在體觀察不同應(yīng)力負(fù)荷對(duì)大鼠膝關(guān)節(jié)軟骨代謝的影響和機(jī)制
發(fā)布時(shí)間：2018-07-14 14:20
【摘要】：目的:骨關(guān)節(jié)炎(Osteoarthritis,OA)的病理生理過(guò)程中力學(xué)因素起到至關(guān)重要的作用。本實(shí)驗(yàn)采用課題組自行設(shè)計(jì)的坡度半定量大鼠跑步設(shè)備獲得大鼠OA模型,通過(guò)研究OA發(fā)生和發(fā)展過(guò)程中其組織和基因水平的改變來(lái)探究在體應(yīng)力負(fù)荷在OA病理生理過(guò)程中的重要作用和相關(guān)機(jī)制。方法:3月齡SD(Sprague Dawley)大鼠84只,采用隨機(jī)數(shù)法分配到各組。大鼠膝關(guān)節(jié)應(yīng)力負(fù)荷采用坡度半定量(0°、10°和20°)方法。長(zhǎng)期應(yīng)力負(fù)荷跑步方案組完成長(zhǎng)達(dá)30d的跑步訓(xùn)練(速度為1km/h,每天跑步1h),頸椎脫臼法處死大鼠,取雙后肢膝關(guān)節(jié)軟骨;急性應(yīng)力負(fù)荷跑步方案組只進(jìn)行一次跑步訓(xùn)練(速度為1km/h,時(shí)長(zhǎng)1h),分別在跑步后0h、2h、6h、12h和24h取雙后肢膝關(guān)節(jié)軟骨。HE、番紅O和甲苯胺藍(lán)染色定性觀察關(guān)節(jié)軟骨大體形態(tài)和聚集蛋白聚糖(aggrecan,AGG)含量的改變,并根據(jù)OOCHAS軟骨損傷評(píng)分系統(tǒng)定量評(píng)價(jià)各組間差異;免疫組化觀察各組軟骨組織中II型膠原、X型膠原、IL-1β、TNF-α、MMP-3、MMP-9和MMP-13等表達(dá)差異;提取軟骨細(xì)胞的總m RNA,反轉(zhuǎn)錄為c DNA,RT-PCR檢測(cè)各組軟骨中II型膠原、AGG、I型膠原、X型膠原、IL-1、IL-6、TNF-α、MMP-3、MMP-9、MMP-13、SOX9和ADAMST5基因表達(dá)水平。采用SPSS統(tǒng)計(jì)軟件進(jìn)行方差分析和組間比較,檢驗(yàn)水平為P0.05。結(jié)果:采用長(zhǎng)期應(yīng)力跑步方案的大鼠,實(shí)驗(yàn)結(jié)束時(shí)膝關(guān)節(jié)軟骨出現(xiàn)了不同程度的損傷表現(xiàn)。平板跑步組大鼠膝關(guān)節(jié)軟骨HE染色、番紅O和甲苯胺藍(lán)染色與普通籠養(yǎng)對(duì)照組差異不明顯,10°和20°上坡組大鼠膝關(guān)節(jié)軟骨番紅O和甲苯胺藍(lán)染色逐漸變淺,軟骨組織中AGG含量逐漸減少,并可見(jiàn)關(guān)節(jié)表面出現(xiàn)不同程度的損傷,20°上坡組的損傷程度要大于10°上坡組,且軟骨細(xì)胞呈現(xiàn)出一定程度的肥大化表現(xiàn),軟骨細(xì)胞數(shù)量減少,胞質(zhì)比例增大,細(xì)胞陷窩明顯變大,排列紊亂,典型的關(guān)節(jié)軟骨層次丟失,軟骨厚度也明顯變薄,潮線消失或變得不明顯。隨著大鼠上坡運(yùn)動(dòng)的角度愈來(lái)愈大,膝關(guān)節(jié)受到的應(yīng)力負(fù)荷越來(lái)越大,采用單因素方差分析可知膝關(guān)節(jié)軟骨OOCHAS組織評(píng)分隨應(yīng)力負(fù)荷增大而增高(F=122.4,P0.0001),組織損傷表現(xiàn)越嚴(yán)重,多組間S-N-K法兩兩比較有統(tǒng)計(jì)學(xué)差異(P0.05)。免疫組化結(jié)果顯示隨著跑步坡度(即應(yīng)力負(fù)荷)的增大,關(guān)節(jié)軟骨中II型膠原的含量較籠養(yǎng)對(duì)照組逐漸減少而X型膠原含量逐漸增加;炎性介質(zhì)如IL-1β、TNF-α、MMP-3、MMP-9和MMP-13的表達(dá)量都有不同程度的增高。RT-PCR的結(jié)果顯示隨著應(yīng)力負(fù)荷的增加,大鼠膝關(guān)節(jié)軟骨II型膠原和AGG的m RNA表達(dá)水平逐漸降低,而IL-1、TNF-α、MMP-3、MMP-9和MMP-13等m RNA表達(dá)水平于對(duì)照組相比明顯增加,其中TNF-α和MMP-3的增高最明顯,IL-1的表達(dá)水平增高不明顯,且10°上坡跑步組IL-1與平板組差異不明顯。急性應(yīng)力跑步組大鼠經(jīng)過(guò)一次過(guò)度應(yīng)力負(fù)荷跑步后分別檢測(cè)其m RNA的表達(dá)水平,II型膠原表達(dá)量在6h時(shí)出現(xiàn)了短暫的升高,之后恢復(fù)到正常水平,且與應(yīng)力負(fù)荷強(qiáng)度呈正相關(guān);AGG、COL I、COL X的表達(dá)水平從6h開始逐漸升高,到24h時(shí)表達(dá)量明顯增高;IL-6在上坡運(yùn)動(dòng)后最早出現(xiàn)增高,之后逐漸降低;IL-1和TNF-α表達(dá)量從12h開始出現(xiàn)升高,但總體表達(dá)量增高不明顯;MMP-3、MMP-9和MMP-13的表達(dá)量從6h時(shí)開始升高,24h時(shí)上坡跑步組的表達(dá)量均明顯高于對(duì)照組和平板跑步組,其中MMP-13的表達(dá)水平增高的最明顯,24h時(shí)20°上坡跑步組的表達(dá)水平升高了近10倍;SOX9早期有輕微的升高,之后與對(duì)照組水平無(wú)差異;ADAMST5基因的表達(dá)在2h時(shí)達(dá)到最高,之后逐漸趨于正常;大多數(shù)基因表達(dá)量的改變幅度隨應(yīng)力負(fù)荷的強(qiáng)度增大而增大。結(jié)論:長(zhǎng)期過(guò)度應(yīng)力負(fù)荷作用于大鼠膝關(guān)節(jié)軟骨可使軟骨發(fā)生退行性改變,軟骨細(xì)胞數(shù)目減少伴肥大化改變,軟骨基質(zhì)損傷和降解,AGG和II型膠原含量減少,X型膠原增加,使關(guān)節(jié)軟骨發(fā)生退行性改變,其力學(xué)性能不再能夠適應(yīng)關(guān)節(jié)活動(dòng),關(guān)節(jié)表面摩擦阻力增大,在過(guò)度應(yīng)力負(fù)荷下發(fā)生撕裂和磨損,最終形成膝關(guān)節(jié)OA,這種改變與過(guò)度應(yīng)力負(fù)荷的強(qiáng)度呈正相關(guān)。過(guò)度應(yīng)力負(fù)荷可能首先通過(guò)提高IL-6的表達(dá)水平,激活I(lǐng)L-1和TNF-α等,促進(jìn)MMPs家族m RNA的合成,導(dǎo)致軟骨基質(zhì)的降解;而SOX9和ADAMST5的表達(dá)上調(diào)可能是軟骨細(xì)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R684.3
本文編號(hào)：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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R684.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李凱;侯志超;陳晉斌;王少偉;衛(wèi)小春;;大鼠跑步裝置的設(shè)計(jì)與應(yīng)用[J];實(shí)用骨科雜志;2010年04期
，本文編號(hào)：2121919
本文鏈接：http://sikaile.net/yixuelunwen/waikelunwen/2121919.html
最近更新
教材專著
