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目的:通过研究不同浓度VEGF作用下离体培养小鼠髁突关节软骨的改变,探讨VEGF对离体培养小鼠髁突骨关节炎的直接影响。方法:取4周龄C57雄性小鼠髁突126个,随机分为21小组,每组6个。加入不同浓度(100 ng/m L、500 ng/m L、1μg/m L、2μg/m L)的VEGF进行离体培养。在不同的时间点(1、2、4、7 d)获取样本后,采用H-E染色、番红O快绿复合染色观察样本髁突的形态结构,并通过Mankin评分评价样本软骨改变;采用免疫组织化学方法观察各组样本中VEGFR2、MMP9、MMP13、TRANCE的表达。采用SPSS 23.0软件包对结果进行统计学分析。结果:H-E染色显示,与不作任何处理的空白对照组相比,加入VEGF离体培养的实验组小鼠髁突软骨结构破坏,肥大细胞增生。番红O快绿复合染色显示,与未加入VEGF的对照组相比,实验组小鼠髁突蛋白多糖含量降低,软骨发生退行性改变。对番红O快绿复合染色采用Mankin评分系统评分,结果表明,不同VEGF刺激时间组内,随着浓度的增高,Mankin评分增高,差异显著(P<0.05)。不同VEGF浓度组内,随着刺激时间的增加,Mankin评分增高,差异显著(P<0.05)。免疫组织化学显示,离体培养时加入VEGF的实验组,VEGFR2、MMP9、MMP13、TRANCE表达量较对照组显著增多。结论:在离体培养条件下,VEGF可直接引起小鼠颞下颌关节发生骨关节炎。
OBJECTIVE: To investigate the direct effect of VEGF on osteoarthritis of the condyles in vitro of mice by studying the changes of the articular cartilage of the condyles in vitro cultured with different concentrations of VEGF. Methods: 126 condyles of 4 weeks old C57 male mice were randomly divided into 21 groups with 6 in each group. Different concentrations of VEGF (100 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL) were cultured in vitro. After taking the samples at different time points (1, 2, 4, 7 d), the condyle morphology of the samples was observed by HE staining and Safranin O fast green complex staining, and the cartilage changes were evaluated by Mankin score. The chemical method was used to observe the expression of VEGFR2, MMP9, MMP13 and TRANCE in each sample. The results were statistically analyzed using SPSS 23.0 software package. Results: H-E staining showed that compared with the blank control group without any treatment, the condylar cartilage structure was destroyed and the mast cell hyperplasia was observed in the experiment group treated with VEGF. Rubescens O fast green complex staining showed that compared with the control group without VEGF, the content of the proteoglycan in the experimental group decreased and the cartilage degenerated. Mankin scoring system was used to evaluate Safranin O fast green complex staining. The results showed that Mankin score increased significantly with different concentrations of VEGF (P <0.05). The Mankin score increased with the increase of stimulation time in different VEGF concentration groups (P <0.05). Immunohistochemistry showed that the expression of VEGFR2, MMP9, MMP13 and TRANCE in VEGF-treated group increased significantly when compared with the control group. CONCLUSION: VEGF can directly induce osteoarthritis of temporomandibular joint in mice under in vitro culture conditions.