【摘要】：鐵參與機(jī)體氧的轉(zhuǎn)運(yùn)、貯存與利用及ATP合成等許多生理過程。血紅蛋白和肌紅蛋白與氧氣的運(yùn)輸和擴(kuò)散有著密切的關(guān)系。運(yùn)動(dòng)訓(xùn)練和低氧環(huán)境均可改變機(jī)體鐵狀態(tài)。低氧訓(xùn)練能發(fā)揮缺氧和運(yùn)動(dòng)雙重刺激，提高機(jī)體運(yùn)載及利用氧的能力。骨骼肌作為運(yùn)動(dòng)中利用氧和鐵的重要組織，低氧刺激下可引起骨骼肌中鐵代謝發(fā)生適應(yīng)性變化。然而國內(nèi)尚缺乏低氧訓(xùn)練對(duì)骨骼肌鐵代謝的機(jī)制研究，因此本研究采用在體和離體實(shí)驗(yàn)結(jié)合分析的方法，以闡明低氧訓(xùn)練對(duì)骨骼肌鐵代謝的影響機(jī)制。 研究?jī)?nèi)容與方法： 在體實(shí)驗(yàn)：選取健康雄性SD大鼠32只，隨機(jī)分為常氧安靜組（normalcontrol，NC）、常氧運(yùn)動(dòng)組(normal exercise，NE)、低氧安靜組(hypoxia control，HC)、低氧運(yùn)動(dòng)組(hypoxia exercise，HE)。每組各8只。常氧安靜組為對(duì)照組，常氧運(yùn)動(dòng)組在常氧下進(jìn)行運(yùn)動(dòng)，強(qiáng)度為從21m/min遞增至25m/min，每周增1m/min，1h/d，6天/周，坡度為0°。低氧安靜組每天在低氧艙安靜8h(氧濃度13.6%,相當(dāng)于3500m海拔)。低氧運(yùn)動(dòng)組在進(jìn)行完常氧運(yùn)動(dòng)后進(jìn)行低氧暴露。共處理5周。最后一次處理24h后安靜常氧時(shí)取材，采用原子吸收法檢測(cè)大鼠骨骼肌總鐵含量；應(yīng)用RT-PCR法分析大鼠骨骼肌低氧誘導(dǎo)因子-1（hypoxiainducible factor1，HIF-1）mRNA相對(duì)表達(dá)變化；應(yīng)用Western Blot檢測(cè)大鼠骨骼肌二價(jià)金屬離子轉(zhuǎn)運(yùn)體(divalent metal transporter l, DMT1),轉(zhuǎn)鐵蛋白受體1(transferring receptor1, TfR1),膜鐵轉(zhuǎn)運(yùn)蛋白1(ferroportin1, FPN1)的表達(dá)的變化離體實(shí)驗(yàn)：培養(yǎng)骨骼肌L6細(xì)胞系，當(dāng)90％以上為貼壁細(xì)胞即可用于實(shí)驗(yàn)。將L6細(xì)胞隨機(jī)分為3組：對(duì)照組（0-h hypoxia），12h低氧處理組（12-h hypoxia），24h低氧處理組（24-h hypoxia），每組6個(gè)重復(fù)。實(shí)驗(yàn)組分別放入低氧培養(yǎng)箱中并通入低氧氣體（1%02）、37℃、95%-98%相對(duì)濕度條件下培養(yǎng)，分別處理12小時(shí)和24小時(shí)。對(duì)照組置于普通培養(yǎng)箱中培養(yǎng)。采用55Fe同位素示蹤檢測(cè)鐵攝取與釋放；細(xì)胞內(nèi)鐵池的測(cè)定運(yùn)用流式細(xì)胞儀法； Western blot檢測(cè)L6細(xì)胞鐵轉(zhuǎn)運(yùn)蛋白（HIF-1，DMT1，TfR1，F(xiàn)PN1）表達(dá)的變化。 研究結(jié)果： 1低氧訓(xùn)練對(duì)大鼠骨骼肌鐵代謝的影響：（1）大鼠骨骼肌總鐵含量的結(jié)果表明，與NC組相比各組骨骼肌總體鐵含量增加（P0.05, P0.01）；而HE組明顯高于各組（P0.05）。（2）HC組和HE組大鼠骨骼肌中HIF-1mRNA的表達(dá)顯著高于NC組和NE組（P0.01）。（3）各組大鼠鐵轉(zhuǎn)運(yùn)蛋白表達(dá)的比較結(jié)果顯示：與NC相比，各組的鐵吸收蛋白（DMT1、TfR1）均顯著性升高(P0.05,P0.01)，，NE組和HC組的鐵釋放蛋白(FPN1)明顯下降（P0.05,P0.01），HE組明顯升高（P0.01）。HE組鐵吸收蛋白（DMT1、TfR1）和鐵釋放蛋白（FPN1）均非常顯著高于NE組和HC組(P0.01)。 2不同時(shí)間低氧暴露對(duì)骨骼肌L6細(xì)胞系鐵代謝的影響：（1）12h組細(xì)胞的鐵攝取能力明顯高于對(duì)照組（P0.05），鐵釋放能力顯著低于對(duì)照組（P0.01）。24h組肌細(xì)胞鐵攝取能力與對(duì)照組相比無明顯差異（P0.05），鐵釋放能力顯著低于對(duì)照組（P0.05）。（2）與0h組相比12h組LIP明顯升高（P0.05），24h組LIP非常明顯降低（P0.01）。（3）與對(duì)照組比較，12h組HIF-1的表達(dá)非常顯著升高（P0.01），DMT1(IRE)表達(dá)顯著升高（P0.05），DMT1(non IRE)表達(dá)顯著升高（P0.05），F(xiàn)PN1表達(dá)非常顯著降低（P 0.01），TfR1表達(dá)無明顯變化（P0.05）。與0h組相比24h組HIF-1的表達(dá)非常顯著升高（P0.01），DMT1(IRE)表達(dá)非常顯著降低（P0.01），DMT1(non IRE)表達(dá)非常顯著降低（P0.01），F(xiàn)PN1表達(dá)非常顯著降低（P0.01），TfR1表達(dá)非常顯著性升高（P0.01）。 研究結(jié)論： （1）在體實(shí)驗(yàn)顯示，大鼠適度運(yùn)動(dòng)和單純的低氧暴露均能增加骨骼肌的鐵貯存，而低氧訓(xùn)練在促進(jìn)骨骼肌鐵吸收的同時(shí)鐵的釋放也顯著增加，這加強(qiáng)了骨骼肌鐵循環(huán)利用的能力從而進(jìn)一步滿足機(jī)體在運(yùn)動(dòng)中對(duì)鐵的需求。 （2）離體實(shí)驗(yàn)顯示，低氧對(duì)骨骼肌鐵代謝的影響與時(shí)間密切相關(guān)，一定時(shí)間的低氧暴露可以提高肌細(xì)胞的鐵吸收能力，降低鐵釋放，增加細(xì)胞內(nèi)的鐵含量，而長(zhǎng)時(shí)間的低氧暴露會(huì)引起細(xì)胞鐵代謝紊亂。 （3）骨骼肌中HIF-1的表達(dá)在低氧中對(duì)調(diào)節(jié)骨骼肌鐵代謝起到關(guān)鍵作用，是低氧對(duì)機(jī)體鐵代謝影響的重要連接紐帶。
[Abstract]:Iron is involved in many physiological processes, such as oxygen transport, storage and utilization, and ATP synthesis. Hemoglobin and myoglobin are closely related to oxygen transport and diffusion. Exercise training and hypoxic environment can change the body's iron status. Hypoxic training can play a dual stimulation of hypoxia and exercise, improve the body's ability to transport and utilize oxygen. Skeletal muscle, as an important tissue of utilizing oxygen and iron during exercise, can cause adaptive changes of iron metabolism in skeletal muscle under hypoxic stimulation. However, there is no study on the mechanism of iron metabolism in skeletal muscle after hypoxic training in China. Therefore, this study uses in vivo and in vitro experiments to clarify the effect of hypoxic training on iron metabolism in skeletal muscle. Influence mechanism.
Research contents and methods:
In vivo experiment: 32 healthy male SD rats were randomly divided into normal control group (NC), normoxic exercise group (NE), hypoxia control group (HC), hypoxia exercise group (HE). Hypoxic exercise group was exposed to hypoxia after normoxic exercise for 5 weeks. Total iron content in skeletal muscle; relative expression of hypoxia inducible factor 1 (HIF-1) mRNA in rat skeletal muscle was analyzed by RT-PCR; divalent metal transporter 1 (DMT1), transferring receptor 1 (TfR1), membrane iron transport was detected by Western Blot. L6 cells were randomly divided into three groups: control group (0-h hypoxia), 12-h hypoxia treatment group (12-h hypoxia), 24-h hypoxia treatment group (24-h hypoxia), 6 replicates in each group. The control group was cultured in a normal incubator. The uptake and release of iron were detected by 55Fe isotope tracer. The iron pool in L6 cells was determined by flow cytometry. The iron transport in L6 cells was detected by Western blot. Changes in expression of white (HIF-1, DMT1, TfR1, FPN1).
Research findings:
1 Effect of hypoxic training on iron metabolism in skeletal muscle of rats: (1) The total iron content in skeletal muscle of rats was higher than that of NC group (P 0.05, P 0.01), while that of HE group was higher than that of each group (P 0.05). (2) The expression of HIF-1 mRNA in skeletal muscle of HC group and HE group was significantly higher than that of NC group and NE group (P 0.01). Compared with NC, the expression of ferritin (DMT1, TfR1) increased significantly (P 0.05, P 0.01), and that of ferritin (FPN1) decreased significantly (P 0.05, P 0.01) in NE and HC groups, and increased significantly (P 0.01) in HE group. Group (P0.01).
2 Effects of hypoxic exposure at different time on iron metabolism of L6 cell line of skeletal muscle: (1) The iron uptake ability of L6 cells in 12h group was significantly higher than that of control group (P 0.05), and the iron release ability was significantly lower than that of control group (P 0.01). The iron uptake ability of L6 cells in 24h group was not significantly different from that of control group (P 0.05), and the iron release ability was significantly lower than that of control group (P 0.05). Compared with the control group, the expression of HIF-1 was significantly increased (P 0.01), the expression of DMT1 (P 0.05), the expression of DMT1 (non-IRE) was significantly increased (P 0.05), the expression of FPN1 was significantly decreased (P 0.01), and the expression of TfR1 was not significantly changed (P 0.05). The expression of HIF-1 was significantly increased (P 0.01), the expression of DMT1 (IRE) was significantly decreased (P 0.01), the expression of DMT1 (non-IRE) was significantly decreased (P 0.01), the expression of FPN1 was significantly decreased (P 0.01), and the expression of TfR1 was significantly increased (P 0.01).
Research conclusions:
(1) In vivo experiments showed that both moderate exercise and pure hypoxic exposure could increase iron storage in skeletal muscle, while hypoxic training promoted iron absorption in skeletal muscle, the release of iron was also significantly increased, which strengthened the ability of iron recycling in skeletal muscle to further meet the body's demand for iron during exercise.
(2) In vitro experiments showed that the effect of hypoxia on iron metabolism of skeletal muscle was closely related to time. Hypoxia exposure for a certain period of time could improve iron absorption capacity of muscle cells, reduce iron release and increase iron content in cells. Long-term hypoxia exposure could cause disorder of iron metabolism.
(3) The expression of HIF-1 in skeletal muscle plays a key role in regulating iron metabolism in skeletal muscle under hypoxia, and is an important link between hypoxia and iron metabolism.
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R87

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