本文主要研究载穿心莲内酯胶原缓释支架对炎症环境下软骨细胞维持表型的作用。通过物理共混结合真空冷冻干燥的方法制备载穿心莲内酯胶原缓释支架，采用环境扫描电子显微镜（ESEM）、真密度仪和紫外可见分光光度计表征载药支架的形貌、开孔率和药物释放情况。将经分离、扩增培养后的兔关节软骨细胞接种于载穿心莲内酯胶原缓释支架上，在常规培养液条件下培养 3 d、白细胞介素 1β（IL-1β）模拟的炎症环境下培养 7 d，期间采用阿尔玛蓝（Alamar Blue）、荧光素二乙酸（FDA）染色、实时定量聚合酶链式反应（RT-qPCR）等方法研究所制备支架对软骨细胞的增殖和表型的影响。实验结果表明，真空冷冻干燥法制备的胶原支架孔连通性良好，平均孔径为（120.7 ± 17.8）μm，开孔率可达 96%，该载药支架可在两周内实现药物的持续释放。Alamar Blue 实验结果表明，质量分数为 2.22% 的载穿心莲内酯胶原缓释支架可显著抑制软骨细胞增殖，而其它载药浓度的胶原缓释支架对炎症环境下软骨细胞的增殖无显著影响。FDA 染色结果表明，质量分数为 0.44% 的载穿心莲内酯胶原缓释支架可降低 IL-1β 对软骨细胞形貌的改变，且该浓度载药支架可显著抑制基质金属蛋白酶-1（MMP-1）和基质金属蛋白酶-13（MMP-13）的转录，促进基质金属蛋白酶抑制剂-1（TIMP-1）、软骨细胞外基质相关基因 II 型胶原（COL II）和聚集蛋白聚糖（Aggrecan）的转录，提高 COL II/I 型胶原（COL I）的比值。基于以上研究结果，表明质量分数为 0.44% 的载穿心莲内酯胶原缓释支架可以抑制 IL-1β 模拟的炎症环境下软骨细胞内不利于维持表型的基因（如：MMP-1、MMP-13）的转录，促进利于维持表型的基因（如：COL II、Aggrecan、TIMP-1）的转录，从而增强炎症环境下的软骨细胞维持其表型的能力，有望用于关节炎软骨损伤修复。
The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1), collagen II (COL II), aggrecan (Aggrecan) and the ratio of COL II/ collagen I(COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.
引用本文： 徐丽女, 刘海蓉, 戴瑶, 李永生, 陈微. 载穿心莲内酯胶原缓释支架促进体外炎症环境下软骨细胞表型维持的研究. 生物医学工程学杂志, 2018, 35(6): 905-913. doi: 10.7507/1001-5515.201804025 复制