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  本文選題：血紅素加氧酶1 + 骨骼肌��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：背景和目的:伴隨機(jī)體老化進(jìn)程,骨骼肌衛(wèi)星細(xì)胞數(shù)量和功能逐步減少減退,這是老年骨骼肌再生能力下降的主要原因。肌衛(wèi)星細(xì)胞微環(huán)境對其增殖分化具有重要影響。課題通過研究改善老年小鼠骨骼肌組織氧化應(yīng)激和炎癥反應(yīng)等微環(huán)境變化,觀察對肌衛(wèi)星細(xì)胞增殖分化潛能和骨骼肌損傷再生的影響,以探討促進(jìn)老年骨骼肌再生能力的新途徑,為增齡性骨骼肌丟失癥等肌萎縮性疾病的防治提供對策。方法:1.分別將正常成年(2月齡)和老年(18月齡)C57BL/6雄性小鼠按照簡單隨機(jī)抽樣法分為對照組及注射鈷原卟啉(Cobalt Protoporphyrin,CoPP)組;CoPP組小鼠連續(xù)3 d腹腔注射CoPP溶液[5 mg/(kg?d)],對照組注射同體積生理鹽水。2.Western Blot檢測成年和老年小鼠骨骼肌組織對照組和誘導(dǎo)劑CoPP處理組血紅素加氧酶-1(Heme Oxygenase-1,HO-1)蛋白表達(dá)變化;3.ELISA檢測成年和老年小鼠骨骼肌對照組、CoPP誘導(dǎo)HO-1組過氧化標(biāo)志分子丙二醛(Malondialdehyde,MDA),過氧化氫(H2O2)含量及抗氧化酶類超氧化物歧化酶(Superoxide Dismutase,SOD),CuZn-超氧化物歧化酶(Cu Zn-Superoxide Dismutase,SOD),谷胱甘肽氧化還原酶(Gluathion Peroxidase,GSH-pX)及過氧化氫酶(Catalase,CAT)活性變化。4.建立老年小鼠腓腸肌損傷模型,髓過氧化物酶(Myeloperoxidase,MPO),CD163免疫組織化學(xué)染色檢測老年小鼠骨骼肌損傷對照及損傷后CoPP誘導(dǎo)HO-1組中性粒細(xì)胞和M2型巨噬細(xì)胞浸潤;Western Blot檢測CD163蛋白表達(dá)變化。5.Western Blot和Real-Time PCR分別從蛋白和RNA水平檢測老年小鼠骨骼肌損傷不同時間點(diǎn)CoPP誘導(dǎo)HO-1組MyoD,Myogenin表達(dá)變化。6.HE染色,胚胎肌球蛋白重鏈(embryonic Myosin Heavy Chain,e MHC)免疫熒光染色觀察老年小鼠骨骼肌損傷對照和Co PP誘導(dǎo)HO-1組骨骼肌組織,特別是新生肌纖維變化。7.westernblot檢測老年小鼠骨骼肌損傷對照及損傷后copp誘導(dǎo)ho-1組Ⅰ型膠原蛋白(collageni,coli)表達(dá)變化;masson三色染色觀察copp誘導(dǎo)ho-1對老年小鼠骨骼肌損傷后纖維沉積的影響。結(jié)果:1.在正常小鼠骨骼肌組織,老年組ho-1表達(dá)高于成年組,老年copp誘導(dǎo)組ho-1表達(dá)升幅顯著低于成年copp誘導(dǎo)組。2.注射copp后,成年copp誘導(dǎo)組和老年copp誘導(dǎo)組t-sod、cuzn-sod酶活性均升高,mda含量降低;老年copp誘導(dǎo)組t-sod,cuzn-sod酶活性低于成年copp誘導(dǎo)組,mda含量高于成年copp誘導(dǎo)組。成年copp誘導(dǎo)組和老年copp誘導(dǎo)組gsh-px、cat酶活性升高,h2o2含量降低;老年copp誘導(dǎo)組gsh-px酶活性高于成年copp誘導(dǎo)組,cat酶活性低于成年copp誘導(dǎo)組,h2o2含量高于成年copp誘導(dǎo)組。3.老年小鼠骨骼肌損傷后,copp誘導(dǎo)組中性粒細(xì)胞浸潤在3d,7d均低于對照組;cd163陽性m2型巨噬細(xì)胞浸潤出現(xiàn)較晚,注射copp后,m2型巨噬細(xì)胞的浸潤顯著高于損傷對照組。4.老年小鼠骨骼肌損傷后,注射copp誘導(dǎo)ho-1表達(dá)3d,7d,14d,實(shí)驗(yàn)組myod,myogenin表達(dá)水平顯著高于損傷對照組,7d時myod有最大表達(dá)量,myogenin的最大表達(dá)量出現(xiàn)在14d。5.老年小鼠骨骼肌損傷后,注射copp5d,7d時新生骨骼肌纖維增多;與對照組比較,copp處理組emhc陽性纖維顯著升高。6.老年小鼠骨骼肌損傷后,注射copp7d,14d,21d,28d,實(shí)驗(yàn)組coli蛋白表達(dá)量顯著低于損傷對照組;膠原纖維數(shù)量同樣低于損傷對照組。21d時coli表達(dá)量和膠原纖維沉積最高。結(jié)論:1.老年小鼠骨骼肌組織ho-1對外界刺激的反應(yīng)性下降,抗氧化應(yīng)激能力下降。2.copp誘導(dǎo)ho-1可以顯著降低成年和老年小鼠骨骼肌組織內(nèi)的過氧化反應(yīng),提高抗氧化酶活性,改善肌組織內(nèi)的氧化應(yīng)激狀態(tài)。3.老年小鼠骨骼肌損傷后,copp誘導(dǎo)ho-1能夠降低早期中性粒細(xì)胞、促進(jìn)m2型巨噬細(xì)胞的浸潤,加快老年小鼠骨骼肌組織的炎癥反應(yīng)進(jìn)程。4.老年小鼠骨骼肌損傷后,CoPP誘導(dǎo)HO-1能夠促進(jìn)肌衛(wèi)星細(xì)胞活化增殖。5.CoPP誘導(dǎo)HO-1改善老年小鼠骨骼肌氧化應(yīng)激和炎癥反應(yīng),可以促進(jìn)老年骨骼肌損傷后再生和修復(fù),降低其纖維化反應(yīng)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R68

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