【作者】 林木南；

【导师】 刘献祥；

【作者基本信息】 福建中医学院 ， 中医骨伤科学， 2008， 博士

【摘要】 目的研制OA护膝,并通过观察OA护膝对骨性关节炎细胞因子、基质溶解素、透明质酸、氧自由基代谢、关节软骨宏微观结构变化和细胞增殖、凋亡的影响,探讨其防治膝骨性关节炎的机制及疗效。方法1 OA护膝的研制:根据中医“寒者热之”、“瘀者通之”的治则,以PIC16F630单片机为核心,电路主要由升压、输出、单片机控制以及电热软膜4部分组成,通过键盘设定单片机产生不同幅度和频率的动态变频疏密波进行治疗,30min后单片机内部定时器中断,自动关机。2实验研究:日本大耳白兔60只,随机分为2组,即:正常组(10只)和手术组(50只)。采用改良Hulth法复制膝骨性关节炎模型,术后随机分为5组,即模型组、对照组(微波组)、实验1组(电组)、实验2组(热组)、实验3组(护膝组),并行对应治疗。8周后,分别测定关节滑液IL-1β、IL-6、TNF-α、MMP-3、HA浓度,及血液MDA、SOD、NO浓度。16周后,分别测定关节滑液IL-1β、IL-6、TNF-α、MMP-3、HA浓度,关节滑膜及血液MDA、SOD、NO浓度,并用CR片、光镜、透射电镜、Tunel法、PCNA进行组织形态学观察,同时用RT-PCR检测关节软骨细胞Bcl-2、P53的mRNA相对表达水平。结果1 OA护膝的研制:护膝输出的疏密波可控制由2～300Hz不同频率刺激脉冲组成,脉冲宽度在100～150μs之间,输出正负向对称的刺激脉冲,采用可充电的手机电池(3.7V)供电,单片机定时,30min自动关机。电热软膜工作时的温度控制在40℃左右。2实验研究:模型组、对照组、实验1组、实验2组、实验3组关节液中IL-1β、IL-6、TNF-α、MMP-3、血液MDA、NO浓度均高表达,含量16周明显高于8周时,而关节液HA和血液SOD浓度均低表达,含量16周明显低于8周时,16周时关节滑膜中MDA、NO浓度、关节软骨细胞P53的mRNA相对表达水平、AI均呈高表达,而SOD浓度、关节软骨细胞Bcl-2的mRNA相对表达水平、PI均低表达,与正常组有显著性差异(P＜0.01,P＜0.05)。8周时,关节液中IL-1β、IL-6、TNF-α、MMP-3、HA,血液中MDA、NO、SOD含量,对照组、实验1组、实验2组、实验3组与模型组有显著性差异(P＜0.01,P＜0.05):对照组、实验1组、实验2组与实验3组有显著性差异(P＜0.05)。16周时,关节液中IL-1β、IL-6、TNF-α、MMP-3、HA,血液和关节滑膜中MDA、NO、SOD含量,关节软骨细胞Bcl-2、P53的mRNA相对表达水平,AI、PI,对照组、实验1组、实验2组、实验3组与模型组有显著性差异(P＜0.01,P＜0.05);对照组、实验1组、实验2组与实验3组有显著性差异(P＜0.05)。16周时与8周时,关节液中IL-1β、IL-6、TNF-α、MMP-3、HA,血液中MDA、NO、SOD含量,实验3组间比较有显著性差异(P＜0.05)。CR片显示:正常组关节间隙正常,关节面平整光滑,无骨赘;模型组内侧间隙明显变窄,有明显骨赘;光镜显示:正常组关节软骨四层结构清晰可辨,软骨细胞排列整齐呈柱状,染色质分布均匀,细胞核清晰;模型组关节软骨层变薄,部分区域软骨细胞核固缩、坏死,软骨细胞排列紊乱,四层结构不易分辨;电镜显示正常组软骨细胞呈椭圆形,细胞及胞膜完整,包质内可见丰富粗面内质网、高尔基体、线粒体,细胞核完整,染色质分布均匀;模型组软骨细胞明显固缩且外形不规则,细胞周晕消失,胞浆内细胞器凝成高电子密度的片状物不易分辨,细胞核不规则,染色质浓聚,散裂于核中;实验3组内侧间隙变窄,关节边缘有轻微骨赘,关节面粗糙变形,软骨细胞轻度萎缩,核内异染色质增多,胞质内可见脂滴及微丝出现。对照组、实验1组与实验2组介于模型组、实验3组之间。免疫组化观察结果:正常组、实验3组、实验1组、实验2组、对照组、模型组TUNEL阳性率逐渐增高,PCNA阳性率逐渐降低。结论1以PIC16F630单片机为系统控制核心的OA护膝,可通过键盘按钮控制输出信号的频率、脉冲宽度、强度和工作时间。2 OA护膝采用低频温热动态变频疏密波,针对膝OA病理过程中的多个环节进行调节,能有效抑制细胞因子、MMP—3、HA、氧自由基、NO的生物学效应,增强SOD的生物学活性,提高关节软骨细胞Bcl-2mRNA表达,减弱软骨细胞P53mRNA表达,从而抑制软骨细胞凋亡,延缓膝关节软骨宏观形态、软骨细胞及软骨基质的退变。更多还原

【Abstract】 Objective To develop an OA kneepad and explore its mechanism and efficacy on the prevention and treatment of osteoarthritis of the knee through observing the effects of OA kneepad on cytokines,matrix-dissolution,hyaluronic acid,oxygen free radical metabolism, articular cartilage micro-structural changes,cell proliferation and apoptosis.Methods1 Development of OA kneepad:Under the principle of"treating cold syndrome with hot-natured drugs" and "treating blood stasis syndrome with promoting blood circulation and removing blood stasis drugs",PIC16F630 SCM was served as the core and mainly composed of four parts including the boost circuit,the output,single-chip microcomputer control,as well as electric soft membrane.Dynamic range and frequency inverter dredging wave of treatment was carried out by settings of SCM via different keyboards.The instrument will be automatically shutdown 30 min later owing to the interruption of internal timer of SCM.2 Experimental Research:Sixty Japan rabbits were randomly divided into two groups, namely:the normal group(10) and surgery group(50).Knee osteoarthritis models were made by using the method of modified Hulth and then randomly divided into five groups,namely, model group and the control group(microwave),experimental group 1(electricity), experimental group 2(thermal),the experimental group 3(kneepad),with corresponding treatment.After 8 weeks,the IL-1β,IL-6,TNF-αand MMP-3,HA concentrations in articular synovial fluid and MDA,SOD,NO concentrations in blood were measured respectively.After 16 weeks,the IL-1β,IL-6,TNF-αand MMP-3,HA concentrations in articular synovial fluid and MDA,SOD,NO concentrations in both blood and synovial were also measured respectively.Besides,tissue morphology were analyzed by using CR tablets,light microscopy, transmission electron microscopy,Tunel,PCNA,and relative mRNA expression levels of Bcl-2 and P53 in articular cartilage cells were detected by real-time quantitative RT-PCR.Results1 Development of OA kneepad:The kneepad density-wave output can be controlled by 2Hz different frequencies to 300 Hz stimulation pulse component,the pulse width of 100 to 150μs between the positive and negative output symmetry to the stimulation pulse.The rechargeable mobile phone batteries(3.7V) were served as power supply and SCM as timer with automatic shutdown after 30 minutes.The temperature of electric soft membrane was controlled in 40℃.2 Experimental Research:IL-1β,IL-6,TNF-αand MMP-3 in synovial fluid,MDA,NO in blood concentrations of model group,the control group,the experimental group 1,group 2,group 3 were high expression levels,16 weeks later was significantly higher than those in 8 weeks,The expression of the HA in synovial fluid and SOD in blood concentration was significantly lower than those in 8 weeks,16 weeks later,MDA and NO concentration of synovial,P53 relative mRNA in articular cartilage cells,the expression level of AI were overexpressed,and SOD concentration,Bcl-2 mRNA expression levels of articular cartilage cells,PI were low expression,and the normal group was significant difference(P＜0.01,P＜0.05).8 weeks later,IL-1β,IL-6,TNF-α,MMP-3 and HA in synovial fluid,MDA,NO, SOD in blood,in the control group,the experimental group 1,group 2,group 3 and Model group there were significant differences(P＜0.01,P＜0.05),there were significant differences (P＜0.05) in the control group,experimental group 1,group 2 and the experimental group.16 weeks later,IL-1β,IL-6,TNF-αand MMP-3,HA in synovial fluid,MDA,NO,SOD in blood and synovial,Bcl-2,P53 mRNA expression in content of articular cartilage cells,AI,PI, there were significant differences(P＜0.01,P＜0.05)in the control group,experimental group 1, group 2,group 3 and model group;In the control group,experimental group 1,group 2 and group 3 there were significant differences(P＜0.05).In 16 weeks and 8 weeks,IL-1β,IL-6, TNF-αand MMP-3,HA in synovial fluid,MDA,NO,SOD in blood in group 3,there was significant difference(P＜0.05).The report of CR corresponds to the sequentiae condition.In the normal group,joint space is normal,and the articular facet is complete and slick with no osteophyte;In the model group,the is obviously narrowing between the arthral wall blank with the obvious osteophyte.The light microscope corresponds to the sequentiae condition.In normal group,the four layers’structures of arthrodial cartilage are clear,distinguishing.The cartilage cells line up in order with the shape of pillar,the disposition of caryotin uniformity,and the caryon is clear,In the model group,the layer of arthrodial cartilage gets to thinningz.In some districts,cartilage cell shows karyopyknosis and cellular necrosis,In the calcifying layer,cartilage cells line up in disorder and four layers’structures are uneasy to tell the differences.In the normal group,Electron microscopy showed normal cartilage cells were Oval,cells and cell memrane are integrity.In intracytoplasm,there are abundant rough endoplasmicsreticulum(PER),dictyosome,The caryon is integrity,and the disposition of caryotin is uniformity;In the model group,cartilage cells obviously pyknosis and their shape shows irregularity.The areolae around cells disappear.In intracytoplasm,cell organs coagulate to falps which show electron dense and it is uneasy to tell the differences.The shape of caryon shows irregularity,and caryotin gathers densely while spalling in caryon.In GroupⅢ,there is narrowing between the arthral wall blank.There is the little osteoplyte at the verge of the articulus and the articular facet is obviously rough and deformation.Cartilage cell shrinks back slightly.Intranuclear different dye-substance is increasing,it is thus clear that there are lipid droplet and microfilament in the intracytoplasm.Changes of morphological organization of control group,experimental group 1 and group 2 between model group and experimental group3.Immunohistochemical study results:normal group,group 3,group 1,group 2,control group and model group,TUNEL-positive rate increased gradually,PCNA-positive rate decreased gradually.Conclusion1 The OA kneepad with PIC16F630 microcontroller core can control the output signal frequency,pulse width,intensity and work time by controlling keyboardbuttons.2 By using low-frequency dynamic frequency density wave,the OA kneepad could regulate many aspects of pathological process of knee OA including:inhibition of biological effects of cytokine,MMP-3,HA,oxygen free radicals and NO;enhancing the biological activity of SOD;up-regulating the mRNA expression of Bcl-2 as well as down-regulating the mRNA expression of P53,thereby to inhibit the apoptosis of cartilage cells and delay the cartilage degeneration of the macro-morphology,cartilage cells and cartilage matrix.更多还原