【作者】 江陟郝；

【导师】 修忠标；

【作者基本信息】 福建中医药大学 ， 中医骨伤科学， 2011， 硕士

【摘要】 目的：探讨鹿瓜多肽促进膝骨性关节炎软骨细胞增殖的机制。方法：实验研究：将50只新西兰大白兔随机分成模型组35只、假模组8只、正常组7只。模型组参照Hulth造模法造骨性关节炎动物模型,假模组仅作左膝关节内侧皮肤切开后缝合,正常组不做任何手术处理。4周后各组随机抽取2只兔子光镜下观察其关节软骨形态变化以及采用ELISA法检测其关节液内细胞因子IL-1β、TNF-α的水平。造模成功后将模型组随机平均分成治疗组、对照组、空白组。治疗组予鹿瓜多肽注射液0.5ml每日1次关节腔注射；对照组予维骨力每天两粒灌胃；空白组予生理盐水0.5ml每日1次关节腔注射。分别于注射后第1、15、30天抽取三组的关节液并获取其关节软骨组织。采用双抗体夹心ELISA法检测其关节液内细胞因子IL-1β、TNF-α的含量和TGF-β1的水平；采用HE染色观察其关节软骨的组织学变化、透射电镜观察其关节软骨细胞内结构变化；采用免疫组织化学法检测关节软骨细胞PCNA的表达,观察鹿瓜多肽促进关节软骨细胞增殖作用的情况。临床观察：将归类好后的50例早中期膝骨性关节炎患者平均随机分成治疗组、对照组。治疗组每日行鹿瓜多肽12ml加入0.9%氯化钠注射液250ml中静脉滴注,共一个疗程(15天)。对照组口服维骨力每次两粒(tid),连续30天。分别于干预后第1、15、30天抽取两组患者的关节液并采用双抗体夹心ELISA法检测其关节液内细胞因子IL-1β、TNF-α的含量和TGF-β1的水平,观察鹿瓜多肽对以上细胞因子的调节作用,进而分析鹿瓜多肽注射液对KOA患者软骨细胞增殖的情况。结果：实验研究：造模4周后,光镜下观察模型组关节软骨形态明显改变,造模成功后对照组与空白组的IL-1β、TNF-α含量在第15、30天不同时期均高于治疗组,两组间比较有较明显的差异(P<0.01)；其TGF-β1水平在第15、30天不同时期均低于治疗组,两组间比较有较明显的差异(P<0.01)；治疗组的软骨细胞增殖指数与对照组、空白组的软骨细胞增殖指数的比较有显著性差(P<0.01)。HE染色：空白组在第30天时能见大部分软骨陷窝内细胞消失,边缘破坏且粗糙不平严重,同时软骨细胞增生现象明显；对照组在第30天时能见部分软骨陷窝内细胞消失,边缘存在破坏且粗糙不平情况,同时也有少量软骨细胞增生现象；治疗组在第30天时软骨结构清晰可辨,软骨细胞排列整齐,潮线完整,没有出现增生现象。透射电镜：空白组在第30天时其胞质萎缩,染色质不均匀明显,细胞可见凋亡小体形成；对照组在第30天时其染色质存在少量不均匀现象,部分细胞可见凋亡小体形成；治疗组在第30天时则可见其软骨细胞表面微绒毛较多,胞膜、胞核较为完整,胞质内有较多的粗面内质网、线粒体等,染色质分布呈较均匀现象。临床观察：治疗组的细胞因子IL-1β、TNF-α含量在药物干预后第15、30天不同时期均低于对照组,两组比较有明显的差异(P<0.01)；其TGF-β1水平在第15、30天不同时期均高于对照组,两组比较有明显的差异(P<0.01)。治疗组的临床症状较对照组的临床症状有明显的好转。结论：鹿瓜多肽可能是通过诱导减少关节液内细胞因子IL-1β、TNF-α的含量及上调转化生长因子TGF-p1的水平这一作用机制来促进膝骨性关节炎软骨细胞增殖的。更多还原

【Abstract】 Objective:The Mechanism of Study On Cervus and Cucumis Polypeptide Injection on Promoting Chondrocytes Proliferation with Knee Osteoarthritis。Methods:The Experimental study:The 50 New Zealand white rabbits were randomly divided into model group of 35. sham module 8, the normal group of 7。Hulth model modeling method group made reference to osteoarthritis animal models, fake modules for the left only after the skin incision medial suture, normal group without any surgical treatment。After 4 weeks in each group randomly selected from two rabbits were observed under light microscope and morphological changes of articular cartilage detected by ELISA in synovial fluid cytokine IL-1β、TNF-αlevels。Successful model after the model group were randomly divided into treatment group and control group and blank group。Treated group Injection 0.5ml Lugua polypeptide articular injection of evey day; The control group for every two-dimensional Glucosamine orally; 0.5ml normal saline blank group articular injection of 1 day。Were taken after injection one day、15 and 30 days and synovial fluid of three groups for the articular cartilage。Double antibody sandwich ELISA assay in synovial fluid cytokine IL-1β、TNF-αlevels and the level of TGF-β1。Observed by HE staining of histological changes of articular cartilage, transmission electron microscopy to observe the structural changes in articular cartilage cells; Detected by immunohistochemistry PCNA expression in articular chondrocytes was observed Lugua peptides promote cell proliferation of articular cartilage situation.。The Clinical observation:After the classification of 50 cases of early and mid knee osteoarthritis were randomly divided into treatment group、the control group on average。Line treatment group Lugua polypeptide 12ml daily in 0.9% sodium chloride injection in the intravenous infusion of 250ml of a course of treatment (15 days)。Glucosamine control group was treated every two-dimensional (tid), in all 30 days。Intervention were taken after 1day、15 and 30 days and synovial fluid of patients with two groups by double antibody sandwich ELISA assay in synovial fluid cytokine IL-1β、TNF-a levels and the level of TGF-β1o Peptides on the above observation Lugua regulation of cytokines, and then analyzes Lugua peptide injection on cartilage cell proliferation in patients with KOA situation。Results:The Experimental study:After 4 weeks of modeling, light microscope morphological changes of articular cartilage in model group, after the success of the modeling group and blank control group. Their IL-1βand TNF-a levels in different periods of 15 and 30 days higher than the treatment group o Between the two groups have a more significant difference (P<0.01); The TGF-β1 levels at different periods of 15 and 30 days were lower than the treatment group, two groups have a more significant difference (P<0.01); the treatment group, the cartilage cell proliferation with the control group and blank group, the proliferation of cartilage cells The comparison of the significant difference (P<0.01)o HE staining:30 days control group visibility in most of the cartilage cell lacunae disappeared, and the rough edge of serious damage, and cartilage cell proliferation significantly; 30 days in the control group some visibility within the cells of cartilage lacunae disappeared, there is destruction and rough edges of the situation, but also a small amount of cartilage cell proliferation; The treatment group in 30 days, clearly visible cartilage, cartilage cells arranged in neat rows, tidal line complete, there was no proliferation。TEM:The blank group in 30 days of its cytoplasmic shrinkage, chromatin was heterogeneous, cell apoptotic body formation; The control group in 30 days, there is a small amount of its uneven chromatin. some cells were apoptotic body formation; Treatment group at 30 days may be reflected in its cell surface microvilli more cartilage in membrane, the nucleus is more complete, more cytoplasm of rough endoplasmic reticulum in mitochondria, chromatin showed a more uniform distribution of the phenomenon。The Clinical observation:the treatment group of cytokines IL-1β、TNF-a levels in 15 and 30 days after drug intervention in different periods were lower than the control group, there were significant differences between the two groups (P<0.01); the TGF-βl level of 15 and 30 days in different periods were higher, there were significant differences between the two groups (P<0.01)。The clinical symptoms of the treatment group better than in the control group, there is a marked improvement in clinical symptoms。Conclusion:CCPI may be reduced by induction of cytokines in synovial fluid IL-1β、TNF-a levels and increased TGF-β1 level mechanisms to promote the role of knee osteoarthritis cartilage cell proliferation。更多还原