中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (5): 729-734.doi: 10.3969/j.issn.2095-4344.3007

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

针刀治疗胶原诱导性关节炎大鼠滑膜组织Bcl-2/Bax的表达

刘  青，万碧江   

  1. 湖北中医药大学针灸骨伤学院，湖北省武汉市   430061
  • 收稿日期:2020-02-10 修回日期:2020-02-19 接受日期:2020-03-18 出版日期:2021-02-18 发布日期:2020-11-28
  • 通讯作者: 万碧江，副教授，副主任医师，硕士生导师，湖北中医药大学针灸骨伤学院，湖北省武汉市 430061
  • 作者简介:刘青，女，1996年生，湖北省汉川市人，汉族，湖北中医药大学在读硕士，主要从事针灸、针刀治疗疼痛的临床相关研究。
  • 基金资助:
    湖北中医药大学研究生创新能力提升项目(1010040103)

Effect of acupotomy therapy on the expression of Bcl-2/Bax in synovial tissue of collagen-induced arthritis rats

Liu Qing, Wan Bijiang   

  1. College of Acupuncture and Moxibustion and Orthopedics, Hubei University of Traditional Chinese Medicine, Wuhan 430061, Hubei Province, China
  • Received:2020-02-10 Revised:2020-02-19 Accepted:2020-03-18 Online:2021-02-18 Published:2020-11-28
  • Contact: Wan Bijiang, Associate professor, Associate chief physician, Master’s supervisor, College of Acupuncture and Moxibustion and Orthopedics, Hubei University of Traditional Chinese Medicine, Wuhan 430061, Hubei Province, China
  • About author:Liu Qing, Master candidate, College of Acupuncture and Moxibustion and Orthopedics, Hubei University of Traditional Chinese Medicine, Wuhan 430061, Hubei Province, China
  • Supported by:
    Postgraduate Innovation Ability Improvement Project of Hubei University of Traditional Chinese Medicine, No. 1010040103

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摘要：

文题释义：
胶原诱导性关节炎：用牛Ⅱ型胶原蛋白和弗氏不完全佐剂制成的Ⅱ型乳剂进行二次免疫，诱导大鼠产生关节炎。由于其免疫炎症反应、病理变化和疾病表现与人类的类风湿关节炎具有高度相似性，是研究类风湿关节炎较理想的动物模型。
滑膜组织Bcl-2/Bax表达：Bcl-2蛋白家族是参与细胞凋亡的关键调节因子，在Bcl-2蛋白家族中Bcl-2是起主要作用的抗凋亡蛋白，Bax是重要的促凋亡蛋白，Bcl-2/Bax是启动细胞凋亡的“分子开关”，滑膜组织中Bcl-2/Bax比值的增高促进了类风湿关节炎的发生发展。

背景：临床研究表明，针刀治疗能够改善类风湿关节炎患者的症状，减轻关节肿胀，改善受损关节的活动功能，但尚无其具体作用机制的实验研究报道。
目的：观察针刀治疗对胶原诱导性关节炎大鼠滑膜组织Bcl-2、Bax表达的影响。
方法：将40只大鼠随机分为正常对照组10只，造模组30只。造模组大鼠采用二次免疫法制备胶原诱导性关节炎模型，随机选取20只造模成功大鼠随机分为模型组和针刀组。正常组和模型组不予处理；针刀组大鼠于左膝关节周围触及髌内外支持带、膝内外侧副韧带、髌底上缘中点及髌韧带的中点，每次选取上述2点予以针刀治疗，1次/周，共治疗3次。实验过程中监测大鼠一般情况，测量大鼠左后足趾厚度，进行关节炎指数评分；苏木精-伊红染色法观察关节滑膜病理形态；应用实时定量PCR法、免疫荧光技术分别检测滑膜组织Bcl-2、Bax的基因表达水平及免疫荧光强度。实验方案经湖北中医药大学动物实验伦理委员会批准(批准号为00127518)。
结果与结论：①与正常组相比，模型组左后足趾厚度及关节炎指数评分明显增加(P < 0.01，P < 0.01)；与模型组相比，针刀组左后足趾厚度及关节炎指数评分减小(P < 0.05，P < 0.05)；②病理形态上，模型组较正常组滑膜层数增厚，大量炎症细胞浸润，针刀组较模型组滑膜层数减少，炎症细胞浸润减轻；③各组间滑膜组织Bax mRNA表达水平比较，差异无显著性意义(P > 0.05)；针刀组Bcl-2 mRNA、Bcl-2/Bax mRNA表达较模型组明显降低(P < 0.01，P < 0.01)；④与模型组比较，针刀组Bcl-2/Bax荧光强度明显降低(P < 0.01)；⑤结果表明，针刀治疗类风湿关节炎的机制可能与下调滑膜组织中Bcl-2表达，降低Bcl-2/Bax比值有关。
https://orcid.org/0000-0001-6777-8616 (刘青) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 针刀, 关节炎, 类风湿关节炎, 滑膜, 细胞凋亡, 动物, 鼠

Abstract: BACKGROUND: It has been shown that acupotomy therapy can improve the symptoms of rheumatoid arthritis, reduce the swelling of the joints, and improve the function of the damaged joints in clinical practice. However, the specific mechanism has not been reported in animal experiments. 
OBJECTIVE: To observe the effect of acupotomy therapy on the expression of Bcl-2 and Bax in synovial tissue of collagen-induced arthritis rats. 
METHODS: Forty rats were prepared for the experiment. Ten of them were randomly selected as normal controls, and the other 30 rats were used to manufacture collagen-induced arthritis models via secondary immunization. After the modeling, 20 successful models were randomly selected for subsequent experiments and randomly divided into a model group and an acupotomy group, with 10 in each group. Normal group and model group remain untreated. The medial and lateral patellofemoral ligament, the tibial and fibular collateral ligament, the midpoint of the upper margin of the patella and the midpoint of the patellar ligament around the left knee joint were selected as points of acupotomy treatment, two of which were selected for each acupotomy session. Rats in the acupotomy group were treated once a week for 3 continuous weeks. The general condition of experimental rats was monitored daily, thickness of the left posterior toe was measured and arthritis index was evaluated. Hematoxylin-eosin staining was executed to observe the pathological morphology of the synovial tissue. Real-time quantitative PCR and immunofluorescence staining were implemented to measure gene expression and immunofluorescence intensity of Bcl-2 and Bax in synovial tissue, respectively. The study protocol was approved by the Animal Ethic Committee of Hubei University of Traditional Chinese Medicine with an approval No. 00127518.
RESULTS AND CONCLUSION: Compared with the normal group, the thickness of the left posterior toe and arthritis index score of the model group were significantly increased (P < 0.01, P < 0.01). Compared with the model group, the left hind toe thickness and arthritis index score of the acupotomy group were decreased (P < 0.05, P < 0.05). Hematoxylin-eosin staining showed that: pathologically, the synovial layers of the model group were thicker, with infiltration of a large number of inflammatory cells. However, the synovial layers of the acupotomy group were fewer with less infiltration of inflammatory cells. RT-PCR showed that there was no significant difference in the expression of Bax mRNA in the synovial tissue between all the groups (P > 0.05). The expressions of Bcl-2 mRNA and Bcl-2/Bax mRNA were significantly reduced in the acupotomy group compared with the model group (P < 0.01, P < 0.01). Immunofluorescence detection indicated that the fluorescence intensity of Bcl-2/Bax in the acupotomy group was significantly reduced compared with the model group (P < 0.01). To conclude, the down-regulation of Bcl-2 expression and Bcl-2/Bax ratio in the synovial tissue may be one of its mechanisms for improving rheumatoid arthritis.

Key words: acupotomy, arthritis, rheumatoid arthritis, synovium, apoptosis, animal, rat

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