Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (16): 2563-2569.doi: 10.3969/j.issn.2095-4344.2014.16.017
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Cui Jing-fu, Xu Yao-zeng, Zhu Shi-jun, Zhu Feng, Fu Wen, Shao Hong-guo, Geng De-chun
Revised:
2014-03-10
Online:
2014-04-16
Published:
2014-04-16
Contact:
Geng De-chun, Associate investigator, Department of Orthopaedics, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
About author:
Cui Jing-fu, Studying for master’s degree, Department of Orthopaedics, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
Supported by:
the Clinical Medicine Specific Fund of Jiangsu Province, No. BL2012004; the National Natural Science Foundation of China, No. 81101399, 81272018, 81372018; the Natural Science Foundation of Jiangsu Province, No. BK2011303; the Innovation Project for Graduate Culture in Jiangsu Province, No. CXZZ13_0835
CLC Number:
Cui Jing-fu, Xu Yao-zeng, Zhu Shi-jun, Zhu Feng, Fu Wen, Shao Hong-guo, Geng De-chun. Icariin inhibits titanium particle-induced inflammatory reaction[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(16): 2563-2569.
2.1 体内实验结果 2.1.1 实验动物一般情况 术后各组小鼠均在1 h内苏醒并自由活动,正常进食,精神状态佳。实验期间小鼠切口愈合佳,无红肿、渗液等不良反应。实验过程中无动物死亡。 2.1.2 淫羊藿苷抑制钛颗粒诱导的炎性细胞浸润 苏木精-伊红染色显示对照组和淫羊藿苷组小鼠颅骨骨膜内细胞总量较少,成纤维细胞较多,炎性细胞较少,骨膜较薄;与对照组相比,钛组小鼠颅骨骨膜增厚明显,骨膜内可见大量细胞浸润,炎性细胞居多,成纤维细胞较少,经淫羊藿苷后情况有所好转(图1)。Paint.NET软件测量结果表明对照组、淫羊藿苷组、钛组、钛+淫羊藿苷组颅骨骨膜厚度为分别为(0.07±0.01),(0.06±0.01),(0.28±0.04),(0.12±0.02) mm。钛组与对照组颅骨骨膜厚度比较差异有显著性意义(P < 0.05),说明钛颗粒的刺激使骨膜炎性增厚明显;钛+淫羊藿苷组与钛组颅骨骨膜厚度相比差异有显著性意义(P < 0.05),说明淫羊藿苷治疗后炎性增厚的骨膜显著变薄;对照组及淫羊藿苷组骨膜厚度相比差异无显著性意义(P > 0.05);淫羊藿苷组与钛+淫羊藿苷组颅骨骨膜厚度相比差异有显著性意义(P < 0.05),这表明淫羊藿苷未能完全抑制钛颗粒诱导的炎症反应。 2.1.3 淫羊藿苷下调肿瘤坏死因子α、白细胞介素1β基因mRNA表达 设定对照组肿瘤坏死因子α、白细胞介素1β mRNA表达量为1,钛组中二者表达量分别升至7.6±0.6和9.8±0.9,两组相比差异有显著性意义(P < 0.05),这表明钛颗粒显著上调肿瘤坏死因子α、白细胞介素1β基因转录水平,钛颗粒的炎症诱导作用在基因层面表现明显;钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA表达量分别降至2.4±0.2和2.1±0.2,与钛组相比差异有显著性意义(P < 0.05),这表明淫羊藿苷抑制肿瘤坏死因子α、白细胞介素1β释放的机制涉及mRNA水平下调,这从基因层面解释了ELISA检测结果;淫羊藿苷组肿瘤坏死因子α、白细胞介素1β基因mRNA表达量分别为0.8±0.1和0.8±0.1,与对照组相比差异无显著性意义(P > 0.05),这表明对于正常小鼠淫羊藿苷不能抑制肿瘤坏死因子α、白细胞介素1β基因表达量(图2A)。 2.1.4 淫羊藿苷抑制肿瘤坏死因子α、白细胞介素1β蛋白的释放 对照组肿瘤坏死因子α、白细胞介素1β蛋白质量浓度分别为(237.1±37.4) ng/L和(133.8±19.6) ng/L;钛颗粒植入后,肿瘤坏死因子α、白细胞介素1β质量浓度在钛组增长明显,分别达到(1447.8±78.2) ng/L和(889.2±45.9) ng/L,与对照组比较差异有显著性意义(P < 0.05),这说明植入的钛颗粒刺激骨组织释放过量的炎症因子;钛+淫羊藿苷组经淫羊藿苷口服治疗后肿瘤坏死因子α、白细胞介素1β蛋白质量浓度下降至(568.8±42.3) ng/L和(279.3±19.4) ng/L,与钛组比较差异有显著性意义(P < 0.05),这表明淫羊藿苷可显著抑制钛颗粒引起的骨组织炎症因子释放;但与淫羊藿苷组相比,钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β蛋白质量浓度仍偏高(P < 0.05),这表明淫羊藿苷的抑制作用是有限的,只是部分削弱了钛颗粒诱导的炎症因子释放但未能彻底消除,尽管抑制作用是不完全的,但炎症因子浓度的减少有利于局部反应的缓解和炎症细胞浸润减少,这与苏木精-伊红染色结果相一致;淫羊藿苷组肿瘤坏死因子α、白细胞介素1β蛋白质量浓度分别为(99.0±26.2) ng/L和(96.0±13.9) ng/L,与对照组比较差异无显著性意义(P > 0.05),这表明在无钛颗粒刺激的情况下,淫羊藿苷对正常小鼠肿瘤坏死因子α、白细胞介素1β炎症因子释放无明显抑制作用(图2B)。 2.2 体外细胞培养实验结果 2.2.1 淫羊藿苷下调钛颗粒诱导的肿瘤坏死因子α、白细胞介素1β mRNA表达 设定对照组RAW264.7细胞中肿瘤坏死因子α、白细胞介素1β mRNA表达量均为1。钛组肿瘤坏死因子α、白细胞介素1β基因转录量分别为4.1±1.0和10.1±2.0,与对照组相比差异有显著性意义(P < 0.05),说明钛颗粒显著上调RAW264.7细胞中肿瘤坏死因子α、白细胞介素1β基因表达量;钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA表达量下调至2.1±0.8和7.0±1.2,与钛组相比差异有显著性意义(P < 0.05),说明淫羊藿苷显著抑制钛颗粒诱导的炎症因子基因表达量;淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA表达量分别为0.9±0.1和0.9±0.1,与对照组相比差异无显著性意义(P > 0.05),这表明无钛颗粒刺激的环境下,淫羊藿苷对RAW264.7细胞肿瘤坏死因子α、白细胞介素1β基因表达量无明显影响,这与体内PCR结果相一致;与淫羊藿苷组相比,钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA水平均显著上调(P < 0.05),这表明钛颗粒刺激环境下淫羊藿苷对炎症因子抑制作用的不完全性(图3A)。 2.2.2 淫羊藿苷抑制钛颗粒诱导的肿瘤坏死因子α、白细胞介素1β蛋白释放 对照组肿瘤坏死因子α、白细胞介素1β蛋白水平分别为(79.0±12.8) ng/L和(66.0±7.8) ng/L。钛组肿瘤坏死因子α、白细胞介素1β蛋白水平为(259.4±17.1) ng/L和(265.8±14.2) ng/L,与对照组相比差异有显著性意义(P < 0.05),说明钛颗粒刺激后炎症因子浓度上升明显;钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β水平分别为(147.2±17.1) ng/L和(132.1±9.2) ng/L,与钛组相比差异有显著性意义(P < 0.05),说明淫羊藿苷干预可显著降低钛颗粒诱导的炎症因子释放;淫羊藿苷组肿瘤坏死因子α、白细胞介素1β蛋白水平分别为(56.0±9.6) ng/L和(47.0±6.3) ng/L,与对照组相比差异无显著性意义(P > 0.05),这说明淫羊藿苷对正常RAW264.7细胞炎症因子释放无明显抑制作用,这一结果与体内ELISA分析结果一致(图3B)。"
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