摘要
退行性骨关节炎(degenerative osteoarthritis OA)病因尚未完全明了,目前认为是多因素致病,临床上采用医用臭氧(O3)关节腔内注射治疗早-中期OA取得了较好的疗效,但O3的作用效应及其治疗机理目前仍不甚明了,本实验采用Hulth模型造模后,在兔膝关节腔内注射臭氧(O3),探讨O3抑制软骨细胞退变作用效应及机理,为医用O3治疗早-中期OA提供实验支持。
一、O3对关节软骨的病理改变的影响
目的：观察O3对Hulth模型OA软骨光镜下改变的影响。方法：32只兔随机分为2组,分别为治疗组16只,模型组16只,采用Hulth模型,自手术当日起肌注普鲁卡因青霉素40万u,每日一次,连续3天；治疗组每日向治疗组兔左膝关节腔内注射40mg/LO2—O3混合气体2ml,连续注射7天；对照组每日向对照组兔左膝关节腔内注射40mg/L022ml,连续注射7天；连续注射7天后,耳缘静脉注入空气处死实验兔,将实验兔的左膝关节备皮,碘伏消毒,切开左膝关节腔,行大体观察,锐利刀片切取左股骨内髁软骨,将软骨标本置于10%甲醛中固定,经一系列脱水、10%EDTA脱钙处理,共10天；软骨组织石蜡包埋；HE染色,生物显微镜观察。结果：O3实验组软骨表面细胞变性、排列紊乱、可见软骨细胞坏死、脱落,少量的炎性渗出物,软骨表面可见糜烂斑,成纤维细胞和毛细血管增生相对少见；OA对照组软骨表面细胞排列紊乱、坏死、变性较实验组明显,可见明显的溃疡,达深层软骨,可见明显的炎性细胞渗出及成纤维细胞和新生的毛细血管增生,可见纤维组织增生于溃疡底部及溃疡底部的软骨细胞变性。结论：O3试验组关节软骨退变的程度较OA对照组减轻,软骨缺损也较轻,表明40mg/LO2-O3的混合气体能有效延缓兔膝关节骨关节炎的软骨退变的病理进程。
目的：观察O3对Hulth模型OA软骨电镜下改变的影响。方法：32只兔随机分为2组,分别为治疗组16只,模型组16只,采用Hu1th模型,自手术当日起肌注普鲁卡因青霉素40万u,每日一次,连续3天；治疗组每日向治疗组兔左膝关节腔内注射40mg/LO2—O3混合气体2ml,连续注射7天；对照组每日向对照组兔左膝关节腔内注射40mg/LO2 2ml,连续注射7天；连续注射7天后,耳缘静脉注入空气处死实验兔,将实验兔的左膝关节备皮,碘伏消毒,切开左膝关节腔,行大体观察,锐利刀片切取左侧股骨内髁关节软骨修剪成1×1×2mm的小条；3%戊二醛酸固定24小时；0.1M磷酸缓冲液冲洗3次；0.1%四氧化锇固定90min；去离子水冲洗3次；不同浓度丙酮逐级脱水；脱钙液脱钙后以61G环氧树脂浸透、包埋、硬化后修块,半薄切片；定位后切0.05mm超薄切片,电子染色后电镜观察。结果：OA对照组大部分的软骨细胞明显肿胀且形态不规则,细胞周晕消失,粗面内质网明显扩张,微绒毛断裂且变短,粗面内质网网膜溶解断裂,胞浆内可见脂滴存在,细胞核形态不规则,细胞核严重变形、凹陷,出现腔隙陷窝及囊泡形成等改变；O3实验组软骨细胞大部分均正常,细胞为椭圆形或梭形,细胞核完整,可见细胞核内异染质轻度边集,线粒体散在分布于胞浆内,粗面内质网较对照组丰富,表面可见许多微小突起。结论：O3试验组的关节软骨细胞退变的程度较OA对照组减轻,软骨缺损也较轻,表明O3能有效延缓关节软骨退变的病理进程。
二、O3对软骨细胞中IL_1β表达的影响
目的：观察O3对Hu1th模型OA软骨细胞中IL_1β表达的影响。方法：将36只兔随机分为3组,分别为治疗组16只,模型组16只,正常组4只,采用Hu1th模型,自手术当日起肌注普鲁卡因青霉素40万u,每日一次,连续3天；治疗组每日向治疗组兔左膝关节腔内注射40mg/LO2—O3混合气体2ml,连续注射7天；对照组每日向对照组兔左膝关节腔内注射40mg/L022ml,连续注射7天；连续注射7天后,耳缘静脉注入空气处死实验兔,将实验兔的左膝关节备皮,碘伏消毒,切开左膝关节腔,耳缘静脉注入空气栓塞处死实验兔,造模的左膝关节备皮,消毒,切开膝关节,刀片切取左侧股骨内髁软骨修剪成1×1×2mm小条,3%戊二醛酸溶液固定24小时；DAB法检测软骨细胞IL_1β；每张切片在软骨破坏的边缘随机选取视野,每个视野计数200个细胞,统计阳性染色细胞的个数,以阳性细胞率作为IL-1β的表达指标。结果：正常组(A组)阳性细胞率为2.815±0.827%,模型组(B组)阳性细胞率为47.159±4.431%①,O3治疗组(C组)阳性细胞率为27.771±4.085%②；模型组(B组)与正常对照组(A组)比较,p<0.01；治疗组(C组)与模型组(B组)比较,p<0.05,有统计学意义。结论：实验组软骨细胞中IL-1β含量低于对照组,提示O3能够降低软骨细胞中IL-1β含量,保护关节软骨。
三、O3对关节液中一氧化氮含量的影响
目的：观察O3对Hulth模型OA关节液中一氧化氮(nrticioxide, NO)的含量的影响。方法：32只兔随机分为2组,分别为治疗组16只,模型组16只,采用Hulth模型,自手术当日起肌注普鲁卡因青霉素40万u,每日一次,连续3天；治疗组每日向治疗组兔左膝关节腔内注射40mmg/LO2—O3混合气体2ml,连续注射7天；对照组每日向对照组兔左膝关节腔内注射40mg/L±2 2ml,连续注射7天；连续注射7天后,将实验兔以速眠新Ⅱ号注射液(0.2ml/kg)肌肉内注射麻醉,麻醉效果满意后,将造模的左膝关节备皮,碘伏消毒,膝关节穿刺,刺入关节腔后,用1ml生理盐水冲洗关节腔,反复注入、回抽3次后,抽尽液体,注入试管,-70℃封存备测；硝酸还原酶法测定关节液中NO的含量。结果：对照组NO的含量为145.57±12.17,实验组为132.33+11.14,经统计学分析,实验组与对照组比较p<0：05,有统计学意义。结论：实验组关节液中NO含量低于对照组,提示O3能够降低关节液中NO含量,保护关节软骨。
四、O3对软骨细胞iNOS表达的影响
目的：观察±3对Hulth模型OA软骨细胞中iNOS表达的影响。方法：32只兔随机分为2组,分别为治疗组16只,模型组16只,采用Hulth模型,自手术当日起肌注普鲁卡因青霉素40万u,每日一次,连续3天；治疗组每日向治疗组兔左膝关节腔内注射40mg/LO2—O3混合气体2m1,连续注射7天；对照组每日向对照组兔左膝关节腔内注射40mg/ L02 2ml,连续注射7天；连续注射7天后,耳缘静脉注入空气,栓塞处死实验兔,造模的左膝关节备皮,消毒,立即切开左侧膝关节,刀片切取股骨内髁关节软骨修剪成1×1×2mm小条,半定量RT-PCR的方法检测软骨中一氧化氮合酶的含量。结果：实验组iNOS mRNA的含量为0.74±0.17,对照组iNOS mRNA的含量为0.83±0.21,统计学分析显示：两组间差异有统计学意义(F=12.1.91,P<0.01),进一步的统计学分析显示,03实验组软骨iNOS mRNA的表达水平显著低于对照组。结论：对照组OA软骨中iNOS高表达,实验组OA软骨中iNOS低表达,实验组软骨iNOS mRNA的表达水平显著低于对照组,O3可以明显地抑制软骨iNOS的表达。
五、O3对软骨细胞MMP-1表达的影响目的：
观察O3对Hulth模型OA软骨细胞中基质金属蛋白酶-1(MMP-1)表达的影响。方法：32只兔随机分为2组,分别为治疗组16只,模型组16只,采用Hulth模型,自手术当日起肌注普鲁卡因青霉素40万u,每日一次,连续3天；治疗组每日向治疗组兔左膝关节腔内注射40mg/LO2—O3混合气体2ml,连续注射7天；对照组每日向对照组兔左膝关节腔内注射40mg/LO2 2ml,连续注射7天；连续注射7天后,耳缘静脉注入空气,栓塞处死实验兔,造模的左膝关节备皮,消毒,立即切开左侧膝关节,刀片切取股骨内髁关节软骨修剪成1×1×2mm小条,半定量RT-PCR的方法检测软骨中MMP-1的含量。结果：实验组MMP-1的含量为0.67±0.13,对照组MMP-1的含量为0.73±0.12,经统计学分析,p<0.05,差异有显著性。结论：对照组OA软骨中MMP-1高表达,实验组OA软骨中MMP-1低表达,实验组软骨MMP-1的表达水平显著低于对照组,O3能明显降低OA软骨中的MMP-1的表达,保护关节软骨。
Degenerative osteoarthritis (OA) are still not fully understood, currently considered many pathogenic factor, people adopt medical ozone (03) joint lumen injections treat early medium-term OA achieved good effect, but the mechanism of 03 and treatment are still not fully understood, this experiment used Hulth model in the rabbit knee lumen injection ozone (03), explore 03 inhibit chondrocytes degeneration mechanism, for medical 03 treatment early, middle OA provide experimental support
First, the pathology change of articular cartilage
Objective:to observe the pathology change of articular cartilage by optical microscope. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date, muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur by razor-sharp blades, fixed cartilage in 10% formaldehyde solution, through a series of dehydration,10% EDTA demineralization processing, in all 10 days; paraffin embedded the Cartilage tissue; HE dyed, biological microscope observated.
Results:in 03 group, the cells of cartilage surface is degeneration, arranging disorderly, visibled chondrocytes necrosis, fell off, a small amount of inflammatory exudated, erosion spot be visible on cartilage surface, fibroblasts and capillary hyperplasia is uncommon; in OA controls group, the cells of cartilage surface are arranged disorder, necrosis and degeneration is the apparent, ulcer can be see obviously, it is visibled obvious of the inflammatory cells and fibroblasts and newborn capillary hyperplasia, visibled fibre hyperplasia in ulcer bottom and ulcer cartilage cells of denaturation. Conclusion: The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
Objective: to observe the pathology change of articular cartilage by electron microscope. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date, muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; Fixed 24 hours in 3% glutaraldehyde; flushed in 0.1 M buffer of phosphoric, in all 3 times; fixed in 0.1% osmium tetroxide, in all 90min; flushed 3 times in deionized water; Dehydrated in different concentrations acetone; Saturated with 61G epoxy resin after demineralizated, embedded, hardened, repaired into semi-thin block; Positioned ultra-thin slices cut 0.05 mm, Electron microscopic observated after electronic dyeing. Results:In OA group, most of the cartilage cells is markedly swollen and form irregularly, the week's halo of cell is disappeared, rough endoplasmic reticulum apparent expansioned, microvilli fractured and shorten, rough endoplasmic reticulum retinal dissolved fractured, lipid drops exists can be see in the cytoplasm, the nucleus is irregularly; In 03 group, cartilage cells are almost normal, cells are elliptic or spindle completely. Conclusion:The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effect ively rel ieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
Second, to observe the IL-1βexpressed change of articular cartilage by DAB method
Objective:to observe the IL-1βexpressed change of articular cartilage by DAB method. Methods:36 rabbit randomly divided into 3 groups, respectively, the treatment group is 16, the model group is 16, the normal group is 4, Used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; Fixed .24 hours in 3% glutaraldehyde; tested IL_1β. in cartilage cells by DAB method; Randomly selected vision in each section of the edge in cartilage damage, counts 200 cells in every vision, statisting, the number of positive dyeing cells with positive cells rate as IL_1βexpression index. Results:the positive cells rate of normal group (group A) is 2.815±0.827%, the positive cells rate of model group (group B) is 47.159±4.431%, the positive cells rate of 03 group is 27.771±4.085%; Compared the model group (group B) and the normal control group (group A), p<0.01; Compared the treatment group (group C) and the model group (group B), p<0.05; with a statistical significance. Conclusion:The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
Three, to observe the nitric oxide (NO) expressed change in joint fluid by nitrate reductase.
Objective: to observe the nitric oxide (NO) expressed change in joint fluid. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected 022ml to the left knee joint lumen of control group rabbit, continuous injected 7 days; the 8th day, anesthesiaed, iodine disinfection, and knee punctured, extracted joint fluid, in all 1 ml, Sealed and saved in -70℃; to observe the nitric oxide (NO) expressed change in joint fluid by nitrate reductase. Results:In control group, the content of NO is 145.57±12.17, In 03 group, the content of NO is 132.33±11.14, by statistical analysis, (p<0.05), with a statistical significance. Conclusion: The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2 - O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
Four, to observe the expression of iNOS in cartilage cells by RT-PCR.
Objective: to observe the expression of iNOS in cartilage cells by RT-PCR. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; to observe the expression of iNOS in cartilage cells by RT - PCR. Results:In control group, the content of iNOS mRNA is 0.74±0.17, In 03 group, the content of iNOS mRNA is 0.83±0.21, by statistical analysis, (F=12.191, P< 0.01), with a statistical significance. Further statistical analysis shows that the expression of iNOS mRNA in 03 group is significantly below than the control group's. Conclusion:The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
Five, the semi-quantitative RT-PCR method in detecting articular cartilage matrix metalloproteinases-1 (matrix metalloproteti-nase-1, MMP-1) expression
Objective:to observe the expression of MMP-1 in cartilage cells by RT-PCR. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; to observe the expression of MMP-1 in cartilage cells by RT-PCR. Results:In control group, the content of iNOS mRNA is 0.73±0.12, In 03 group, the content of iNOS mRNA is 0.67±0.13, by statistical analysis, (P<0.05), with a statistical significance. Conclusion: The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
引文
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[28]Kobayashi K,Mishima H, Hashimoto S, et al.Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy. J Orthop Res,2001,19:802-808.
[29]Stefanovic-Racic M, Stadler J, Evans CH, et al. Nitric oxide and arthritis. Arthritis Rheum [J],1993,36 (8):1036-1044.
[30]Moilanen E. Nitric oxide in inflammation and immune response. Ann Med[J],1995,27(3):359-367.
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[37]Roberts S, Caterson B, Menage J, et al. Matrix metall-oproteinases and aggrecanase. Their role in disorders of the human intervertebral disc.Spine,2000,25:3005
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