诱导HO-1对老年骨骼肌微环境及肌卫星细胞增殖分化的影响

发布时间：2018-05-26 15:09

本文选题：血红素加氧酶1 + 骨骼肌　；参考：《第三军医大学》2015年硕士论文

【摘要】：背景和目的:伴随机体老化进程,骨骼肌卫星细胞数量和功能逐步减少减退,这是老年骨骼肌再生能力下降的主要原因。肌卫星细胞微环境对其增殖分化具有重要影响。课题通过研究改善老年小鼠骨骼肌组织氧化应激和炎症反应等微环境变化,观察对肌卫星细胞增殖分化潜能和骨骼肌损伤再生的影响,以探讨促进老年骨骼肌再生能力的新途径,为增龄性骨骼肌丢失症等肌萎缩性疾病的防治提供对策。方法:1.分别将正常成年(2月龄)和老年(18月龄)C57BL/6雄性小鼠按照简单随机抽样法分为对照组及注射钴原卟啉(Cobalt Protoporphyrin,CoPP)组;CoPP组小鼠连续3 d腹腔注射CoPP溶液[5 mg/(kg?d)],对照组注射同体积生理盐水。2.Western Blot检测成年和老年小鼠骨骼肌组织对照组和诱导剂CoPP处理组血红素加氧酶-1(Heme Oxygenase-1,HO-1)蛋白表达变化;3.ELISA检测成年和老年小鼠骨骼肌对照组、CoPP诱导HO-1组过氧化标志分子丙二醛(Malondialdehyde,MDA),过氧化氢(H2O2)含量及抗氧化酶类超氧化物歧化酶(Superoxide Dismutase,SOD),CuZn-超氧化物歧化酶(Cu Zn-Superoxide Dismutase,SOD),谷胱甘肽氧化还原酶(Gluathion Peroxidase,GSH-pX)及过氧化氢酶(Catalase,CAT)活性变化。4.建立老年小鼠腓肠肌损伤模型,髓过氧化物酶(Myeloperoxidase,MPO),CD163免疫组织化学染色检测老年小鼠骨骼肌损伤对照及损伤后CoPP诱导HO-1组中性粒细胞和M2型巨噬细胞浸润;Western Blot检测CD163蛋白表达变化。5.Western Blot和Real-Time PCR分别从蛋白和RNA水平检测老年小鼠骨骼肌损伤不同时间点CoPP诱导HO-1组MyoD,Myogenin表达变化。6.HE染色,胚胎肌球蛋白重链(embryonic Myosin Heavy Chain,e MHC)免疫荧光染色观察老年小鼠骨骼肌损伤对照和Co PP诱导HO-1组骨骼肌组织,特别是新生肌纤维变化。7.westernblot检测老年小鼠骨骼肌损伤对照及损伤后copp诱导ho-1组Ⅰ型胶原蛋白(collageni,coli)表达变化;masson三色染色观察copp诱导ho-1对老年小鼠骨骼肌损伤后纤维沉积的影响。结果:1.在正常小鼠骨骼肌组织,老年组ho-1表达高于成年组,老年copp诱导组ho-1表达升幅显著低于成年copp诱导组。2.注射copp后,成年copp诱导组和老年copp诱导组t-sod、cuzn-sod酶活性均升高,mda含量降低;老年copp诱导组t-sod,cuzn-sod酶活性低于成年copp诱导组,mda含量高于成年copp诱导组。成年copp诱导组和老年copp诱导组gsh-px、cat酶活性升高,h2o2含量降低;老年copp诱导组gsh-px酶活性高于成年copp诱导组,cat酶活性低于成年copp诱导组,h2o2含量高于成年copp诱导组。3.老年小鼠骨骼肌损伤后,copp诱导组中性粒细胞浸润在3d,7d均低于对照组;cd163阳性m2型巨噬细胞浸润出现较晚,注射copp后,m2型巨噬细胞的浸润显著高于损伤对照组。4.老年小鼠骨骼肌损伤后,注射copp诱导ho-1表达3d,7d,14d,实验组myod,myogenin表达水平显著高于损伤对照组,7d时myod有最大表达量,myogenin的最大表达量出现在14d。5.老年小鼠骨骼肌损伤后,注射copp5d,7d时新生骨骼肌纤维增多;与对照组比较,copp处理组emhc阳性纤维显著升高。6.老年小鼠骨骼肌损伤后,注射copp7d,14d,21d,28d,实验组coli蛋白表达量显著低于损伤对照组;胶原纤维数量同样低于损伤对照组。21d时coli表达量和胶原纤维沉积最高。结论:1.老年小鼠骨骼肌组织ho-1对外界刺激的反应性下降,抗氧化应激能力下降。2.copp诱导ho-1可以显著降低成年和老年小鼠骨骼肌组织内的过氧化反应,提高抗氧化酶活性,改善肌组织内的氧化应激状态。3.老年小鼠骨骼肌损伤后,copp诱导ho-1能够降低早期中性粒细胞、促进m2型巨噬细胞的浸润,加快老年小鼠骨骼肌组织的炎症反应进程。4.老年小鼠骨骼肌损伤后,CoPP诱导HO-1能够促进肌卫星细胞活化增殖。5.CoPP诱导HO-1改善老年小鼠骨骼肌氧化应激和炎症反应,可以促进老年骨骼肌损伤后再生和修复,降低其纤维化反应。
[Abstract]:Background and purpose: with the aging process, the number and function of skeletal muscle satellite cells decrease and decrease gradually. This is the main reason for the decline of skeletal muscle regeneration ability in the elderly. The microsatellite cell microenvironment has an important influence on its proliferation and differentiation. The effects on the proliferation and differentiation potential of muscle satellite cells and the regeneration of skeletal muscle injury were observed in order to explore a new way to promote the regeneration of skeletal muscle, and to provide countermeasures for the prevention and treatment of amyotrophic diseases such as skeletal muscle loss. Method 1. the male mice of normal adult (2 month old) and aged (18 month old) were in accordance with normal adult (1.). The simple random sampling was divided into the control group and the injection of Cobalt Protoporphyrin (CoPP) group, and the mice in the CoPP group were injected with CoPP solution [5 mg/ (kg? D) intraperitoneally 3 D in the CoPP group, and the control group was injected with the same volume physiological saline for.2.Western Blot to detect the skeletal muscle group in adult and the aged mice and the inducer to treat the heme oxygenase. Oxygenase-1, HO-1) protein expression changes; 3.ELISA detection of adult and elderly mice skeletal muscle control group, CoPP induced HO-1 group peroxidation marker malondialdehyde (Malondialdehyde, MDA), hydrogen peroxide (H2O2) content and antioxidant enzyme superoxide dismutase (Superoxide Dismutase, SOD), CuZn- superoxide dismutase ASE, SOD), glutathione oxidoreductase (Gluathion Peroxidase, GSH-pX) and catalase (Catalase, CAT) activity change.4. to establish the gastrocnemius injury model in old mice, myeloperoxidase (Myeloperoxidase, MPO), CD163 immunohistochemical staining for the detection of skeletal muscle damage in aged mice and CoPP induced neutral particles after injury. Cell and M2 type macrophage infiltration, Western Blot detection of CD163 protein expression change.5.Western Blot and Real-Time PCR from protein and RNA level detection of skeletal muscle damage at different time points in old mice CoPP induced HO-1 group MyoD. The skeletal muscle injury control and Co PP induction of skeletal muscle tissue in the elderly mice were observed by immunofluorescence staining, especially in the HO-1 group of HO-1 group, especially the changes of the muscle fibers of the newborn muscles were detected by.7.westernblot. The expression of type I collagen (CollagenI, coli) in the HO-1 group induced by copp was changed after the injury, and the Masson tricolor staining was used to observe the HO-1 to the elderly by copp. The effect of fibrous deposition on skeletal muscle injury in mice. Results: 1. in the skeletal muscle tissue of normal mice, the expression of HO-1 in the aged group was higher than that in the adult group. The increase of HO-1 expression in the elderly copp induced group was significantly lower than that of the adult copp induced group.2. injected with copp. The T-SOD in the adult copp induction group and the old copp induction group increased, the CuZn-SOD enzyme activity increased and the MDA content decreased. The activity of T-SOD, CuZn-SOD enzyme in copp induction group was lower than that of adult copp induction group, and the content of MDA was higher than that of adult copp induction group. The activity of cat enzyme in adult copp induction group and old copp induction group increased, H2O2 content decreased, and the activity of senile copp induction group was higher than that of adult induction group, and the activity of the enzyme was lower than that of adult induction group. The infiltration of neutrophils in copp induction group was 3D, 7d was lower than that of control group, and CD163 positive M2 type macrophage infiltrated later. After copp, the infiltration of M2 type macrophages was significantly higher than that of the control group. After copp, the infiltration of M2 type macrophages was significantly higher than that of the control group. After copp, the infiltration of CD163 positive M2 type macrophages was significantly higher than that of the skeletal muscle in the control group of.4. old mice. The expression level of MyoD, myogenin in the experimental group was significantly higher than that in the control group, and the maximum expression of MyoD was found at 7d. The maximum expression of myogenin appeared in the skeletal muscle injury of 14d.5. old mice, and the increase of skeletal muscle fibers was increased at copp5d and 7d. Compared with the control group, the copp treatment group emhc positive fiber significantly increased the skeletal muscle damage in the.6. old mice. After injection of copp7d, 14d, 21d, 28d, the expression of coli protein in the experimental group was significantly lower than that in the control group, and the number of collagen fibers was also lower than that of the control group.21d coli expression and the highest deposition of collagen fibers. Conclusion: 1. the reverse response of HO-1 to the external stimuli in the skeletal muscle tissue of the aged mice decreased and the antioxidant stress decreased.2.copp induced HO-1. In order to significantly reduce the peroxidation in the skeletal muscle tissue of adult and old mice, improve the activity of antioxidant enzymes and improve the oxidative stress in the muscle tissue, the copp induced HO-1 can reduce the early neutrophils, promote the infiltration of M2 type macrophages and accelerate the inflammatory reaction in the skeletal muscle tissue of the aged mice. After the skeletal muscle injury of.4. aged mice, CoPP induced HO-1 can promote the activation and proliferation of muscle satellite cells to induce HO-1 to improve the oxidative stress and inflammatory response of skeletal muscle in old mice. It can promote the regeneration and repair of skeletal muscle injury in the aged and reduce the fibrosis reaction.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R68

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