摘要
目的采用免疫组化法测定兔膝骨关节炎(KOA)膝关节软骨中IGF-Ⅰ(胰岛素样生长因子)及Ⅱ型胶原在软骨细胞的表达,探讨麝香乌龙丸对关节软骨修复和保护的可能作用机制,并进一步阐明该药在改善关节功能及保护关节软骨等方面的作用,为临床治疗KOA提供实验及理论依据。
方法1分组、造模本实验分4组,将40只体重2.5Kg(±150g)健康成年雄性大耳白兔按体重从小到大进行编号,按随机数字表随机分成四组:正常对照组为A组,模型组为B组,麝香乌龙丸组为C组,抗骨增生片组为D组,每组各10只。A组兔常规饲养,B、C、D三组每只兔于实验的第1、4、7天在右后肢膝关节腔内注射1.6%木瓜蛋白酶生理盐水溶液0.5ml建立兔膝骨关节炎模型。
2给药实验开始起第8天给药,C、D组每日各一次,按成人每日每公斤体重用药量的6倍作为实验兔每日每公斤体重的等效剂量,即C组给予每只兔麝香乌龙丸0.6g/kg/d,D组给予每只兔抗骨增生片0.36g/kg/d。溶于10ml生理盐水中灌胃。A、B两组灌入等剂量生理盐水。每组灌胃4周。期间常规饲养,正常饮水。
3灌胃结束后即实验第38天,用锐利刀片切开右膝关节前正中区滑膜组织,以矢状位切取胫骨内侧髁软骨,取出软骨组织。
4软骨组织切片免疫组化法检测关节软骨中IGF-Ⅰ及Ⅱ胶原表达的水平。
5光镜观察软骨组织病理学改变A组软骨细胞排列规则整齐,未见裂纹及缺损,未见软骨细胞增生、变性的改变,潮线完整;B组软骨细胞排列紊乱,数量明显减少,可见明显溃疡病变,少数见潮线断裂不完整;C组、D组可见轻度的软骨表面缺损现象,软骨细胞较B组排列较整齐,数量增多,潮线均完整。
结果1 IGF-Ⅰ阳性表达指数:B组软骨IGF-Ⅰ阳性表达指数较A组明显减低(P<0.05),C组及D组IGF-Ⅰ阳性表达指数较B组均增高(P<0.05),C组阳性表达指数高于D组(P>0.05)。
2 B组软骨Ⅱ型胶原阳性表达指数较A组明显升高(P<0.05),C、D组Ⅱ型胶原阳性表达指数较B组明显降低(P<0.05),C组阳性表达指数低于D组(P<0.05)。
结论麝香乌龙丸能增加修复性生长因子IGF-Ⅰ及Ⅱ型胶原在兔实验性膝骨关节炎软骨细胞中的表达,实验结果表明该药在兔膝骨关节炎的作用机理与调节软骨细胞因子水平有关,为麝香乌龙丸在临床治疗KOA奠定了实验基础。
Objective: Determined by immunohistochemical staining of rabbit knee osteoarthritis (KOA) knee cartilage IGF-Ⅰ(insulin-like growth factor) and typeⅡcollagen expression in chondrocytes , illustrating. Musk-Aconite-Earthworm-pill have a certain restorative and protective effect on articular cartilage, and to further clarify the drug in improving joint function and protection of articular cartilage in areas of possible mechanism, in order to provide experimental basis for clinical treatment.
Methods:
1.Divide group and build model: 40 healthy male rabbits (weight 2.5Kg±150g) were randomly divided into four groups: A. normal control group, B. model group, C. Musk-Aconite-Earthworm pill group, D. Anti-bone hyperostosis tablet group .Each group of 10. A group of rabbits convention raising .B, C, D group of each rabbit in the experiment 1st, 4th, 7th days injects 1.6% papain physiological saline solution 0.5ml establishment rabbit knee osteoarthritis model in the right hind legs knee joint cavity.
2.Give medicine: Tests the 8th day to start for the medicine, C, D two groups of daily each one time, According to the adult each kilogram body weight every day medication quantity's 6 time of achievement rabbit each kilogram every day body weight's equivalent dosage, group C of each rabbit gives in the musk-aconite-earthworm pill 0.6g/kg /d, group D of each rabbit gives in anti-bone hyperostosis tablet 0.36g/kg /d. Dissolves fills the stomach in the 10ml physiological saline after the stomach tube. Groups A、B pour into the isometric physiological saline. Each group fills in the stomach for 4 weeks. Period convention raising , normal potable water.
3 After filling in the stomach finished namely-tests the 38th day, open the right knee joint to observe generally, cut and take the shinbone platform.
4 detected the expression levels of IGF-Ⅰand typeⅡcollagen in cartilage tissue by Cartilage tissue section immunohistochemistry.
Results:
1. Observe the changes of pathomorphology by light microscope: Cartilage: Group A cartilage cell arranged orderly, there was no crazing and coloboma , no cell degeneration , hyperplasia, tidal line was integrity. Group B quantity of cartilage cell decreased obviously ,arranged disorderly, there was obvious ulcer in cartilage, tidal line were not integrity or broken. Group C and D coloboma were lesser than group B, quantity of cartilage cell increased than group B, arranged relatively orderly, tidal line was integrity.
2 IGF-Ⅰ,Ⅱcollagen positive expression rate: B Group cartilage IGF-Ⅰ,Ⅱcollagen positive expression rate significantly lower than the A group (P <0.05), C group and D group of IGF-Ⅰ,Ⅱcollagen positive expression rate than the B group were significantly higher (P <0.05), C positive expression group was higher than D group (P> 0.05).
Conclusion: Musk-Aconite-Earthworm-pill can increase the effective of the reparative growth factor type -ⅠandⅡin Experimental osteoarthritis of rabbit knee cartilage cell, and further clarify the drug in curing the osteoarthritis of the knee mechanism and the regulation of cytokine levels in cartilage have some relationship, moreover,provide directive function in the use of clinical medicine.
引文
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