【作者】 赵征；

【导师】 黄烽；

【作者基本信息】 中国人民解放军军医进修学院 ， 风湿病学， 2009， 博士

【摘要】 第一部分洛伐他汀对大鼠胶原性关节炎的治疗作用及免疫学机制目的:本研究以胶原诱导性关节炎(CIA)大鼠为模型,评价洛伐他汀对CIA的抗炎疗效及其免疫学机制,为探索炎性关节炎新的治疗药物提供理论基础。方法:本研究首先利用牛Ⅱ型胶原建立Wistar大鼠CIA模型,造模成功后随机分为正常对照组、CIA模型对照组、布洛芬阳性药物对照组、甲氨蝶呤阳性药物对照组和洛伐他汀治疗组。分别观察各给药组大鼠的体重、测量不同时间点大鼠的后足容积,并对关节炎的肿胀程度进行评分;同时以ELISA检测各组血清的TNF-α,IL-6,IL-8及IP-10的水平;用实时荧光定量PCR方法检测大鼠IL-1β及IL-17 mRNA的表达量;对各治疗组大鼠的踝关节拍摄X线片,评价CIA大鼠的软骨和骨的破坏程度;同时对各组大鼠踝关节的滑膜进行HE染色及免疫组化染色,用免疫组织化学的方法,分析大鼠踝关节滑膜中单核细胞趋化蛋白(MCP)-1和调节激活正常T细胞表达和分泌因(RANTES)的表达情况,分析其在CIA发病中的作用。结果:1.CIA模型的建立:在初次免疫Ⅱ型胶原后的第11～13d,大鼠后足趾开始发红,轻度肿胀,第17d后足背及踝关节红肿加重,大鼠足垫增厚,活动量减少,食欲下降。2.踝关节肿胀程度的变化:布洛芬及洛伐他汀组大鼠的踝关节肿胀程度于第3W开始下降,第4W时洛伐他汀组的后足容积较CIA组下降显著(P＜0.05)。关节炎评分在第5w时较CIA组显著降低(P＜0.01),两组有统计学显著性差异。3.细胞因子的变化:免疫后第5w,洛伐他汀组、布洛芬组和MTX组显著低于CIA组大鼠的血清TNF-α,IL-6及IP-10的水平(P＜0.05),而洛伐他汀组和MTX组的IL-8水平较CIA组大鼠无显著下降(P＞0.05)。洛伐他汀及MTX组IL-1β及IL-17 mRNA的表达量显著低于CIA组(P＜0.05)4.影像学改变:CIA组大鼠X线显示大鼠后足关节软组织肿胀,骨质疏松伴有骨质破坏,洛伐他汀组、布洛芬组和MTX组大鼠的X线显示关节周围软组织肿胀、无骨质疏松和骨质破坏。5.组织病理学改变:与CIA组相比,洛伐他汀组、布洛芬组和MTX组关节滑膜组织充血水肿较CIA组明显减轻,滑膜细胞轻度增生,炎性细胞浸润较少,血管增生和血管翳形成大大减少。未见关节软骨变形、坏死或剥脱。6.关节滑膜MCP-1和RANTES的表达:洛伐他汀组、布洛芬组和MTX组大鼠表达MCP-1和RANTES阳性的细胞较CIA组显著减少。结论:1.洛伐他汀对胶原诱导性大鼠关节炎具有缓解症状、抑制炎症肿胀的作用,与CIA模型组相比,疗效显著。2.洛伐他汀能够抑制TNF-α,IL-6,IL-1β,IL-17,IP-10,MCP-1和RANTES的水平,其作用机制可能是通过抑制这些细胞因子的表达,而达到阻断免疫复合物的形成及维持细胞因子网络的平衡,从而抑制滑膜的增生、炎性细胞的浸润和软骨侵蚀的。3.CIA组大鼠关节滑膜中可见MCP-1和RANTES阳性细胞的表达,且在洛伐他汀治疗后其表达量减少,说明MCP-1和RANTES在CIA的发病机制中有重要意义。第二部分巴曲酶对大鼠胶原性关节炎的治疗作用及免疫学机制目的:本研究以胶原诱导性关节炎(CIA)大鼠为模型,评价巴曲酶对CIA的抗炎疗效及其免疫学机制,为探索炎性关节炎新的治疗药物提供理论基础。方法:本研究首先利用牛Ⅱ型胶原建立Wistar大鼠CIA模型,造模成功后随机分为正常对照组、生理盐水CIA模型对照组、布洛芬阳性药物对照组、甲氨蝶呤阳性药物对照组和巴曲酶大、小剂量治疗组。分别观察各给药组大鼠的体重变化、测量不同时间点大鼠的后足容积,并对关节炎的肿胀程度进行评分;检测血浆纤维蛋白原水平;并用ELISA检测各组血清的TNF-α,IL-6,IL-8,IP-10,单核细胞趋化蛋白(MCP)-1和调节激活正常T细胞表达和分泌因子(RANTES)的水平;用实时荧光定量PCR方法检测大鼠IL-1β及IL-17mRNA的表达量;对各治疗组大鼠的踝关节拍摄X线片,评价CIA大鼠的软骨和骨的破坏程度;同时对各组大鼠踝关节的滑膜进行HE染色,分析其在CIA发病中的作用。结果:1.CIA模型的建立:在初次免疫Ⅱ型胶原后的第11～13d,Wistar大鼠后足趾开始发红,轻度肿胀,第17d后足背及踝关节红肿加重,大鼠足垫增厚,活动量减少,食欲下降。2.踝关节肿胀程度的变化:免疫后第5w,巴曲酶大、小剂量组大鼠的后足容积较CIA组显著下降(P＜0.05)。大、小剂量组的关节炎评分自19d显著下降,在第5w时与CIA组相比显著降低(P＜0.05)。3.血浆纤维蛋白原(Fg)的检测:第2w时测定Fg,巴曲酶大剂量组即可见显著降低(P＜0.05),第5w时,各治疗组Fg均较CIA组显著降低(P＜0.05)。4.细胞因子水平的检测:免疫后第5w,巴曲酶大、小剂量组、布洛芬组和MTX组显著低于CIA组大鼠的TNF-α,IL-6及RANTES的水平(P＜0.05),IP-10的水平在巴曲酶大剂量组、布洛芬组及MTX组的水平较CIA组显著降低(P＜0.05),MCP-1和IL-8的水平在巴曲酶大剂量组及布洛芬组的表达显著低于CIA组。巴曲酶大、小剂量组IL-17 mRNA的表达显著低于CIA组(P＜0.05),但IL-1βmRNA的表达与CIA组相比无统计学意义。5.影像学改变:巴曲酶大、小剂量组、布洛芬组和MTX组大鼠的X线显示关节周围软组织肿胀、无骨质疏松和骨质破坏。6.组织病理学改变:与CIA组相比,巴曲酶大、小剂量组、布洛芬组和MTX组关节滑膜组织充血水肿较CIA组明显减轻,滑膜细胞轻度增生,炎性细胞浸润较少,未见关节软骨变形、坏死或剥脱。结论:1.巴曲酶可显著缓解CIA大鼠关节炎的症状、抑制炎症肿胀,与CIA模型组相比,疗效显著。2.巴曲酶对纤维蛋白原有显著的抑制作用,可以有效的抑制炎症急性时相反应蛋白。3.巴曲酶能够抑制TNF-α,IL-6,IL-8,IP-10,IL-17,MCP-1和RANTES的水平,其作用机制可能是通过抑制这些细胞因子的表达,而达到阻断免疫复合物的形成及维持细胞因子网络的平衡,从而抑制滑膜的增生、炎性细胞的浸润和软骨侵蚀的。更多还原

【Abstract】 PartⅠTreatment and Immunological Mechanism of the Protective Effect of Lovastatin on Collagen-induced Arthritis in RatsObjective:To evaluate the efficacy and immunological mechanism of lovastatin on the treatment of collagen-induced arthritis（CIA） in rats and to supply theoretical basis for exploring new treatments of inflammatory arthritis.Methods:Arthritis of rat was induced injecting bovineⅡcollagen（BIIC） to Wistar rats.The rats were randomly divided into five groups:normal control group,CIA control group treated with normal saline,ibuprofen group,Methotrexate（MTX） group and Lovastatin group.The body weight,the hind paw volume,the arthritic index of all rats at different time points were observed and measured.The therapeutic effects of different reagents were assessed by measuring the change of serum cytokine levels including TNF-α,IL-6,IL-8 and IP-10 level..At the same time,the expression of IL-1βand IL-17mRNA’were detected by QRT-PCR.At the end of experiment,the radiographic changes and the synovial pathology score of rat ankle joints were evaluated.The expressions of MCP-1 and RANTES in the synovium of ankle joint were evaluated and the pathogenic roles of MCP-1 and RANTES in CIA in rats were analysed.Results:1.The model of CIA was constituted successfully at 11～13 days after first immunization of typeⅡcollagen.At the 17^th day,the swelling of dorsum of hind feet and ankle joints deteriorated.The sole of the rats were thick.The mobility and appetite of the rats decreased.2.The degree of swelling of ankle joints in Lovastatin group was decreased since the 3^th week.At week 4,the volume of hind paw in Lovastatin group was lower than that of CIA group.At the 5^th week,the arthritis index of Lovastatin groups was lower than that of the CIA group.3.The serum levels of TNF-α,IL-6 and IP-10 in Lovastatin group were decreased since 5^th week.The expressions of IL-1βand IL-17mRNA’ in Lovastatin group were less than that of the CIA group.4.Radiologic finding:The soft tissue around the ankle joint was swollen. Osteoporosis and and bone erosion were not found in Lovastatin group.5.Histopathologic results:Comparing with CIA group,the degree of synovial swelling in Lovastatin group was decreased.The synovial proliferation and inflammatory cell infiltration were mild.The vascular hyperplasia and pannus were significantly declined.6.At the 5^th week,the cells expressing MCP-1 and RANTES were found in the interface layer of synovium and cartilage in CIA group.Few positive cells of MCP-1 and RANTES were found in Lovastatin group.Conclusion:1.Lovastatin significantly ameliorates the symptoms of CIA rats,and inhibits the development of inflammatory swelling.2.Lovastatin can decrease the expression of TNF-α,IL-1β,IL-6,IP-10, IL-17,MCP-1 and RANTES.It indicates Lovastatin may play a protective role on joint erosion through regulating the expression of these cytokine.3.MCP-1 and RANTES have important roles in CIA rats,because they expressed on CIA rats’ joints. PartⅡTreatment and Immunological Mechanism of the Protective Effect of Batroxobin on Collagen-induced Arthritis in RatsObjective:To evaluate the efficacy and Immunological Mechanism of Batroxobin on the treatment of CIA in rats and to supply theoretical basis for exploring new treatments of inflammatory arthritis.Methods:The CIA rats were randomly divided into different groups:normal control group,CIA group treated with normal saline,Ibuprofen group, methotrexate（MTX） group,high dose and low dose Batroxobin groups.Several indexes（weight,volume of hind paw,arthritic index,radiography of ankle joint） were observed in different groups.The plasma fibrinogen level in each group was detected.The levels of TNF-α,IL-6,IL-8,IP-10,MCP-1 and RANTES in each group were measured by ELISA.The expressions of IL-1βand IL-17mRNA were detected by QRT-PCR.The histopathology of synovium was scored.Results:1.The model of collagen-induced arthritis was constituted successfully at 11～13 days after first immunization of typeⅡcollagen.At the 17^th day,the swelling of dorsum of hind feet and ankle joints deteriorated.The sole of the rat was thick.The mobility and appetite of the rats decreased.2.The degrees of swelling of ankle joints in high dose and low dose of Batroxobin groups were decreased since the 3^th week.At week five,the volume of hind paw in high dose Batroxobin group was lower than that of the CIA group.At the 5^th week,the arthritis indexes in both Batroxobin groups were lower than CIA group.3.The plasma fibrinogen level of in each group wae detected at 2^th and 5^th week.The plasma fibrinogen level in high dose of Batroxobin group was lower than that of the CIA group at 2^th week.And the plasma fibrinogen levels in both high and low dose Batroxobin groups were lower than that of the CIA group at 5^th week.4.The levels of TNF-α,IL-6 and RANTES in both high and low dose Batroxobin groups were decreased since 5^th week.The levels of IP-10,IL-8 and MCP-1 in high dose of Batroxobin were lower than the CIA group.The expressions of IL-17mRNA’s in both Batroxobin groups were less than that of the CIA group.The expressions of IL-1βmRNA in both Batroxobin groups were decrease.But there were no stastistically difference when compared with CIA control group at 5^th week.5.Radiologic finding:There was soft tissue swelling around the ankle joint but osteoporosis and bone erosion were not found in Batroxobin group.6.Histopathologic results:Comparing with CIA group,the degree of synovial swelling in Batroxobin group was decreased.The synovial proliferation and inflammatory cell infiltration were mild.The vascular hyperplasia and pannus were significantly declined.Concluion:1.Batroxobin significantly ameliorates the symptoms of CIA rats,and inhibits the development of inflammatory swelling.2.Batroxobin can decrease the plasma fibrinogen level and inhibit the actue reaction protein.3.Batroxobin can decrease the expression of TNF-α,IL-1β,IL-6,IP-10, IL-17,MCP-1 and RANTES.Indicates Batroxobin may play a protective role on joint erosion through regulating the expression of these cytokine.更多还原