低强度脉冲超声波对兔膝骨性关节炎的软骨修复作用及机制研究

【作者】 李雪萍；

【导师】 励建安；

【作者基本信息】 南京医科大学 ， 运动医学， 2011， 博士

【摘要】 目的:研究低强度脉冲超声波(Low intensity pulsed ultrasound, LIPUS)对体外培养兔膝关节软骨细胞增殖、软骨细胞中金属蛋白酶-13(matrix metalloproteinase,MMP-13)及Ⅱ型胶原的影响,探讨LIPUS对软骨细胞活性的促进作用及机制。方法:选用6只1月龄的新西兰白兔膝关节软骨进行体外培养,体外培养每只兔的软骨细胞传至第二代后,均分为对照组与4个LIPUS照射组,LIPUS强度分别是20 mW /cm~2、30 mW /cm~2、40 mW /cm~2、50 mW /cm~2,每天照射20min,连续10天。每天进行细胞计数;分别在第0天、5天及第10天收集细胞,提取软骨细胞全蛋白及RNA,采用Western-blot技术、qRT-PCR技术检测软骨细胞中MMP-13、Ⅱ型胶原的含量。结果:1.软骨细胞计数:与对照组相比,各LIPUS照射组软骨细胞数均明显增高(P<0.05),其中40 mW /cm~2组软骨细胞数最高,与其他各照射组比较有显著性差异(P<0.05); 2. MMP-13含量检测:细胞培养5天和10天后,各组MMP-13含量均较第0天明显升高(P<0.05),但各照射组MMP-13含量均较其对照组显著降低(P<0.05),其中40 mW /cm~2组MMP-13含量最低(P<0.05); 3.Ⅱ型胶原表达量检测:细胞培养5天和10天后,各组Ⅱ型胶原表达量均较0天显著降低(P<0.05)。但各照射组Ⅱ型胶原表达量均显著高于对照组(P<0.05)。其中40 mW /cm~2组Ⅱ型胶原表达量最高(P<0.05); 4. MMP-13与Ⅱ型胶原相关性:分析各组细胞培养0天、5天和10天的MMP-13与Ⅱ型胶原含量,两者呈负相关(r=-0.757, P <0.05)。结论:不同强度的LIPUS照射可促进体外培养兔软骨细胞的活性,其作用与软骨细胞增殖、MMP-13、Ⅱ型胶原的表达变化相关。目的:通过对兔膝早、中期骨性关节炎进行LIPUS干预,观察低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)对兔早中期OA关节软骨损伤、软骨细胞增殖及PI3K信号转导通路的影响,并探讨其相关的作用机制。方法:36只健康雄性新西兰兔平均分成6组,早期对照组(early control,EC组),早期OA组(early OA, EO组),早期治疗组(early treatment,ET组),中期对照组(medium-term control,MC组),中期OA组(medium-term OA,MO组)和中期治疗组(medium-term treatment,MT组)。对EO、ET、MO及MT组兔右膝关节行前交叉韧带切断(Anterior Cruciate Ligament Transection,ACLT)术,ET组术后第三天、MT组术后第5周行LIPUS治疗,频率为3MHz,照射强度为40mW/cm~2,每次20min,每天1次,6天/周,持续6周。治疗6周后,取兔右侧膝关节行大体组织学观察,进行改良Mankin评分;应用蛋白质印迹(western-blot)法检测关节软骨细胞增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及磷脂酰肌醇-3激酶(phosphatidylinositol-3-kinase,PI3K)含量。结果:1.组织病理学检查:EO组关节软骨表面不规则、甲苯胺蓝染色减少、裂隙形成,与EC组相比,Mankin评分显著升高(P <0.01);MO组关节软骨损伤明显,Mankin评分较MC组显著升高(P<0.01)。与EO组相比,ET组组织病理学改变程度轻,Mankin评分显著降低(P<0.01);而MT组Mankin评分较MO组无显著降低(P >0.05)。2. PCNA检测:EO组PCNA较EC组显著升高(P <0.01),ET组PCNA较EO组显著升高(P <0.01);与EO组相比,MO、MT组PCNA均无升高(P >0.05)。3. PI3K检测: EO组PI3K与EC组相比,无显著增高(P >0.05);ET组PI3K较EO组显著升高(P <0.01);而MT组PCNA较MO组无明显升高(P >0.05)。结论:研究提示LIPUS早期干预更有利于促进兔骨关节炎软骨修复,其作用机制与LIPUS促进了软骨细胞增殖,并激活PI3K信号转导通路密切相关。目的:研究LIPUS对兔膝OA早期关节软骨Ⅱ型胶原、MMP-13及MAPKs信号通路的影响,探讨在OA早期应用LIPUS延缓关节软骨退变的作用机制。方法:新西兰白兔18只,随机分为照射组(operation plus LIPUS,O+L组)、假照射组(operation without LIPUS,O-L组)和假手术组( sham operation, SO组),每组6只。O+L组,右膝接受ACLT术,术后第3天起进行LIPUS照射,频率为3MHz,照射强度为40mW/cm~2,每次20min,每天1次,6天/周,持续6周。O-L组,手术及照射方案与O+L组一致,但无超声输出。SO组仅行关节囊切开术。治疗6周后将兔处死,取兔右侧膝关节行大体组织学观察,进行改良Mankin评分;应用蛋白质印迹法(Western-blot)检测Ⅱ型胶原蛋白、基质金属蛋白酶13(matrix metalloproteinase-13,MMP-13)及细胞外信号调节激酶1/2 (extracellular signal-regulated kinase1/2,ERK1/2)、丝裂原活化蛋白激酶38(Mitogen-activated protein kinase38,p38)、C6N末端激酶(c-Jun N-terminal kinase,JNK)的表达。结果:1.关节软骨Mankin评分:与SO组相比, O+L组、O-L组显著高于SO组(P<0.05,P<0.01);与O+L组相比,O-L组显著增高(P<0.05)。2.Ⅱ型胶原检测:与SO组相比,O+L组、O-L组Ⅱ型胶原均有降低,但O-L组下降更为明显(P<0.05);O+L组与O-L组相比无显著性差异(P >0.05)。3. MMP-13检测:与SO组相比,O+L组、O-L组均有增高(P<0.05,P<0.01),但O-L组增高更为明显;与O+L组相比,O-L组MMP-13显著增高(P<0.05)。4.磷酸化ERK1/2、p38、JNK检测:与SO组相比,O+L组、O-L组磷酸化ERK1/2、p38均有增高(P<0.05,P<0.01),但O-L组增高更为明显;与O+L组相比,O-L组磷酸化ERK1/2、p38显著增高(P<0.05,P<0.05)。在O+L组、O-L组、SO组之间,磷酸化JNK均无显著性差异。结论:OA早期应用LIPUS可减轻关节软骨的损伤程度,其作用与LIPUS干预后关节软骨中MMP-13、p38、ERK1/2表达下调有关。更多还原

【Abstract】 Objective:To study the effects of low intensity pulsed ultrasound (LIPUS) on chondrocytes proliferation and MMP-13 and typeⅡcollagen of chondrocytes of rabbit’s knee in vitro and discuss the effect and Mechanism of LIPUS to chondrocytes activity.Methods: The chondrocytes were obtained from knee joints of six one-month old rabbits. Each rabbit’s chondrocytes were divided into 5 groups: control group and four LIPUS groups with the intensity of LIPUS is 20 mW /cm2, 30 mW /cm2, 40 mW /cm2, 50 mW /cm2 respectively. During the second cultured generation, the chondrocytes were irradiated by LIPUS for 20 minutes and cells were counted each day for 10 days. On the 0, 5th and 10th day, chondrocytes were collected and their protein and RNA were extracted for test of MMP-13 and typeⅡcollagen content with Western-blot and qRT-PCR technique.Result: 1.Contrasting with control groups, the chondrocytes number were higher in each LIPUS groups(p<0.05) and the numbers were significantly higher in the 40 mW /cm2 group (p<0.05). 2. MMP-13 contents were higher in the 5th day and 10th day than in the 0 day(p<0.05). After irradiated 5 days and 10 days, the contents of MMP-13 in each LIPUS group were remarkable lower than control group(p<0.05) and 40 mW /cm2 group was the lowest (p<0.05). 3. After cultured 5 days and 10 days, typeⅡcollagen contents were lower than the 0 day(p<0.05). The contents of typeⅡcollagen in each LIPUS groups were remarkable higher than control groups(p<0.05) and 40 mW /cm2 group was the highest (p<0.05). 4. There was significantly negative correlation between MMP-13 and typeⅡcollagen (r=-0.757,p<0.05).Conclusion: The 4 kinds of intensity of LIPUS could promote the chondrocyte activities of rabbit’s knee and the effect is related to the chondrocyte proliferation and changes of MMP-13 and typeⅡcollagen content in chondrocytes. Objective: This study was designed to establish rabbit knee OA models by Anterior Cruciate Ligament Transection (ACLT) surgery, and observed the effect of LIPUS irradiation on articular cartilage repair, chondrocytes proliferation and PI3K signal transduction pathway.Method: Thirty-six healthy New Zealand rabbits were divided into six groups: early control group (EC group), early OA group (EO group), early treatment group (ET group), medium-term control group (MC group), medium-term OA group (MO group) and medium-term treatment group (MT group). ACLT was performed in EO, ET, MO and MT groups. Animals in ET group received LIPUS irradiation 3 days after surgery, while the fifth week after surgery in MT group. Six weeks after irradiation, the rabbits were sacrificed respectively and observed for knee gross microscopy with Modified Mankin score, proliferating cell nuclear antigen (PCNA) and phosphatidylinositol-3-kinase (PI3K) with Western-blot technique.Result: Toluidine blue stain showed in ET group, the surface of articular cartilage were regular, normally stained and chondrocytes proliferation were observed. While in MT group, the surface of articular cartilage were irregular, TBS was reduced significantly, and the number of chondrocytes was decreased. Total Mankin score was significantly increased in EO group (EO vs. EC group, P <0.01) and reduced in ET group (ET vs. EO group, P <0.01). Total Mankin score in MO group was significantly higher than that in MC group (P <0.01). However, total Mankin score in MT group didn’t significantly decrease compared with that in MO group (P >0.05). Western blot analysis showed that PCNA in EO group was significantly increased as compared with EC group (P <0.01), PCNA in ET group was significantly higher than that in EO group (P <0.01). PCNA in MO group and MT group was not significantly increased compared with that in MC group (P >0.05). PCNA in ET group was significantly increased as compared with that in MT group (P <0.01). Western blot analysis showed that phosphorylation of PI3K (p-PI3K) in EO group didn’t significantly increase as compared with EC group (P >0.05). Phosphorylation of PI3K in ET group was significantly higher than that in EO group (P <0.01). There was no significant difference in PI3K phosphorylation between MT group and MO group (P >0.05).Conclusion: Early LIPUS intervention is beneficial to articular cartilage repair through promotion of chondrocytes proliferation in OA. The mechanism of action is closely associated with the effect of LIPUS in PI3K signal transduction pathway. Objective: This study was designed to observed the effect of LIPUS irradiation on typeⅡcollagen, matrix metalloproteinase-13(MMP-13), mitogen-activated protein kinases(MAPKs) signal transduction pathway and articular cartilage repair on rabbits models in early stage of OA progression.Method: 18 healthy New Zealand rabbits were divided randomly into three groups: treat group (O+L group), control group (O+L group) and sham operation group (SO group), 6 in each group. In O+L group and O-L group, Surgical ACLT was performed in the right knee of rabbits. 3 days after surgery, all of the rabbits were received LIPUS irradiation, and sacrificed respectively 6 week later. Modified Mankin score, typeⅡcollagen, MMP-13 and MAPKs were observed.Result: Modified Mankin score: compared with SO group, O+L group, O-L group were significant high (P<0.05,P<0.01); and compared with O+L group score in O-L group was also significant high (P<0.05). Western-blot analysis: typeⅡc ollagen in O+L group and O-L group were significant lower than that in SO group (P<0.05), but no difference between O+L group and O-L group. Compared with SO group, MMP -13 in both O+L and O-L group were significant high and compared with O+L group, MMP-13 in O-L was significant high (P<0.05). Compared with SO group, p-ERK1/2 and p-p38 in both O+L and O-L group were significant high, and higher in O-L group (P<0.01); compared with O+L group, p-ERK1/2, p-p38 in O-L group was significant high (P<0.05); p-JNK showed no difference in all three groups.Conclusion: Early LIPUS intervention relieved damage in articular cartilage of OA. The mechanism may closely associate with the effect of LIPUS reducing MMP-13, p38 and ERK1/2 contents of articular cartilage.更多还原