推拿干预膝骨关节炎兔模型
Summary
该协议描述了一种在膝骨关节炎的兔子模型中进行推拿干预的方法。
Abstract
膝骨关节炎（KOA）的主要特征是膝关节软骨和周围软组织的退行性变化。推拿治疗KOA的疗效已得到证实，但其潜在机制有待研究。本研究旨在建立推拿治疗的KOA兔模型，以揭示其潜在机制。为此，将 18 只 6 个月大的正常级雄性新西兰兔随机分为假手术组、模型组和推拿组，每组 6 只兔子。将4%木瓜蛋白酶溶液注入膝关节腔中，建立KOA模型。推拿组采用推拿联合膝关节旋转矫正法干预4周。在假手术组和模型组中仅进行标准抓握和固定。在1周的干预结束时，观察膝关节关节活动度（ROM），并进行软骨苏木精-伊红（HE）染色。研究表明，推拿可以抑制软骨细胞凋亡，修复软骨组织，恢复膝关节活动度。综上所述，本研究证明了推拿治疗KOA模型兔的科学可行性，凸显了其在KOA和类似膝关节相关疾病研究中的潜在应用。
Introduction
膝骨关节炎（KOA）是一种膝关节退行性疾病，主要表现为膝关节疼痛、肿胀、变形、活动受限等，致残率高，女性患病率较高，2019年全球骨关节炎患者5.2781亿，其全球患病率占OA全球总患病率的60.6%1.临床上，KOA的治疗通常分为非手术治疗和手术治疗。非手术治疗包括物理治疗、药物治疗和富血小板血浆注射治疗 2,3。推拿是中医常用、安全、可靠、有效的治疗方法。本研究采用推拿结合膝关节旋转矫正法治疗KOA。推拿技术，如旋转揉捏和按压法，可以平衡肌肉组织，减轻疼痛，调节炎症因子水平，改善组织代谢，抑制关节软骨退化4,5。膝关节旋转矫正法可以调整下肢骨骼和关节的对齐方式，改善膝关节间隙，恢复正常受力线，平衡下肢生物力学6,7,8,9。抗阻运动可以增强肌肉质量和力量，促进软骨组织更新10,11。一项初步研究发现，这种推拿方案在治疗 KOA 方面明显比口服硫酸氨基葡萄糖胶囊更有效，起效更快，并显着抑制软骨细胞变性和受损软骨组织的修复12。在KOA的治疗中，与推拿治疗相比，非甾体抗炎药有不良反应和不理想的长期疗效，手术风险和费用相对较高，并且需要一定的适应证才能进行手术治疗，并伴有术后问题和假体周围并发症13,14,15.与药物治疗和手术相比，推拿治疗 KOA 具有多种优势，包括减少副作用、降低风险、增强安全性、成本效益和更持久的疗效。此外，它还可以有效缓解膝关节疼痛、肿胀、爆裂和活动受限 6,13,16,17。
然而，推拿治疗KOA的机制有待明确，这限制了KOA治疗方案的改进和完善。因此，通过动物实验研究推拿干预KOA的机制是一种有效的方法。与老鼠相比，兔子具有温顺的气质和较大的膝关节。其解剖结构和软骨生化指标与人类相似，是推拿18研究膝关节病机制的合适课题。将木瓜蛋白酶注射到兔膝关节腔中建立的KOA模型具有建模时间短、创伤小、成功率高、存活率高、病理机制与KOA19相似等优点。本研究旨在建立推拿干预KOA的科学可行的动物实验方案，并探讨推拿的机制。
Protocol
该研究获得山东中医药大学附属医院伦理委员会批准（批准文号：2020-29）。 1.实验动物 在标准单笼（12小时光/暗循环，温度20-24°C，空气湿度40%-60%）中饲养18,6个月大的正常级雄性新西兰兔（2.75±0.25kg）。 2.分组方式 使用随机数法从 18 只新西兰兔子中选择 6 只作为假组，并将剩余的 12 只兔子分配到建模组。</…
Representative Results
膝关节活动受限和软骨组织损伤的程度反映了 KOA 的严重程度。膝关节的 ROM 反映了膝关节运动的限制程度。膝关节活动度越小，膝关节活动受限越严重。相反，膝关节ROM越大，膝关节运动程度越正常。HE染色观察软骨组织的形态和结构，反映软骨组织损伤程度。软骨组织表面越不规则，裂缝和缺损的存在越多，软骨细胞数量越少，软骨层厚度越薄，细胞排列越无序，细胞分布越不均匀，层越不清?…
Discussion
实验方案的设计对于研究推拿治疗KOA的机制尤为重要。通过在Waixiyan注射木瓜蛋白酶对兔子进行KOA建模。外显妍位于髌韧带外隐窝内，易于定位，膝关节屈曲时股骨与胫骨的关节间隙较大，便于注入膝关节腔，防止损伤周围组织，因此易于建立KOA模型34。在KOA兔推拿干预期间，模型兔被适当定位以利于推拿给药。兔子被放在兔子固定盒中，身体健康，头部固定，情绪得到安抚，全…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了山东省中医药科技计划项目（2021Q080）和齐鲁中医药学院学校传承项目[鲁维信（2022）93]的支持。
Materials
| 0.9 % sodium chloride injection | Sichuan Keren Pharmaceutical Co. | Z22121903 | |
| -20°C refrigerator | Haier | BD-328WL | |
| 4 % fixative solution | Solarbio | P1110 | |
| 4°C refrigerator | Haier | SC-315DS | |
| Anhydrous ethanol | Sinopharm | ||
| Automatic tissue dewatering machine | Dakowei (Shenzhen) Medical Equipment Co. | HP30 | |
| Blast drying oven | Shanghai Yiheng Scientific Instruments Co. | DHG-9070A | |
| Coverslip | Biyuntian | FCGF50 | |
| Electric thermostat water bath | Shanghai Yiheng Scientific Instruments Co. | HWS-26 | |
| Embedding freezing table | Changzhou Paishijie Medical Equipment Co. | BM450 | |
| Embedding machine | Changzhou Paishijie Medical Equipment Co. | BM450A | |
| Ethylenediaminetetraacetic acid decalcification solution | Servicebio | G1105-500ML | |
| Fluorescent inverted microscope | Leica | Leica DM IL LED | |
| Hematoxylin-eosin staining kit | Cisco Jet | EE0012 | |
| Hydrochloric acid | Laiyang Economic and Technological Development Zone Fine Chemical Plant | ||
| Medical joint goniometer | KOSLO | ||
| Neutral gum | Cisco Jet | EE0013 | |
| Normal-grade male New Zealand rabbit | Jinan Xilingjiao Breeding and Breeding Center | SCXK (Lu) 2020 0004 | |
| Papain(3000 U/mg) | Bioss | D10366 | |
| Pathological tissue bleaching and drying instrument | Changzhou Paishijie Medical Equipment Co. | PH60 | |
| Pet electric clippers | Codos | CP-3180 | |
| Rabbit fixing box | any brand | ||
| Rotating Slicer | Leica | 531CM-Y43 | |
| Tuina technique parameter determination instrument | Shanghai DuKang Instrument Equipment Co. Ltd. | ZTC- |
|
| Ventilator | TALY ELECTRIC | C32 | |
| Xylene | Fuyu Reagent |
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Cite This Article
Zhang, S., Zhang, X., Sun, G., Wang, K., Qiao, Y., He, Y., Li, M., Li, H., Zheng, L. Tuina Intervention in Rabbit Model of Knee Osteoarthritis. J. Vis. Exp. (198), e65763, doi:10.3791/65763 (2023).