运动性骨疲劳大鼠骨代谢与骨调因子的研究
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摘要
研究目的:运动性骨疲劳是年轻运动员的常见现象,也是一直困扰运动员的一个棘手问题,但目前关于运动性骨疲劳的发生机理尚不清楚。本研究从建立运动性骨疲劳动物模型入手,从骨密度、骨生物力学、骨代谢生化标志物、骨调因子的蛋白和基因表达等不同层面,分析运动性骨疲劳发生时骨变化的特点,探讨运动性骨疲劳的发生机理。
研究方法:66只8周龄雌性SD大鼠,随机分为三组:运动性骨疲劳动物模型组(36只),对照组(20只,其中10只笼养8周),一次性大强度运动组(10只);其中运动性骨疲劳动物模型组进行4周大强度跑台运动,造模期间每周对大鼠进行99mTc-MDP骨显影监测,并进行骨刚度(stiffness)、血清碱性磷酸酶(ALP)、抗酒石酸酸性磷酸酶(TRAP)、雌激素(E2)指标变化的测定,当确诊达到运动性骨疲劳标准后,完成运动性骨疲劳动物模型的建立过程。将运动性骨疲劳大鼠随机分为两组,分别进行自由恢复和积极运动干预恢复,时间均为4周,采用DXA法测定胫骨BMD、BMC含量,在Anuda微机控制电子万能试验机上用三点弯曲法测定胫骨生物力学指标,双抗体夹心酶标免疫分析法测定血清E2含量,比色法测定血清ALP、TRAP含量,免疫组化法检测胫骨组织ER和TGF- ?1蛋白表达,RT-PCR法测定胫骨组织ER、TGF-?1、BMP、OPG和OPGL基因转录水平的表达。
研究结果:(1)4周大强度跑台运动导致胫骨刚度显著下降(-33%),骨99mTc-MDP显影显著上升(P<0.05),血清E2显著下降(P<0.05),ALP含量极显著下降(P<0.01),TRAP极显著升高(P<0.01),根据骨疲劳标准证实本研究成功建立运动性骨疲劳动物模型;一次性大强度跑台运动没有引起大鼠发生上述指标的显著改变。(2)运动性骨疲劳雌性大鼠血清ALP极显著下降(P<0.01),E2含量显著低于对照组(P<0.05),血清TRAP含量显著高于对照组(P<0.01)。(3)运动性骨疲劳雌性大鼠胫骨BMD、最大载荷、刚度、最大应力、弹性模量均极显著低于对照组(P<0.01),BMC、最大能量吸收均显著低于对照组(P<0.05),一次性大强度跑台运动组大鼠骨量和生物力学指标均无显著改变。(4)运动性骨疲劳雌性大鼠骨组织ER、TGF-?1蛋白表达均极显著低于对照组(P<0.01),ER、TGF-?1、BMP基因转录水平表达均显著低于对照组(P<0.05),OPG基因转录水平极显著下降(P<0.01),OPGL基因转录水平表达显著高于对照组(P<0.05),一次性大强度运动组大鼠蛋白和基因表达均未发生显著改变。(5)运动性骨疲劳雌性大鼠上述指标,经过4周适宜运动和自由恢复后,组间比较无统计学意义。
研究结论:经过4周大强度跑台运动,本研究成功建立大鼠运动性骨疲劳模型,而一次性大强度跑台运动不能引起骨疲劳;运动性骨疲劳大鼠雌激素水平下降,骨形成指标下降,骨吸收指标升高,发生骨代谢紊乱,胫骨骨密度降低,胫骨生物力学性能降低,是运动性骨疲劳大鼠骨机械性能下降的基础;运动性骨疲劳大鼠ER表达的降低是运动性骨疲劳发生机理的关键环节,骨组织TGF-?1蛋白和转录水平的降低、BMP表达降低、OPG表达降低、OPGL表达升高是影响运动性骨疲劳大鼠骨代谢的重要分子生物学因素;运动性骨疲劳对雌性大鼠骨变化的抑制是可逆的。
Objective: exercise induced bone fatigue is a common phenomenon and is one of the trobulesome problems for athletes.It is not completely known by us about the mechanism of sport bone fatigue.The study started establishing repeatable animal model of sport bone fatigue and investigated bone mineral density, bone biomechanics, serum biochemical markers of bone and modulative factors of bone to analyse characteristics of bone changes and discuss the mechanism on exercise induced bone fatigue.
Methods: 66 female SD rats(8weeks old)were randomly assigned to three groups:animal model group of sport bone fatigue(36), quiet control group (20,10 of them captive for 8 weeks)and a bout of high-intensity exercise group (10);animal model groupof sport bone fatigue run high intensity treadmill exercise for 4 weeks. During the modeling, rats’bone 99mTc-MDP were monitored by SPECT; bone stiffness were measured;serum E2 level were measured by double-antibody sandwich enzyme immunoassay;blood serum ALP and TRAP level were measured by colorimetry; When they were diagnosed to achieve bone fatigue according to standards.So we complete the animal model of the exercise induced bone fatigue. animal model group of the exercise induced bone fatigue were randomly assigned to two groups:free recovery group and exercise intervention recovery group.All rats recover for 4 weeks.serum E2 level were measured by double-antibody sandwich enzyme immunoassay;blood serum ALP and TRAP level were measured by colorimetry tibiaes’parameters of BMD and BMC were measured by DXA method;bone biomechanics were measured by anuda microcomputer control electronic universal testing machine; protein expression of ER and TGF-?1 in bone tissue were measured through immunohistochemistry;gene expression of ER,TGF-?1,BMP,OPG and OPGL in bone tissue were examined RT-PCR.
Results:(1) After heavy treadmill running of four weeks, bones’stiffness of rat decreases 33%; the technetium-99m-methylene diphosphonate has obviously rising(P<0.05); concentration of tartrate-resistant acid phosphatase in blood serum has obviously increased(P<0.01); the level of estrogen and alkaline phosphatase in blood serum has obvious decreased(P<0.05 or P<0.01).These has proved that the building up of the animal experimental mode is successful, A bout of high-intensity treadmill exercise did not give rise to changes on these indicators significantly. (2)Alkaline phosphatase of serum were significantly lower(P<0.01) and serum estrogen lower(P<0.05)in exercise induced bone fatigue;tartrate-resistant acid phosphatase of serum significantly higher in exercise induced bone fatigue than group(P<0.01); (3) bone mineral density,maximum load, stiffness, maximum stress and modulus were significantly lower(P<0.01);bone mass content and maximum energy absorption were significantly lower in exercise induced bone fatigue rats; (4) Protein expression of estrogen receptor and transforming growth factor ?1 was significantly lower in exercise induced bone fatigue group than in control group(P<0.01), gene expression of estrogen receptor, transforming growth factor ?1, bone morphogenetic protein-2 , osteoprotegerin were significantly lower in exercise induced bone fatigue group(P<0.05 or P<0.01);gene expression of osteoprotegerin ligand was significantly higher(P<0.05); (5)the above-mentioned indicators are all no statistically significant difference after free recovery and exercise recovery of four weeks.
Conclusion: This study established rat model of exercise induced bone fatigue after 4 weeks of high-intensity treadmill exercise successfully; while one-time high-intensity treadmill exercise does not cause the occurrence of bone fatigue.rats of exercise induced bone fatigue decrease estrogen levels, decline in bone formation markers and increased bone resorption markers, leading to the occurrence of bone metabolism disorder finally; tibial bone mineral density decrease and biomechanical properties of tibia reduce, they are the basis of falling on bone mechanical properties in rat of exercised bone fatigue. Decreasing of ER expression is the key to exercise induced bone fatigue pathogenesis in rats. Decreasing of Expression on TGF-?1、BMP and OPG and increasing of OPGL expression in bone tissue was the important factor in molecular biology in rats of exercise induced bone fatigue; suppression of bone changes on exercise induced bone fatigue in female rats is reversible.
研究方法:66只8周龄雌性SD大鼠,随机分为三组:运动性骨疲劳动物模型组(36只),对照组(20只,其中10只笼养8周),一次性大强度运动组(10只);其中运动性骨疲劳动物模型组进行4周大强度跑台运动,造模期间每周对大鼠进行99mTc-MDP骨显影监测,并进行骨刚度(stiffness)、血清碱性磷酸酶(ALP)、抗酒石酸酸性磷酸酶(TRAP)、雌激素(E2)指标变化的测定,当确诊达到运动性骨疲劳标准后,完成运动性骨疲劳动物模型的建立过程。将运动性骨疲劳大鼠随机分为两组,分别进行自由恢复和积极运动干预恢复,时间均为4周,采用DXA法测定胫骨BMD、BMC含量,在Anuda微机控制电子万能试验机上用三点弯曲法测定胫骨生物力学指标,双抗体夹心酶标免疫分析法测定血清E2含量,比色法测定血清ALP、TRAP含量,免疫组化法检测胫骨组织ER和TGF- ?1蛋白表达,RT-PCR法测定胫骨组织ER、TGF-?1、BMP、OPG和OPGL基因转录水平的表达。
研究结果:(1)4周大强度跑台运动导致胫骨刚度显著下降(-33%),骨99mTc-MDP显影显著上升(P<0.05),血清E2显著下降(P<0.05),ALP含量极显著下降(P<0.01),TRAP极显著升高(P<0.01),根据骨疲劳标准证实本研究成功建立运动性骨疲劳动物模型;一次性大强度跑台运动没有引起大鼠发生上述指标的显著改变。(2)运动性骨疲劳雌性大鼠血清ALP极显著下降(P<0.01),E2含量显著低于对照组(P<0.05),血清TRAP含量显著高于对照组(P<0.01)。(3)运动性骨疲劳雌性大鼠胫骨BMD、最大载荷、刚度、最大应力、弹性模量均极显著低于对照组(P<0.01),BMC、最大能量吸收均显著低于对照组(P<0.05),一次性大强度跑台运动组大鼠骨量和生物力学指标均无显著改变。(4)运动性骨疲劳雌性大鼠骨组织ER、TGF-?1蛋白表达均极显著低于对照组(P<0.01),ER、TGF-?1、BMP基因转录水平表达均显著低于对照组(P<0.05),OPG基因转录水平极显著下降(P<0.01),OPGL基因转录水平表达显著高于对照组(P<0.05),一次性大强度运动组大鼠蛋白和基因表达均未发生显著改变。(5)运动性骨疲劳雌性大鼠上述指标,经过4周适宜运动和自由恢复后,组间比较无统计学意义。
研究结论:经过4周大强度跑台运动,本研究成功建立大鼠运动性骨疲劳模型,而一次性大强度跑台运动不能引起骨疲劳;运动性骨疲劳大鼠雌激素水平下降,骨形成指标下降,骨吸收指标升高,发生骨代谢紊乱,胫骨骨密度降低,胫骨生物力学性能降低,是运动性骨疲劳大鼠骨机械性能下降的基础;运动性骨疲劳大鼠ER表达的降低是运动性骨疲劳发生机理的关键环节,骨组织TGF-?1蛋白和转录水平的降低、BMP表达降低、OPG表达降低、OPGL表达升高是影响运动性骨疲劳大鼠骨代谢的重要分子生物学因素;运动性骨疲劳对雌性大鼠骨变化的抑制是可逆的。
Objective: exercise induced bone fatigue is a common phenomenon and is one of the trobulesome problems for athletes.It is not completely known by us about the mechanism of sport bone fatigue.The study started establishing repeatable animal model of sport bone fatigue and investigated bone mineral density, bone biomechanics, serum biochemical markers of bone and modulative factors of bone to analyse characteristics of bone changes and discuss the mechanism on exercise induced bone fatigue.
Methods: 66 female SD rats(8weeks old)were randomly assigned to three groups:animal model group of sport bone fatigue(36), quiet control group (20,10 of them captive for 8 weeks)and a bout of high-intensity exercise group (10);animal model groupof sport bone fatigue run high intensity treadmill exercise for 4 weeks. During the modeling, rats’bone 99mTc-MDP were monitored by SPECT; bone stiffness were measured;serum E2 level were measured by double-antibody sandwich enzyme immunoassay;blood serum ALP and TRAP level were measured by colorimetry; When they were diagnosed to achieve bone fatigue according to standards.So we complete the animal model of the exercise induced bone fatigue. animal model group of the exercise induced bone fatigue were randomly assigned to two groups:free recovery group and exercise intervention recovery group.All rats recover for 4 weeks.serum E2 level were measured by double-antibody sandwich enzyme immunoassay;blood serum ALP and TRAP level were measured by colorimetry tibiaes’parameters of BMD and BMC were measured by DXA method;bone biomechanics were measured by anuda microcomputer control electronic universal testing machine; protein expression of ER and TGF-?1 in bone tissue were measured through immunohistochemistry;gene expression of ER,TGF-?1,BMP,OPG and OPGL in bone tissue were examined RT-PCR.
Results:(1) After heavy treadmill running of four weeks, bones’stiffness of rat decreases 33%; the technetium-99m-methylene diphosphonate has obviously rising(P<0.05); concentration of tartrate-resistant acid phosphatase in blood serum has obviously increased(P<0.01); the level of estrogen and alkaline phosphatase in blood serum has obvious decreased(P<0.05 or P<0.01).These has proved that the building up of the animal experimental mode is successful, A bout of high-intensity treadmill exercise did not give rise to changes on these indicators significantly. (2)Alkaline phosphatase of serum were significantly lower(P<0.01) and serum estrogen lower(P<0.05)in exercise induced bone fatigue;tartrate-resistant acid phosphatase of serum significantly higher in exercise induced bone fatigue than group(P<0.01); (3) bone mineral density,maximum load, stiffness, maximum stress and modulus were significantly lower(P<0.01);bone mass content and maximum energy absorption were significantly lower in exercise induced bone fatigue rats; (4) Protein expression of estrogen receptor and transforming growth factor ?1 was significantly lower in exercise induced bone fatigue group than in control group(P<0.01), gene expression of estrogen receptor, transforming growth factor ?1, bone morphogenetic protein-2 , osteoprotegerin were significantly lower in exercise induced bone fatigue group(P<0.05 or P<0.01);gene expression of osteoprotegerin ligand was significantly higher(P<0.05); (5)the above-mentioned indicators are all no statistically significant difference after free recovery and exercise recovery of four weeks.
Conclusion: This study established rat model of exercise induced bone fatigue after 4 weeks of high-intensity treadmill exercise successfully; while one-time high-intensity treadmill exercise does not cause the occurrence of bone fatigue.rats of exercise induced bone fatigue decrease estrogen levels, decline in bone formation markers and increased bone resorption markers, leading to the occurrence of bone metabolism disorder finally; tibial bone mineral density decrease and biomechanical properties of tibia reduce, they are the basis of falling on bone mechanical properties in rat of exercised bone fatigue. Decreasing of ER expression is the key to exercise induced bone fatigue pathogenesis in rats. Decreasing of Expression on TGF-?1、BMP and OPG and increasing of OPGL expression in bone tissue was the important factor in molecular biology in rats of exercise induced bone fatigue; suppression of bone changes on exercise induced bone fatigue in female rats is reversible.
引文
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