C in a humidified atmosphere of 5% CO2/95% air. The chondrocytes from each of the three donors were left untreated (control) and treated with tunicamycin (500ng/ml for 20 hours).Total RNA was extracted from the chondrocytes and processed for qPCR according to the manufacturer's instructions (SA Biosciences RT2 Profiler PCR Array PAHS-089Z). Overall design: qPCR gene expression profiling: chondrocytes from three OA donors were used and treated separately as indicated in the summary. Equal amount total RNA from each donor was used for gene expression analysis. The average of the values obtained from the three treated cells were compared to the average of the three untreated control chondrocytes.

人类骨关节炎（osteoarthritis, OA）软骨细胞取自3例行全膝关节置换术患者的股骨髁与胫骨平台软骨组织。将软骨细胞从软骨组织中解离后，以高密度接种于杜氏改良伊格尔培养基（Dulbecco’s modified Eagle’s medium, DMEM）中，该培养基添加10%热灭活胎牛血清及含100单位/ml青霉素、100μg/ml链霉素的抗生素混合液，置于37℃、5%CO₂与95%空气的饱和湿度环境中培养。
将3名供者来源的软骨细胞分为两组：一组不予处理作为空白对照组，另一组以500ng/ml衣霉素处理20小时。
从软骨细胞中提取总RNA，按照试剂盒制造商的操作说明（SA Biosciences公司RT2 Profiler PCR Array PAHS-089Z）进行实时定量聚合酶链反应（qPCR）检测。
实验整体设计：实时定量PCR基因表达谱分析。本研究使用3名骨关节炎供者来源的软骨细胞，按前述方案分别处理；每名供者提取的总RNA用量一致，均用于基因表达分析。将3例衣霉素处理组软骨细胞的检测值平均值，与3例未处理对照组软骨细胞的检测值平均值进行比较。