本文選題：類風(fēng)濕性關(guān)節(jié)炎 + 炎癥��； 參考：《蘇州大學(xué)》2015年碩士論文

【摘要】：目的:觀察Ⅱ型大麻素受體(cannabinoid receptor 2,CB2)在類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis,RA)疾病進程中的表達變化,分析CB2選擇性激動劑JWH-133對RA炎癥性骨侵蝕的治療作用。方法:雄性DBA/1小鼠50只,采用隨機數(shù)字表法分為5組,即正常對照組(Control)、模型組(Vehicle)、JWH-133低劑量治療組(L-JWH-133)、JWH-133高劑量治療組(H-JWH-133)和陽性對照組(甲氨蝶呤治療組,MTX),每組10只。采用牛Ⅱ膠原誘導(dǎo)的小鼠CIA模型,藥物治療組于初次免疫后第28天(D28)經(jīng)腹腔分別給予JWH133(1mg/kg/d和10mg/kg/d)和MTX(20mg/kg,每周2次),連續(xù)給藥6周。模型組經(jīng)腹腔注射生理鹽水0.1ml/kg/d,空白組小鼠常規(guī)飼養(yǎng),不予處理。分別觀察各組小鼠的體重、后足足掌厚度變化,并對關(guān)節(jié)炎的腫脹程度進行評分。Micro-CT檢查評估骨組織破壞程度,HE染色評估滑膜增生和炎性細胞浸潤程度,番紅O染色檢測軟骨破壞程度,抗酒石酸酸性磷酸酶(TRAP)染色檢測成熟破骨細胞,免疫組織化學(xué)染色檢測CB2、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-1β、白介素-6(IL-6)、基質(zhì)金屬蛋白酶-3(matrix metallo proteinases-3,MMP-3)、環(huán)氧化酶-2(Cyclooxygenase-2,COX-2)、誘導(dǎo)性一氧化氮合酶(hadueed-nitric oxide syathase,i NOS)以及核因子-κB受體活化因子配體(receptor activators of nuclear factor-kappa B ligand,RANKL)和骨保護素(osteoprotegerin,OPG)的表達變化。結(jié)果:DBA/1小鼠初次免疫21天(D21)開始,體重逐漸下降,最低為(D35,17.99±1.44)g,明顯低于Control組(D35,21.81±1.23)g比較差異有統(tǒng)計學(xué)意義(P0.05)給予JWH-133后,小鼠體重逐漸增加,與Vehicle組比較,差異有統(tǒng)計學(xué)意義(P0.05)。CIA模型小鼠后足于D21開始出現(xiàn)腫脹,靶向激活CB2后小鼠足掌厚度開始減少,H-JWH-133組D42足掌厚度為(2.52±0.12)mm與Vehicle組(2.63±0.07)mm比較,差異有統(tǒng)計學(xué)意義(P0.05);D42時H-JWH-133組關(guān)節(jié)炎評分(13.6±1.14)與Vehicle組(15.6±0.55)比較,差異有統(tǒng)計學(xué)意義(P0.05)。Micro-CT掃描結(jié)果顯示Vehicle組骨質(zhì)破壞嚴(yán)重,關(guān)節(jié)結(jié)構(gòu)消失,經(jīng)選擇性CB2受體激動劑治療后骨質(zhì)破壞逐漸減輕,關(guān)節(jié)結(jié)構(gòu)得到恢復(fù);Control組骨體積(BV)、骨體積分?jǐn)?shù)(BV/TV)、骨面積分?jǐn)?shù)(BS/BV)、骨小梁厚度(Tb.Th)分別為(5.90±0.12)mm3、(32.52±0.89)%、(9.26±0.62)mm、(0.36±0.02)mm;Vehicle組為(3.77±0.22)mm3、(17.81±0.95)%、(17.78±1.03)mm、(0.21±0.01)mm,H-JWH-133組為(3.91±0.23)mm3、(19.45±0.98)%、(16.82±0.56)mm、(0.24±0.01)mm;H-JWH-133組為(5.22±0.19)mm3、(24.94±1.12)%、(11.81±0.60)mm、(0.30±0.02)mm,與Vehicle組相比差異有統(tǒng)計學(xué)意義(P0.01)。HE染色顯示Vehicle組滑膜組織滑膜組織顯著增生,大量炎癥細胞浸潤,靶向活化CB2后滑膜組織炎癥明顯減輕。番紅O染色顯示Vehicle組軟骨細胞大量壞死,局部軟骨層完全消失,H-JWH-133組軟骨細胞顯著增多,軟骨層結(jié)構(gòu)明顯恢復(fù)。TRAP染色結(jié)果顯示,CIA模型組關(guān)節(jié)有大片紫紅色區(qū)域,H-JWH-133治療后破骨細胞數(shù)量明顯減少。免疫組織化學(xué)染色結(jié)果顯示CB2在CIA小鼠的滑膜組織和骨組織均有表達;Vehicle組TNF-?、IL-1?、IL-6、MMP-3、COX-2和i NOS的表達明顯增高,選擇性CB2受體激動劑治療后上述因子表達顯著降低;H-JWH-133組RANKL表達強度較Vehicle組顯著降低,OPG表達則高于Vehicle組。結(jié)論:CB2選擇性激動劑JWH-133能抑制炎性細胞浸潤,減少炎性因子分泌;JWH-133能下調(diào)RANKL表達,促進OPG表達,抑制軟骨和骨組織的破壞。CB2有望成為治療RA炎癥性骨侵蝕的新靶點。
[Abstract]:Objective: To observe the expression changes of cannabinoid receptor 2 (CB2) in the process of rheumatoid arthritis (rheumatoid arthritis, RA), and to analyze the therapeutic effect of CB2 selective agonist JWH-133 on RA inflammatory bone erosion. Methods: 50 male DBA/1 rats were divided into 5 groups by random digital table, that is, normal control group (normal control group). Control), model group (Vehicle), JWH-133 low dose treatment group (L-JWH-133), JWH-133 high dose treatment group (H-JWH-133) and positive control group (methotrexate treatment group, MTX), 10 rats in each group. The mouse CIA model induced by bovine collagen was used, and the drug treatment group was given JWH133 (1mg/kg/d and 10mg/kg/d) in the abdominal cavity twenty-eighth days after primary immunization (D28). And MTX (20mg/kg, 2 times a week) for 6 weeks. The model group was injected with saline 0.1ml/kg/d by intraperitoneal injection, and the blank group was fed with normal feeding, no treatment. The weight of the mice in each group and the thickness of hind foot palmar were observed, and the degree of swelling of the arthritis was evaluated by.Micro-CT examination to evaluate the destruction of bone tissue, and HE staining was used to evaluate the increase of synovial membrane. The degree of infiltration of raw and inflammatory cells, the damage degree of cartilage by O staining, anti tartaric acid phosphatase (TRAP) staining to detect mature osteoclasts, immunohistochemical staining for detection of CB2, tumor necrosis factor - alpha (tumor necrosis factor- alpha, TNF- a), interleukin, IL -1 beta, interleukin--6 (IL-6), matrix metalloproteinase X metallo proteinases-3, MMP-3), cyclooxygenase -2 (Cyclooxygenase-2, COX-2), inducible nitric oxide synthase (hadueed-nitric oxide Syathase, I NOS), and the expression changes of nuclear factor kappa activation factor ligand and osteoprotegerin. /1 mice first immunized 21 days (D21), the body weight gradually decreased, the lowest was (D35,17.99 + 1.44) g, obviously lower than the Control group (D35,21.81 1.23) g, the difference was statistically significant (P0.05), the mice weight gradually increased, and compared with the Vehicle group, the difference has statistical significance (P0.05).CIA model mice began to appear swelling after the onset. The thickness of the foot of the mouse was reduced after the target activation of CB2, and the thickness of D42 foot palms in group H-JWH-133 was (2.52 + 0.12) mm and Vehicle group (2.63 + 0.07) mm, the difference was statistically significant (P0.05); at D42, the score of H-JWH-133 (13.6 + 1.14) was compared with the Vehicle group (15.6 + 0.55), and the difference was statistically significant (P0.05). In group ehicle, bone destruction was serious, joint structure disappeared, bone destruction gradually lessen and joint structure recovered after selective CB2 receptor agonist; Control group bone volume (BV), bone volume fraction (BV/TV), bone area fraction (BS/BV), bone small Liang Houdu (Tb.Th) were (5.90 + 0.12) mm3, (32.52 + 0.89)%, (9.26 + 0.62) mm, (0.36 + 0.02) mm; V Group ehicle was (3.77 + 0.22) mm3, (17.81 + 0.95)%, (17.78 + 1.03) mm, (0.21 + 0.01) mm, H-JWH-133 group was (3.91 + 0.23) mm3, (19.45 + 0.98)%, (16.82 + 0.98) mm, mm, H-JWH-133 +% mm3, mm, mm, and mm. The synovial tissue of the synovium of group E was significantly proliferated, a large number of inflammatory cells infiltrated, and the inflammation of the synovial tissue was obviously reduced after the target activation of CB2. The red O staining showed that the chondrocytes in the group Vehicle were necrotic, the local cartilaginous layer completely disappeared, the cartilage cells in the H-JWH-133 group increased significantly, the cartilage structure was obviously restored to the.TRAP staining results, and the CIA model group was displayed. The number of osteoclasts in the joint was significantly reduced after H-JWH-133 treatment. The results of immunohistochemical staining showed that CB2 was expressed in the synovium and bone tissue of CIA mice, and the expression of TNF-? IL-1?, IL-6, MMP-3, COX-2 and I NOS increased in the Vehicle group, and the expression of the above factors was significant after the selective CB2 agonist. The expression intensity of RANKL in group H-JWH-133 was significantly lower than that in group Vehicle, and the expression of OPG was higher than that in group Vehicle. Conclusion: CB2 selective agonist JWH-133 can inhibit inflammatory cell infiltration and reduce inflammatory factor secretion; JWH-133 can down regulate the expression of RANKL, promote OPG expression, and inhibit the destruction of cartilage and bone tissue..CB2 is expected to be the treatment of inflammatory bone erosion of RA. New targets.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R593.22

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本文編號：1990284