“短刺加电针法”对膝骨关节炎模型兔软骨细胞外基质的修复作用研究

发布时间：2018-05-19 17:19

本文选题：短刺法 + 膝骨关节炎　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的观察“短刺加电针法”对膝骨关节炎模型兔血清中CTX-II、PIICP含量与膝关节软骨中II型胶原(Collagen Type II)—盘状结构域受体(discoidin domain receptor,DDR)-2—基质金属蛋白酶(matrix metalloproteinase,MMP)-13信号通路的影响,探讨该治疗方法修复膝关节软骨细胞外基质的可能作用机制。方法将40只新西兰大白兔随机分为正常组(10只)和造模组(30只),造模组动物采用Hulth-Telhag法手术复制膝骨关节炎模型,Lequesne MG膝关节级别评估法对各组兔进行评价,X线评估造模结果。将造模成功的动物随机分为3组,即模型组、短刺组、普通针刺组,每组10只。短刺组采用短刺加电针法,普通针刺组采用常规针刺加电针法治疗,均选取“内膝眼”、“犊鼻”、“阴陵泉”、“足三里”和“梁丘”进行针刺。正常组和模型组正常抓取、固定,不予以干预。每次治疗20min,每日1次,5d为一疗程,每个疗程间隔2d,共治疗4个疗程。疗程结束后,Lequesne MG法再次评估各组兔膝关节级别,并对各组动物进行膝关节半月板及软骨取材。行HE染色,于光镜下观察软骨细胞的变化;行铅铀双重染色,于透射电镜下观察关节软骨超微结构和细胞器结构的变化。采用蛋白免疫印迹法检测软骨DDR2、II型胶原、MMP13的蛋白表达;免疫组化法检测软骨DDR2、II型胶原、MMP13的表达;逆转录聚合酶链式反应检测DDR2和MMP13的m RNA表达;酶联免疫吸附测定法检测血清PIICP、CTX-II含量。结果1.X线结果显示,造模组兔膝关节较正常组改变明显,造模组兔膝关节对线不良,内侧关节间隙狭窄,关节面粗糙变形,关节边缘有骨赘形成。2.光镜下观察HE染色结果显示,与正常组相比,模型组软骨细胞排列紊乱,细胞成簇出现,细胞核裂解退化明显;与模型组相比,短刺组和普通针刺组软骨细胞排列尚齐,成簇存在。透射电镜检查结果显示,与正常组相比,模型组软骨细胞核固缩,细胞外基质中胶原纤维排列不齐或降解;与模型组相比,短刺组和普通针刺组软骨细胞核形态较正常,基质中胶原纤维较丰富。3.Lequesne MG评分结果显示,造模后6周,模型组、短刺组和普通针刺组兔膝关节均出现强烈的局部疼痛反应,关节肿胀明显且活动度多在15-45度。与正常组相比,模型组、短刺组和普通针刺组Lequesne MG评分均显著增高(p0.01);造模后10周,短刺组和普通针刺组兔膝关节局部疼痛刺激反应、膝关节肿胀程度均减轻,活动度可达45-90度。与造模后6周同组别兔Lequesne MG评分相比均明显降低(p0.01)。4.Western-Blot、免疫组化、RT-PCR、ELISA结果显示,与正常组相比,模型组、短刺组和普通针刺组兔血清中PIICP含量与膝关节软骨中II型胶原的蛋白表达等均明显减少(均p0.01),而血清中CTX-II含量与膝关节软骨中DDR2、MMP13的蛋白及m RNA表达均明显增加(均p0.01);治疗后,短刺组和普通针刺组兔血清中PIICP含量与膝关节软骨中II型胶原的蛋白表达等较模型组明显增多(均p0.01),而血清中CTX-II含量与膝关节软骨中DDR2、MMP13的蛋白及m RNA表达较模型组均明显减少(均p0.01);与普通针刺组相比,短刺组兔血清中PIICP含量与膝关节软骨中II型胶原的蛋白表达增多(p0.01),而血清中CTX-II含量与膝关节软骨中DDR2、MMP13的蛋白及m RNA表达减少(均p0.01)。结论本次研究采用Hulth-Telhag法成功复制了膝骨关节炎兔模型。普通电针法和短刺加电针法均能调整软骨细胞排列方式,抑制细胞外基质的异常降解,恢复细胞外基质的合成,促进兔膝关节软骨修复;且短刺加电针法较普通电针法有一定的疗效优势。其作用机制可能与其阻断II型胶原/DDR2/MMP13通路的病理循环有关。
[Abstract]:Objective To observe the effect of "short prickly plus Electroacupuncture" on CTX-II, PIICP content in the serum of knee osteoarthritis model rabbits and the effect of II collagen (Collagen Type II) in the cartilage of knee joint (discoidin domain receptor, DDR) -2 matrix metalloproteinase. The possible mechanism of repairing the cartilage extracellular matrix of the knee joint. Methods 40 New Zealand white rabbits were randomly divided into the normal group (10) and the model group (30). The model animal model was replicated by Hulth-Telhag method. The Lequesne MG knee assessment method was used to evaluate the rabbits in each group. The results of the X-ray evaluation were made. The results of the model were made. The results of the model were made. The animals were randomly divided into 3 groups, namely, model group, short stab group, common acupuncture group, 10 in each group. Short thorn group was treated with short stab plus Electroacupuncture, and ordinary acupuncture group was treated with conventional acupuncture plus Electroacupuncture. All of them selected "inner knee eye", "calf nose", "Yin Ling spring", "foot three li" and "Liang Qiu" by acupuncture. Normal group and model group were normal grasp. After the treatment of 20min, 1 times a day, 5D was a course of treatment, each course was 2D, and 4 courses were treated. After the end of the course, the knee joint level of each group was evaluated by Lequesne MG method again, and the meniscus and cartilage of the knee joint were taken from each group. The changes of cartilage cells were observed under the light microscope, and the lead and uranium were observed under the light microscope. Double staining was used to observe the changes of ultrastructure and organelle structure of articular cartilage under transmission electron microscope. The protein expression of cartilage DDR2, type II collagen and MMP13 was detected by protein immunoblotting; immunohistochemical method was used to detect the expression of cartilage DDR2, II collagen, MMP13; reverse transcription polymerase chain reaction was used to detect the m RNA expression of DDR2 and MMP13; enzyme linked immunosorbent assay The results of 1. X-ray results showed that the knee joint of the model rabbit model was more obvious than that of the normal group, the knee joint of the model rabbit model was bad, the medial joint space was narrow, the joint surface was rough, and the joint edge had the osteophyte formation under the.2. light microscope, and the HE staining results showed that the model group was soft compared with the normal group. Compared with the model group, the cartilage cells in the short stings group and the common acupuncture group were arranged and clustered. The results of transmission electron microscopy showed that compared with the normal group, the nuclei of the cartilage cells in the model group were fixed and the collagen fibers in the extracellular matrix were inhomogeneous or degraded; and the model group was the same as the model group. Compared with the short prickle group and the common acupuncture group, the morphology of the cartilage nuclei was more normal. The.3.Lequesne MG score of the collagen fiber in the matrix showed that the knee joint of the model group, the short spiny group and the common acupuncture group had strong local pain response 6 weeks after the model, and the joint swelling was obvious and the activity was 15-45 degrees more. Compared with the normal group, the model was compared with the normal group. The score of Lequesne MG in the short spines group and the common acupuncture group increased significantly (P0.01), and the knee joint pain stimulation reaction in the short stings group and the common acupuncture group after 10 weeks of modeling was reduced and the activity of the knee joint was 45-90 degrees. Compared with the Lequesne MG score of the group of the rabbits in the same group at 6 weeks after making the model, the.4.Western-Blot was significantly reduced (P0.01).4.Western-Blot. The results of phytophthoraczation, RT-PCR and ELISA showed that compared with the normal group, the content of PIICP in the serum of the rabbits in the model group, the short spiny group and the common acupuncture group and the expression of the protein expression of type II collagen in the cartilage of the knee were significantly decreased (P0.01), but the content of CTX-II in the serum and the expression of DDR2, MMP13 and m RNA in the cartilage of the knee joint were significantly increased (P0.01). After the treatment, the content of PIICP in the serum and the protein expression of type II collagen in the articular cartilage of the short spiny group and the common acupuncture group increased significantly (P0.01), while the CTX-II content in the serum and the expression of DDR2, MMP13 and m RNA in the cartilage of the knee joint were significantly less than those in the model group (all P0.01). Compared with the common acupuncture group, the blood of the short spiny group was compared with the normal acupuncture group. The content of PIICP and the protein expression of type II collagen in the cartilage of the knee joint increased (P0.01), while the CTX-II content in the serum and the expression of DDR2, MMP13 and m RNA in the cartilage of the knee joint decreased (P0.01). Conclusion the rabbit model of knee osteoarthritis was successfully replicated by Hulth-Telhag method. The common electric needle method and the short spiny acupuncture and electroacupuncture were all adjustable. The arrangement of the whole cartilage cells can inhibit the abnormal degradation of the extracellular matrix, restore the synthesis of extracellular matrix and promote the repair of the cartilage of the knee joint of rabbits, and the short spiny and electroacupuncture method has a certain therapeutic advantage over the common electroacupuncture method. Its mechanism may be related to the pathological cycle of blocking the II collagen /DDR2/MMP13 pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245;R-332

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