递增负荷运动对大鼠胸腺细胞膜电位及相关凋亡基因表达的影响
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摘要
目的探讨6周递增负荷运动对大鼠胸腺细胞膜电位及相关凋亡基因表达的影响。方法将96只雄性SD大鼠随机分为对照组和运动组,运动组进行跑台训练6周,分别于第0、2、4、6周末,用JC-1染色流式细胞术检测胸腺细胞形成的单体和聚合体的平均荧光强度,并计算线粒体膜电位(△Ψm);利用FQ-RT-PCR技术测定BaxmRNA和Bcl-2mRNA表达水平;免疫组化法测定Bax、Bcl-2和Cyt-C等蛋白表达水平。结果递增负荷运动过程中,WK2、WK4和WK6组大鼠胸腺细胞的△Ψm都显著低于WK0组;WK6组的大鼠胸腺细胞△Ψm显著低于WK4组;WK4、WK6组BaxmRNA表达显著高于WK0组;WK2、WK4和WK6组Bax蛋白表达显著高于WK0组;WK6组的Bax蛋白表达显著高于WK2组;WK4、WK6组大鼠胸腺组织Bcl-2mRNA表达显著低于WK0组;WK4、WK6组大鼠胸腺组织Bcl-2蛋白表达显著低于WK0组;WK6组的大鼠胸腺组织Bcl-2蛋白表达显著低于WK2组;WK4和WK6组Cyt-C蛋白表达都显著高于WK0组;WK6组的Cyt-C蛋白表达显著高于WK2组;胸腺细胞凋亡指数(SI)与△Ψm存在高度负相关,与Bcl-2mRNA和Bcl-2蛋白表达存在中度负相关,与BaxmRNA和Bax蛋白表达存在高度正相关,与Cyt-C蛋白表达和Caspase-3存在中度正相关。结论 6周递增负荷运动诱导胸腺细胞凋亡的过程中激活了线粒体凋亡信号途径,表明线粒体凋亡途径可能是运动诱导胸腺细胞凋亡增加,导致运动性免疫抑制的机制之一。
Objective The purpose of this study was to explore the mitochondria signal transduction pathway mechanism of thymocyte apoptosis during long-term incremental exercise. Method 96 SD rats,aged 8 weeks,were divided into Control Group and Exercise Group. The experiment lasted 6 weeks. Samples were taken in the end of weeks 0,2,4 and 6 respectively. The J-aggregate( —FL2) and J-monomer( —FL1) were measured by JC-1. △Ψm was calculated by( —FL2/—FL1). The BaxRNA and Bcl-2 mRNA were analyzed by FQ-RT-PCR,and the protein expressions of Bax、Bcl-2 and Cyt-C in thymocyte tissues was detected by Immunocytochemistry. Results In WK2,WK4 and WK6,the △Ψm was much lower than WK0. In WK6,the △Ψm was much lower than WK4. In WK4 and WK6,the expression of BaxmRNA was much higher than WK0. In WK2,WK4 and WK6,The protein expressions of Bax was much higher than WK0. In WK6,The protein expressions of Bax was much higher than WK2. In WK4 and WK6,the expression of Bcl-2 mRNA was much lower than WK0,and The protein expressions of Bcl-2 was much lower than WK0. In WK6,The protein expressions of Bcl-2 was much higher than WK2. The protein expressions of Cyt-C was much higher than WK0 in WK4 and WK6. SI and △Ψare high negative correlation,SI and BaxmRNA and Bax are highly positively correlated,SI and Bcl-2 mRNA and Bcl-2 are moderate negative correlation,SI and Cyt-C and Caspase-3 are moderate positive correlation. Conclusion The mitochondria apoptotic signal pathway was activated during Long-term incremental exercise. It is indicated that the mitochondrial apoptosis pathway is one of the mechanisms of exercise induced apoptosis in the thymus.
Objective The purpose of this study was to explore the mitochondria signal transduction pathway mechanism of thymocyte apoptosis during long-term incremental exercise. Method 96 SD rats,aged 8 weeks,were divided into Control Group and Exercise Group. The experiment lasted 6 weeks. Samples were taken in the end of weeks 0,2,4 and 6 respectively. The J-aggregate( —FL2) and J-monomer( —FL1) were measured by JC-1. △Ψm was calculated by( —FL2/—FL1). The BaxRNA and Bcl-2 mRNA were analyzed by FQ-RT-PCR,and the protein expressions of Bax、Bcl-2 and Cyt-C in thymocyte tissues was detected by Immunocytochemistry. Results In WK2,WK4 and WK6,the △Ψm was much lower than WK0. In WK6,the △Ψm was much lower than WK4. In WK4 and WK6,the expression of BaxmRNA was much higher than WK0. In WK2,WK4 and WK6,The protein expressions of Bax was much higher than WK0. In WK6,The protein expressions of Bax was much higher than WK2. In WK4 and WK6,the expression of Bcl-2 mRNA was much lower than WK0,and The protein expressions of Bcl-2 was much lower than WK0. In WK6,The protein expressions of Bcl-2 was much higher than WK2. The protein expressions of Cyt-C was much higher than WK0 in WK4 and WK6. SI and △Ψare high negative correlation,SI and BaxmRNA and Bax are highly positively correlated,SI and Bcl-2 mRNA and Bcl-2 are moderate negative correlation,SI and Cyt-C and Caspase-3 are moderate positive correlation. Conclusion The mitochondria apoptotic signal pathway was activated during Long-term incremental exercise. It is indicated that the mitochondrial apoptosis pathway is one of the mechanisms of exercise induced apoptosis in the thymus.
引文
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[13]王雪芹.T细胞活性与巨噬细胞吞噬能力对长期递增负荷运动的应答和适应特征[D].广州:华南师范大学,2005.
[14]王雪芹,郝选明.6周递增负荷运动对大鼠小肠集合淋巴结细胞线粒体凋亡途径的影响[J].体育学刊,2013,21(2):141-144.
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[19]HAN Y T,CHEN X H,XIE J,et al.Purple batatopigmentoj scavenge ROS,redukti p53 kaj modular Bcl-2/Bax por deteni radiado induktita apoptozo en murine thymocytes[J].elo Physiol Biochem,2011,28(5):865-872.
[20]崔玉芳,丁彦青,徐菡.致死剂量γ射线照射小鼠淋巴结淋巴细胞凋亡动力学及其与相关蛋白表达的关系[J].辐射研究与辐射工艺学报,2005,2(1):57-61.
[21]XIN M G,DENG X M.Protein phosphatase-A enhances the proapoptotic function of Bax through D ephosphorylation[J].J Biol Chem,2006,281:18859-18867.
[22]CAIN K,BRATTON S B,COHEN G M.The Apaf-1 apoptosis:a large caspase-activating complex[J].BioChimie,2002,9(2/3):203.
[23]GARRIDO C,GALLUZZI L,BRUNET M,et al.Mechanisms of cytochrome c release from mitochondria[J].Cell Death Differ,2006,13(9):1423-1433.
[24]FERRI K F,JACOTOT E,BLANCO J,et al.Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex:role of mitochondria and caspases[J].J Exp Med,2000,192(8):1081-1092.
[2]ESKES R,DESAGHER S,ANTONSSON B,et al.Bid induces the oligomerization and insertion of Bax into the outermitochondrial membrane[J].Mol Cell Biol,2000,20(3):929-935.
[3]PERCHELLET E M,WANG Y,WEBER R L,et al.Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and-9 in HL-60 cells by a mechanism which does not involve Fas signaling[J].Anticancer Drugs,2004,15(10):929-946。
[4]TAN K O,FUN Y,SUKUMARAN S K,et al.MAP-1 is am itochondrial effectors of Bax[J].Proc Natl A cad SciUSA,2005,102(41):14623-14628.
[5]DAUGAS E,SUSIN S A,ZAM I N,et al.Mitochond rionuclear translocation of AIF in apoptosis and necrosis[J].FASEB J,2000,14(5):729-739.
[6]CAIN K,BRATTON S B,LANGLAIS C,et al.Apaf-1oligomerizes into biologically active approximately 700-k Da and inactive approximately 1.4-MD aapoptosome complexes[J].J Biol Chem,2000,275(9):6067-6070.
[7]NAVALTA J W,MCFARLIN B K,LYONS T S.Does exercise really induce lymphocyte apoptosis[J]Front Biosci(Elite Ed),2010(2):478-488.
[8]王雪芹.长期递增负荷运动对大鼠胸腺细胞凋亡及caspaes-3表达的影响[J].北京体育大学学报,2014,37(4):68-71.
[9]王雪芹.长期递增负荷运动对大鼠脾淋巴细胞增殖活性、凋亡相关基因表达的影响[J].中国体育科技,2013,49(6):100-104.
[10]覃飞,郝选明.长期递增负荷运动对小肠集合淋巴结结构及淋巴细胞凋亡的影响[J].体育学刊,2012,19(1):134-138.
[11]张琳,郝选明.胸腺细胞转录因子TCF-1、Nur77以及mRNA对长期递增负荷运动的应答性特征[J].北京体育大学学报,2012,35(3):56-59.
[12]曹伟,郝选明.六周递增负荷运动对大鼠胸腺蛋白组表达的影响[J].体育科学,2013,33(6):64-68.
[13]王雪芹.T细胞活性与巨噬细胞吞噬能力对长期递增负荷运动的应答和适应特征[D].广州:华南师范大学,2005.
[14]王雪芹,郝选明.6周递增负荷运动对大鼠小肠集合淋巴结细胞线粒体凋亡途径的影响[J].体育学刊,2013,21(2):141-144.
[15]ADRAIN C,MARTIN S J.The mitochondrial apoptosome:a killer unleashed by the cytochrome seas[J].Trends Biochem Sci,2001,26(6):390-397.
[16]LY J D,GRUBB D R,LAWEN A.The mitochondrial membrane potential(deltapsi(m))in apoptosis;an update[J].Apoptosis,2003,8(2):115-128.
[17]PAPINAZATH T,MIN W,SYJITHTHA S,et al.Effects of purine nucleoside phosphorylase deficiency on thymocyte development[J].J Allergy Clin Immunol,2011,128(4):854-863.
[18]TUAN T C,HSU T G,FONG M C,et al.Deleterious effects of short-term,high-intensity exercise on immune function:evidence from leucocyte mitochondrial alterations and apoptosis[J].Br J Sports Med,2008,42(1):11-5.
[19]HAN Y T,CHEN X H,XIE J,et al.Purple batatopigmentoj scavenge ROS,redukti p53 kaj modular Bcl-2/Bax por deteni radiado induktita apoptozo en murine thymocytes[J].elo Physiol Biochem,2011,28(5):865-872.
[20]崔玉芳,丁彦青,徐菡.致死剂量γ射线照射小鼠淋巴结淋巴细胞凋亡动力学及其与相关蛋白表达的关系[J].辐射研究与辐射工艺学报,2005,2(1):57-61.
[21]XIN M G,DENG X M.Protein phosphatase-A enhances the proapoptotic function of Bax through D ephosphorylation[J].J Biol Chem,2006,281:18859-18867.
[22]CAIN K,BRATTON S B,COHEN G M.The Apaf-1 apoptosis:a large caspase-activating complex[J].BioChimie,2002,9(2/3):203.
[23]GARRIDO C,GALLUZZI L,BRUNET M,et al.Mechanisms of cytochrome c release from mitochondria[J].Cell Death Differ,2006,13(9):1423-1433.
[24]FERRI K F,JACOTOT E,BLANCO J,et al.Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex:role of mitochondria and caspases[J].J Exp Med,2000,192(8):1081-1092.