在体观察不同应力负荷对大鼠膝关节软骨代谢的影响和机制
发布时间：2018-07-14 14:20
【摘要】：目的:骨关节炎(Osteoarthritis,OA)的病理生理过程中力学因素起到至关重要的作用。本实验采用课题组自行设计的坡度半定量大鼠跑步设备获得大鼠OA模型,通过研究OA发生和发展过程中其组织和基因水平的改变来探究在体应力负荷在OA病理生理过程中的重要作用和相关机制。方法:3月龄SD(Sprague Dawley)大鼠84只,采用随机数法分配到各组。大鼠膝关节应力负荷采用坡度半定量(0°、10°和20°)方法。长期应力负荷跑步方案组完成长达30d的跑步训练(速度为1km/h,每天跑步1h),颈椎脱臼法处死大鼠,取双后肢膝关节软骨;急性应力负荷跑步方案组只进行一次跑步训练(速度为1km/h,时长1h),分别在跑步后0h、2h、6h、12h和24h取双后肢膝关节软骨。HE、番红O和甲苯胺蓝染色定性观察关节软骨大体形态和聚集蛋白聚糖(aggrecan,AGG)含量的改变,并根据OOCHAS软骨损伤评分系统定量评价各组间差异;免疫组化观察各组软骨组织中II型胶原、X型胶原、IL-1β、TNF-α、MMP-3、MMP-9和MMP-13等表达差异;提取软骨细胞的总m RNA,反转录为c DNA,RT-PCR检测各组软骨中II型胶原、AGG、I型胶原、X型胶原、IL-1、IL-6、TNF-α、MMP-3、MMP-9、MMP-13、SOX9和ADAMST5基因表达水平。采用SPSS统计软件进行方差分析和组间比较,检验水平为P0.05。结果:采用长期应力跑步方案的大鼠,实验结束时膝关节软骨出现了不同程度的损伤表现。平板跑步组大鼠膝关节软骨HE染色、番红O和甲苯胺蓝染色与普通笼养对照组差异不明显,10°和20°上坡组大鼠膝关节软骨番红O和甲苯胺蓝染色逐渐变浅,软骨组织中AGG含量逐渐减少,并可见关节表面出现不同程度的损伤,20°上坡组的损伤程度要大于10°上坡组,且软骨细胞呈现出一定程度的肥大化表现,软骨细胞数量减少,胞质比例增大,细胞陷窝明显变大,排列紊乱,典型的关节软骨层次丢失,软骨厚度也明显变薄,潮线消失或变得不明显。随着大鼠上坡运动的角度愈来愈大,膝关节受到的应力负荷越来越大,采用单因素方差分析可知膝关节软骨OOCHAS组织评分随应力负荷增大而增高(F=122.4,P0.0001),组织损伤表现越严重,多组间S-N-K法两两比较有统计学差异(P0.05)。免疫组化结果显示随着跑步坡度(即应力负荷)的增大,关节软骨中II型胶原的含量较笼养对照组逐渐减少而X型胶原含量逐渐增加;炎性介质如IL-1β、TNF-α、MMP-3、MMP-9和MMP-13的表达量都有不同程度的增高。RT-PCR的结果显示随着应力负荷的增加,大鼠膝关节软骨II型胶原和AGG的m RNA表达水平逐渐降低,而IL-1、TNF-α、MMP-3、MMP-9和MMP-13等m RNA表达水平于对照组相比明显增加,其中TNF-α和MMP-3的增高最明显,IL-1的表达水平增高不明显,且10°上坡跑步组IL-1与平板组差异不明显。急性应力跑步组大鼠经过一次过度应力负荷跑步后分别检测其m RNA的表达水平,II型胶原表达量在6h时出现了短暂的升高,之后恢复到正常水平,且与应力负荷强度呈正相关;AGG、COL I、COL X的表达水平从6h开始逐渐升高,到24h时表达量明显增高;IL-6在上坡运动后最早出现增高,之后逐渐降低;IL-1和TNF-α表达量从12h开始出现升高,但总体表达量增高不明显;MMP-3、MMP-9和MMP-13的表达量从6h时开始升高,24h时上坡跑步组的表达量均明显高于对照组和平板跑步组,其中MMP-13的表达水平增高的最明显,24h时20°上坡跑步组的表达水平升高了近10倍;SOX9早期有轻微的升高,之后与对照组水平无差异;ADAMST5基因的表达在2h时达到最高,之后逐渐趋于正常;大多数基因表达量的改变幅度随应力负荷的强度增大而增大。结论:长期过度应力负荷作用于大鼠膝关节软骨可使软骨发生退行性改变,软骨细胞数目减少伴肥大化改变,软骨基质损伤和降解,AGG和II型胶原含量减少,X型胶原增加,使关节软骨发生退行性改变,其力学性能不再能够适应关节活动,关节表面摩擦阻力增大,在过度应力负荷下发生撕裂和磨损,最终形成膝关节OA,这种改变与过度应力负荷的强度呈正相关。过度应力负荷可能首先通过提高IL-6的表达水平,激活IL-1和TNF-α等,促进MMPs家族m RNA的合成,导致软骨基质的降解;而SOX9和ADAMST5的表达上调可能是软骨细胞肥大化和凋亡的原因。
[Abstract]:Objective: in the pathophysiological process of Osteoarthritis (OA), the mechanical factors play an important role. In this experiment, the rat OA model was obtained by a self-designed slope semi quantitative rat running equipment. By studying the changes of the tissue and gene levels in the course of the occurrence and development of OA, the stress load in the body was explored in OA The important role and mechanism in the pathophysiological process. Methods: 3 month old Sprague Dawley (Sprague Dawley) rats were assigned to each group by random number method. The stress load of the knee joint was semi quantified (0, 10 and 20). The long-term stress load running program completed the 30d running training (the speed was 1km/h, running 1H every day). The rats were killed by the dislocated cervical vertebra, and the knee joint cartilage of the two hind limbs was taken. The acute stress load running program group had only one running training (speed of 1km/h, long 1H). The knee cartilage.HE of the hind limbs was taken after running 0h, 2h, 6h, 12h and 24h respectively. The gross morphology of the articular cartilage and the aggregation of proteoglycan (aggr) were observed by the red O and toluidine blue dye (aggr). The changes in the content of ECAN, AGG) and the quantitative evaluation of the differences between each group according to the OOCHAS cartilage damage scoring system. The differences in the expression of type II collagen, X collagen, IL-1 beta, TNF- a, MMP-3, MMP-9 and MMP-13 in cartilage tissue were observed by immunohistochemistry; the total M RNA of cartilage cells was extracted and the reverse transcription was detected. Collagen, type X collagen, IL-1, IL-6, TNF- alpha, MMP-3, MMP-9, MMP-13, SOX9, and ADAMST5 gene expression level. Using SPSS statistics software for variance analysis and comparison between groups, the test level is P0.05. results: the rats with long-term stress running program have different degrees of injury in the knee joint cartilage at the end of the experiment. The flat running group rats HE staining of articular cartilage in the knee joint, the difference between the red O and toluidine blue staining and the general cage control group was not obvious. The cartilage of the knee joint of the 10 and 20 degrees group was gradually shallower, the AGG content in the cartilage tissue decreased gradually, and the injury of the articular surface appeared on the surface of the articular surface, and the damage degree of the 20 degree slope group was more than 10. The cartilaginous cells showed a certain degree of hypertrophy, the number of chondrocytes decreased, the proportion of cytoplasm increased, the cell lacunae became larger, the arrangement was disorderly, the typical articular cartilage was lost, the cartilage thickness also became thinner, the tide line disappeared or became unclear. With the angle of the upper slope movement of the rat, the knee joint became bigger and bigger, knee joint. The stress load is getting bigger and bigger. The OOCHAS tissue score of the knee cartilage increases with the stress load increasing (F=122.4, P0.0001), the more serious the tissue damage is, and the difference between the multiple groups of S-N-K method 22 is statistically different (P0.05). The results of immunohistochemistry show that the increase of the running slope (that is stress load) The content of type II collagen in articular cartilage decreased gradually and the content of type X collagen increased gradually, and the expression of IL-1 beta, TNF- a, MMP-3, MMP-9 and MMP-13 increased to varying degrees of.RT-PCR in inflammatory mediators, and the results showed that the expression level of II type collagen and AGG m RNA expression in the knee cartilage of the rat was increased with the increase of stress load. The expression level of M RNA in IL-1, TNF- a, MMP-3, MMP-9 and MMP-13 increased obviously, and the increase of TNF- A and MMP-3 was most obvious, the expression level of IL-1 increased not obviously, and the difference between the IL-1 and the flat group in the running group of 10 degrees was not obvious. The rats in the acute stress running group were tested after an excessive stress load. The expression level of M RNA was measured. The expression of type II collagen appeared a short rise at 6h, then returned to the normal level, and was positively correlated with the stress load intensity; the expression level of AGG, COL I, COL X increased gradually from 6h to 24h; IL-6 was the first increase after the uphill movement, and then gradually decreased. The expression of F- alpha increased from 12h, but the overall expression increased not obviously. The expression of MMP-3, MMP-9 and MMP-13 increased from 6h, and the expression levels of the running groups on the upslope of 24h were significantly higher than those of the control group and the flat running group. The expression level of MMP-13 was the most obvious, and the expression level of the running group in the 20 degree slope at 24h was increased by the time of 24h. 10 times; there was a slight increase in early SOX9 and no difference between the control group and the control group. The expression of ADAMST5 gene reached the highest at 2h, and then gradually tended to normal; the change amplitude of most gene expression increased with the increase of stress load. Conclusion: long term overstress load on the cartilage of the knee joint of rats can induce cartilage degeneration. The number of chondrocytes decreased with hypertrophy, cartilage matrix damage and degradation, the content of AGG and II collagen decreased, type X collagen increased, the articular cartilage was degenerative, its mechanical properties no longer adapted to joint activity, the friction resistance of the joint surface increased, and the tear and wear occurred under excessive stress load. Finally, tear and wear occurred under excessive stress load. Finally, tear and wear occurred under overstress load. The formation of OA in the knee joint is positively related to the intensity of excessive stress load. Over stress load may first increase the expression level of IL-6, activate IL-1 and TNF- alpha, and promote the synthesis of M RNA in the MMPs family, and lead to the degradation of cartilage matrix, and the up regulation of SOX9 and ADAMST5 may be the cause of chondrocyte hypertrophy and apoptosis.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3
本文编号：2121919
[Abstract]:Objective: in the pathophysiological process of Osteoarthritis (OA), the mechanical factors play an important role. In this experiment, the rat OA model was obtained by a self-designed slope semi quantitative rat running equipment. By studying the changes of the tissue and gene levels in the course of the occurrence and development of OA, the stress load in the body was explored in OA The important role and mechanism in the pathophysiological process. Methods: 3 month old Sprague Dawley (Sprague Dawley) rats were assigned to each group by random number method. The stress load of the knee joint was semi quantified (0, 10 and 20). The long-term stress load running program completed the 30d running training (the speed was 1km/h, running 1H every day). The rats were killed by the dislocated cervical vertebra, and the knee joint cartilage of the two hind limbs was taken. The acute stress load running program group had only one running training (speed of 1km/h, long 1H). The knee cartilage.HE of the hind limbs was taken after running 0h, 2h, 6h, 12h and 24h respectively. The gross morphology of the articular cartilage and the aggregation of proteoglycan (aggr) were observed by the red O and toluidine blue dye (aggr). The changes in the content of ECAN, AGG) and the quantitative evaluation of the differences between each group according to the OOCHAS cartilage damage scoring system. The differences in the expression of type II collagen, X collagen, IL-1 beta, TNF- a, MMP-3, MMP-9 and MMP-13 in cartilage tissue were observed by immunohistochemistry; the total M RNA of cartilage cells was extracted and the reverse transcription was detected. Collagen, type X collagen, IL-1, IL-6, TNF- alpha, MMP-3, MMP-9, MMP-13, SOX9, and ADAMST5 gene expression level. Using SPSS statistics software for variance analysis and comparison between groups, the test level is P0.05. results: the rats with long-term stress running program have different degrees of injury in the knee joint cartilage at the end of the experiment. The flat running group rats HE staining of articular cartilage in the knee joint, the difference between the red O and toluidine blue staining and the general cage control group was not obvious. The cartilage of the knee joint of the 10 and 20 degrees group was gradually shallower, the AGG content in the cartilage tissue decreased gradually, and the injury of the articular surface appeared on the surface of the articular surface, and the damage degree of the 20 degree slope group was more than 10. The cartilaginous cells showed a certain degree of hypertrophy, the number of chondrocytes decreased, the proportion of cytoplasm increased, the cell lacunae became larger, the arrangement was disorderly, the typical articular cartilage was lost, the cartilage thickness also became thinner, the tide line disappeared or became unclear. With the angle of the upper slope movement of the rat, the knee joint became bigger and bigger, knee joint. The stress load is getting bigger and bigger. The OOCHAS tissue score of the knee cartilage increases with the stress load increasing (F=122.4, P0.0001), the more serious the tissue damage is, and the difference between the multiple groups of S-N-K method 22 is statistically different (P0.05). The results of immunohistochemistry show that the increase of the running slope (that is stress load) The content of type II collagen in articular cartilage decreased gradually and the content of type X collagen increased gradually, and the expression of IL-1 beta, TNF- a, MMP-3, MMP-9 and MMP-13 increased to varying degrees of.RT-PCR in inflammatory mediators, and the results showed that the expression level of II type collagen and AGG m RNA expression in the knee cartilage of the rat was increased with the increase of stress load. The expression level of M RNA in IL-1, TNF- a, MMP-3, MMP-9 and MMP-13 increased obviously, and the increase of TNF- A and MMP-3 was most obvious, the expression level of IL-1 increased not obviously, and the difference between the IL-1 and the flat group in the running group of 10 degrees was not obvious. The rats in the acute stress running group were tested after an excessive stress load. The expression level of M RNA was measured. The expression of type II collagen appeared a short rise at 6h, then returned to the normal level, and was positively correlated with the stress load intensity; the expression level of AGG, COL I, COL X increased gradually from 6h to 24h; IL-6 was the first increase after the uphill movement, and then gradually decreased. The expression of F- alpha increased from 12h, but the overall expression increased not obviously. The expression of MMP-3, MMP-9 and MMP-13 increased from 6h, and the expression levels of the running groups on the upslope of 24h were significantly higher than those of the control group and the flat running group. The expression level of MMP-13 was the most obvious, and the expression level of the running group in the 20 degree slope at 24h was increased by the time of 24h. 10 times; there was a slight increase in early SOX9 and no difference between the control group and the control group. The expression of ADAMST5 gene reached the highest at 2h, and then gradually tended to normal; the change amplitude of most gene expression increased with the increase of stress load. Conclusion: long term overstress load on the cartilage of the knee joint of rats can induce cartilage degeneration. The number of chondrocytes decreased with hypertrophy, cartilage matrix damage and degradation, the content of AGG and II collagen decreased, type X collagen increased, the articular cartilage was degenerative, its mechanical properties no longer adapted to joint activity, the friction resistance of the joint surface increased, and the tear and wear occurred under excessive stress load. Finally, tear and wear occurred under excessive stress load. Finally, tear and wear occurred under overstress load. The formation of OA in the knee joint is positively related to the intensity of excessive stress load. Over stress load may first increase the expression level of IL-6, activate IL-1 and TNF- alpha, and promote the synthesis of M RNA in the MMPs family, and lead to the degradation of cartilage matrix, and the up regulation of SOX9 and ADAMST5 may be the cause of chondrocyte hypertrophy and apoptosis.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3
【参考文献】
相关期刊论文 前1条
1 李凯;侯志超;陈晋斌;王少伟;卫小春;;大鼠跑步装置的设计与应用[J];实用骨科杂志;2010年04期
，本文编号：2121919
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