缺血后適應(yīng)對(duì)老齡大鼠心肌缺血再灌注損傷的保護(hù)作用

楊 藝，張存泰，李彩萍，全小慶，阮 磊

?

缺血后適應(yīng)對(duì)老齡大鼠心肌缺血再灌注損傷的保護(hù)作用

楊 藝，張存泰，李彩萍*，全小慶，阮 磊

（華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院綜合科，武漢 430030）

研究缺血后適應(yīng)能否減輕老齡大鼠急性心肌缺血再灌注損傷，比較缺血后適應(yīng)的心臟保護(hù)作用在成年和老齡大鼠之間有無差別。32只雄性F344大鼠分為成年（6～8月齡）和老齡（20～22月齡）組，每組再分為缺血再灌注（I/R）組（缺血30min，再灌注2h，8只）和缺血后適應(yīng)（IPost）組（缺血30min，給予4輪10s再通/10s缺血后再灌注2h，8只），監(jiān)測(cè)平均動(dòng)脈壓（MAP）、心率收縮壓乘積（RPP）等血流動(dòng)力學(xué)參數(shù)和心電圖，測(cè)定心肌梗死面積，再灌注2h后檢測(cè)血清肌酸激酶（CK）和乳酸脫氫酶（LDH）濃度。再灌注2h時(shí)，成年IPost組MAP和RPP均高于I/R組（＜0.05），而老齡IPost組與I/R組間差異無統(tǒng)計(jì)學(xué)意義（＞0.05）。再灌注初30min內(nèi)，成年IPost組和I/R組心律失常評(píng)分分別為1分和3.5分，兩者間差異有統(tǒng)計(jì)學(xué)意義（＜0.05）；老齡IPost組和I/R組心律失常評(píng)分分別為2分和3分，兩者間差異無統(tǒng)計(jì)學(xué)意義（＞0.05）。成年和老齡IPost組心肌梗死面積較I/R組分別減少52%和44%（均＜0.05）。成年和老齡IPost組CK和LDH濃度較I/R組均有明顯降低（＜0.05）。缺血后適應(yīng)能縮小老齡大鼠心肌梗死面積、減少心肌酶釋放，且其程度與成年大鼠相似；同時(shí)缺血后適應(yīng)能改善成年大鼠心肌頓抑、減少再灌注心律失常，但此作用在老齡大鼠未觀察到。

大鼠；再灌注損傷；缺血后適應(yīng)；心肌保護(hù)

2003年Zhao等[1]發(fā)現(xiàn)在長(zhǎng)時(shí)間缺血后、恢復(fù)再灌注前進(jìn)行數(shù)次短暫重復(fù)的心肌缺血再灌注能保護(hù)心肌對(duì)抗隨后的再灌注損傷，進(jìn)而提出了缺血后適應(yīng)（ischemic post-conditioning，IPost）這一概念。多種動(dòng)物模型已經(jīng)證明其在減少梗死面積和保護(hù)內(nèi)皮細(xì)胞功能等方面與缺血預(yù)適應(yīng)（ischemic pre-conditioning，IP）有相同的功效，但它有更強(qiáng)的臨床可操作性，成為近來急性心肌梗死（acute myocardial infarction，AMI）治療的研究熱點(diǎn)。然而，目前關(guān)于IPost對(duì)衰老心臟是否具有保護(hù)作用國(guó)內(nèi)外相關(guān)報(bào)道較少，本課題以20～22月齡的F344老齡大鼠建立在體心臟急性缺血再灌注（ischemia/reperfusion，I/R）損傷模型，觀察IPost對(duì)老齡大鼠心臟再灌注損傷有無保護(hù)作用，為IPost在老年AMI患者冠狀動(dòng)脈介入治療中的應(yīng)用提供動(dòng)物實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 材料

1.1.1 實(shí)驗(yàn)動(dòng)物 成年雄性F344大鼠（6～8月齡），體質(zhì)量190～220g；老齡雄性F344大鼠（18～20月齡），體質(zhì)量310～340g，華中科技大學(xué)同濟(jì)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心。本實(shí)驗(yàn)遵循《實(shí)驗(yàn)動(dòng)物保護(hù)條例》。

1.1.2 主要儀器及試劑 HX-300小型動(dòng)物呼吸機(jī)，成都泰盟，GY-6328生理參數(shù)分析記錄儀，華南醫(yī)電，血管擴(kuò)張球囊（￠2.5-L14），伊文思藍(lán)和氯化三苯基四氮唑（triphenyltetrazolium chloride，TTC）Sigma，肌酸激酶（creatine kinase，CK）和乳酸脫氫酶（lactate dehydrogenase，LDH）試劑盒，南京凱基。

1.2 動(dòng)物建模及分組

1.2.1 建立模型 大鼠以3%戊巴比妥鈉（50mg/kg）腹腔麻醉后固定，行右側(cè)股動(dòng)脈插管，監(jiān)測(cè)心率（heart rate，HR）、收縮壓（systolic blood pressure，SBP）、舒張壓（diastolic blood pressure，DBP）和平均動(dòng)脈壓（mean arterial pressure，MAP），皮下埋入針形電極記錄心電圖。行氣管插管，連接小動(dòng)物呼吸機(jī)，調(diào)節(jié)潮氣量2～3ml/100g，呼吸頻率50～60次/min，呼吸比2∶1。經(jīng)左側(cè)第四肋間開胸暴露心臟，于左心耳下方2mm進(jìn)針，以6-0線在左冠狀動(dòng)脈前降支后方穿過，將充水球囊墊于血管與結(jié)扎線之間，拉緊縫線后可見心電圖ST段明顯抬高，松開后ST段下降＞1/2，表明結(jié)扎位置正確。缺血30min后，抽空球囊，再灌注2h。

1.2.2 分組 32只大鼠分為4組，每組8只：（1）老齡缺血再灌注組（I/R aged）及成年缺血再灌注組（I/R adult）：大鼠造模，結(jié)扎冠狀動(dòng)脈左前降支缺血30min后，直接恢復(fù)再灌注2h；（2）老齡缺血后適應(yīng)組（IPost aged）及成年缺血后適應(yīng)組（IPost adult）：大鼠造模，結(jié)扎冠狀動(dòng)脈左前降支缺血30min后，于再灌注即刻給予4輪10s再灌注/10s缺血（4×10）的IPost，再持續(xù)灌注2h。

1.3 觀察指標(biāo)

1.3.1 記錄缺血和再灌注 記錄缺血1min、30min和再灌注1h、2h大鼠的HR、SBP、DBP和MAP，以相應(yīng)的心率收縮壓乘積（rate pressure product，RPP）作為心肌耗氧量指標(biāo)。

1.3.2 采用評(píng)分系統(tǒng)對(duì)再灌注初30min內(nèi)心律失常的嚴(yán)重程度進(jìn)行定量分析[2]無心律失常為0分；偶發(fā)室性期前收縮（premature ventricular beats，PVB）為1分；PVB二聯(lián)律或PVB連發(fā)為2分；1～2次室性心動(dòng)過速（ventricular tachycardia，VT）為3分；≥3次VT或一過性心室顫動(dòng)（ventricular fibrillation，VF）為4分；頻發(fā)或持續(xù)性VF甚至導(dǎo)致動(dòng)物死亡為5分。每個(gè)心臟以其出現(xiàn)的最嚴(yán)重的心律失常所對(duì)應(yīng)的分?jǐn)?shù)作為其評(píng)分。

1.3.3 再灌注后處理 再灌注2h后，原位結(jié)扎冠狀動(dòng)脈左前降支，推注伊文思藍(lán)，染色5min后處死動(dòng)物，立即取心臟，剪除心房、大血管、右心室，將左心室凍存于-80℃冷凍20min。從心尖至心底垂直長(zhǎng)軸水平切片，每片厚2～3mm。將切片浸泡于1%TTC溶液中，37℃避光水浴30min后置于10%甲醛溶液過夜。經(jīng)伊文思藍(lán)和TTC雙染色后[3]，正常非缺血心肌為藍(lán)色，缺血未梗死心肌為磚紅色，梗死心肌為白色。采用Image-Pro Plus 6.0圖像分析軟件分析各區(qū)域面積。缺血危險(xiǎn)區(qū)面積（area at risk，AAR）（%）＝（紅色+白色區(qū)域面積）/（藍(lán)色+紅色+白色區(qū)域面積）×100%；心肌梗死面積（infarct size，IS）（%）＝（白色區(qū)域面積）/（紅色+白色區(qū)域面積）×100%。

1.3.4 再灌注2h后采動(dòng)脈血 采動(dòng)脈血1ml，3000r/min離心15min后收集血清，凍存于-80℃，使用商業(yè)試劑盒按說明書步驟檢測(cè)血清CK和LDH濃度。

1.4 統(tǒng)計(jì)學(xué)處理

采用SPSS16.0統(tǒng)計(jì)軟件，符合正態(tài)分布數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差表示，組間比較采用方差分析；不符合正態(tài)分布數(shù)據(jù)以中位數(shù)（四分位間距）表示，組間比較采用Kruskal-Wallis檢驗(yàn)和Fisher精確檢驗(yàn)。＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 一般情況

老齡組大鼠體質(zhì)量明顯高于成年組大鼠[（327±11）（208±10）g，＜0.05]，老齡組大鼠心臟質(zhì)量也是成年組大鼠的1.5倍[（1.14±0.04）（0.77±0.03）g，＜0.05]，但是心重指數(shù)（＝心臟質(zhì)量/體質(zhì)量×100%）老齡大鼠顯著低于成年大鼠[（0.35±0.004）%（0.37±0.004）%，＜0.05]。

2.2 血流動(dòng)力學(xué)變化

結(jié)扎前4組大鼠間HR、MAP和RPP差異無統(tǒng)計(jì)學(xué)意義。缺血1min時(shí)4組大鼠HR均明顯加快，MAP均明顯降低（＜0.05），除I/R aged組外余3組RPP明顯降低。在缺血30min時(shí)，各組HR、MAP、RPP又回到基線水平。在再灌注過程中，4組大鼠HR有下降趨勢(shì)，但各組間無差異，與結(jié)扎前也無差異；而MAP和RPP與結(jié)扎前相比均明顯下降。再灌注2h結(jié)束時(shí)，IPost adult組的MAP和RPP均高于I/R adult組（＜0.05），而IPost aged組與I/R aged組間差異無統(tǒng)計(jì)學(xué)意義。在缺血30min及再灌注1，2h時(shí)，IPost aged組的MAP都低于IPost adult組（＜0.05；表1）。

2.3 再灌注心律失常發(fā)生情況

再灌注初30min內(nèi)，I/R aged組和I/R adult組分別有7例（87.5%）和6例（75.0%）發(fā)生了VT或VF，心律失常評(píng)分分別為3.0（1.75）和3.5（1.75）。IPost aged組和IPost adult組分別發(fā)生了3例（37.5%）和1例（12.5%）VT或VF，心律失常評(píng)分為2.0（2.0）和1.0（1.5）?？梢钥闯觯琁Post組VT、VF的發(fā)生例數(shù)和心律失常評(píng)分均較I/R組降低，但僅成年組大鼠兩者間差異有統(tǒng)計(jì)學(xué)意義（＜0.05）。成年組大鼠和老齡組大鼠間差異無統(tǒng)計(jì)學(xué)意義（＞0.05；表2）。

2.4 缺血危險(xiǎn)區(qū)面積及梗死面積

AAR在4組大鼠之間是可比的[I/R adult（54.7±5.6）%；IPost adult（52.5±7.3）%；I/R aged（53.2±5.1）%；IPost aged（55.7±3.7）%；所有＞0.05]。IPost adult組的IS較I/R adult組減少52%[分別為（15.2±1.8）%和（31.8±2.7）%，＜0.05]，IPost aged組IS較I/R aged組減少44%[分別為（16.1±2.6）%和（28.6±3.4）%，＜0.05]。成年大鼠和老齡大鼠間IS差異無統(tǒng)計(jì)學(xué)意義（＞0.05；圖1）。

2.5 心肌酶學(xué)

再灌注2h結(jié)束時(shí)，IPost組CK和LDH濃度較I/R組明顯降低（均＜0.05），但I(xiàn)/R adult組和I/R aged組之間、IPost adult組和IPost aged組之間差異無統(tǒng)計(jì)學(xué)意義（＞0.05；圖2）。

表1 缺血/再灌注過程中血流動(dòng)力學(xué)變化

HR: heart rate; MAP: mean arterial pressure; RPP: rate pressure product. 1mmHg=0.133kPa. Compared with baseline,*＜0.05; compared with I/R adult group,#＜0.05; compared with IPost adult group,△＜0.05

表2 4組大鼠心律失常發(fā)生例數(shù)和評(píng)分

VT: ventricular tachycardia; VF: ventricular fibrillation. Compared with I/R adult group,*＜0.05

3 討 論

2003年，Zhao等[1]首次在犬心肌I/R模型上提出IPost的概念，后被應(yīng)用到其他物種[4]，繼而推廣到臨床研究[5]中以期減少AMI患者的IS。相對(duì)預(yù)適應(yīng)，IPost有更好的臨床可操作性，因此研究其對(duì)衰老心臟是否具有保護(hù)作用有重要的臨床價(jià)值。

IPost減少IS的程度與血管再閉塞的次數(shù)和持續(xù)時(shí)間密切相關(guān)。李敏等[6]對(duì)成年大鼠（8～10月齡）行3輪5s I/5s R（3×5）、3輪10s I/10s R（3×10）或3輪30s I/30s R（3×30）IPost均能減少IS；對(duì)老齡大鼠（22～24月齡）3×10和3×30的方法有保護(hù)作用，而3×5的方法卻沒有，提示對(duì)衰老心臟刺激強(qiáng)度的重要性。本實(shí)驗(yàn)選擇的是4輪10s I/10s R（4×10）的IPost方式，結(jié)果顯示該方式能明顯減少成年及老齡大鼠在體心臟IS（52%和44%），與國(guó)內(nèi)外相關(guān)報(bào)道一致[7]。

除IS外，本研究同時(shí)選擇IPost臨床研究常用的終點(diǎn)指標(biāo)心肌酶CK和LDH間接反映IS的大小[8]。與IS染色結(jié)果一致，本研究發(fā)現(xiàn)IPost較I/R能明顯降低血清CK和LDH釋放水平，此效果在成年大鼠和老齡大鼠中均存在，提示CK和LDH可作為評(píng)估IPost對(duì)衰老心臟保護(hù)作用的良好間接指標(biāo)。IPost的心肌保護(hù)作用可能由ROS、NO、Ca2+、腺苷、緩激肽、ANP/BNP等介導(dǎo)[9]，其具體機(jī)制尚有待進(jìn)一步研究。

I/R導(dǎo)致的心肌可逆性或不可逆性損傷均造成心肌舒縮功能降低，其中可逆性的心功能降低稱為心肌頓抑（myocardial stunning）。本實(shí)驗(yàn)通過觀察再灌注過程中HR、MAP、RPP等血流動(dòng)力學(xué)參數(shù)的變化間接反映心功能的改變。恢復(fù)再灌注2h后，IPost組MAP和RPP水平較I/R組有所升高，但僅在成年組大鼠兩者間有統(tǒng)計(jì)學(xué)差異，對(duì)老齡組大鼠無明顯作用。提示IPost抗梗死和抗頓抑效果在不同年齡組不盡一致。盡管IPost對(duì)改善老齡大鼠心肌頓抑沒有明確效果，但I(xiàn)Post減少IS仍可能間接改善左室功能。大鼠心臟離體實(shí)驗(yàn)表明IPost對(duì)心功能恢復(fù)的作用與其減少IS的能力是平行的[10]。臨床實(shí)驗(yàn)中也觀察到，能減少IS的IPost干預(yù)對(duì)左室功能改善能獲得短期及長(zhǎng)期的益處[11]。

圖1 成年及老齡大鼠缺血危險(xiǎn)面積和梗死面積

Figure 1 AAR and IS in adult and aged rats

NS: not statistically significant; AAR: area at risk; LV: left ventricle; IS: infarct size. Compared with I/R group,*＜0.05

圖2 再灌注2h時(shí)成年組和老齡組大鼠血清CK和LDH濃度

Figure 2 Serum levels of CK and LDH in adult and aged rats at 2 hours post reperfusion

CK: creatine kinase; LDH: lactate dehydrogenase. Compared with I/R group,*＜0.05

目前已有研究報(bào)道IPost抗再灌注心律失常作用。Galagudza等[12]將離體大鼠心臟缺血30min后，于再灌注15min后行單次全心缺血2min的IPost可使持續(xù)性VF轉(zhuǎn)復(fù)為竇律。Kloner等[13]采用在體大鼠模型，在缺血5min后行更經(jīng)典的IPost方法（4輪20s I/20s R），發(fā)現(xiàn)IPost能夠降低室性心律失常的發(fā)生率，并且VT的平均持續(xù)時(shí)間也明顯減少。本研究采用全程心電監(jiān)護(hù)，通過心律失常評(píng)分系統(tǒng)對(duì)心律失常發(fā)生的嚴(yán)重程度進(jìn)行定量分析，發(fā)現(xiàn)IPost可使成年大鼠再灌注30min內(nèi)VT、VF的發(fā)生率和心律失常評(píng)分降低，但老齡大鼠未獲得相似的效果，推測(cè)可能與老齡大鼠心肌老化、本身心律失常發(fā)生率升高[14]有關(guān)；也可能本研究樣本數(shù)量偏少，未能發(fā)生差異。既往研究[15,16]提示IPost抗心律失常作用不依賴于抗心肌細(xì)胞凋亡作用，具體機(jī)制尚有待進(jìn)一步研究。

[1] Zhao ZQ, Corvera JS, Halkos ME,. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning[J]. Am J Physiol Heart Circ Physiol, 2003, 285(2): H579?H588.

[2] Xiong J, Wang Q, Xue FS,. Comparison of cardioprotective and anti-inflammatory effects of ischemia pre- and postconditioning in rats with myocardial ischemia-reperfusion injury[J]. Inflamm Res, 2011, 60(6): 547?554.

[3] Schwarz ER, Somoano Y, Hale SL,. What is the required reperfusion period for assessment of myocardial infarct size using triphenyltetrazolium chloride staining in the rat[J]? J Thromb Thrombolysis, 2000, 10(2): 181?187.

[4] Yang XM, Liu Y, Liu Y,. Attenuation of infarction in cynomolgus monkeys: preconditioning and postconditioning[J]. Basic Res Cardiol, 2010, 105(1): 119?128.

[5] Hahn JY, Song YB, Kim EK,. Ischemic postconditioning during primary percutaneous coronary intervention: the effects of postconditioning on myocardial reperfusion in Patients with ST-segment Elevation Myocardial Infarction (POST) randomized trial[J]. Circulation, 2013, 128(17): 1889?1896.

[6] Li M, Zhang J, Liang YH,. Effects of ischemic postconditioning on myocardial infarction sizes and protein kinase C expression in aged rats with post-ischemia reperfusion injury[J]. Chin J Geriatr, 2010, 29(5): 420?423. [李 敏, 張 健, 梁艷紅, 等. 缺血后處理對(duì)心肌缺血再灌注大鼠心肌梗死面積和細(xì)胞蛋白激酶Cα表達(dá)的影響[J]. 中華老年醫(yī)學(xué)雜志, 2010, 29(5): 420?423.]

[7] Yin Z, Gao H, Wang H,. Ischaemic post-conditioning protects both adult and aged Sprague-Dawley rat heart from ischaemia-reperfusion injury through the phosphatidylinositol 3-kinase-AKT and glycogen synthase kinase-3beta pathways[J]. Clin Exp Pharmacol Physiol, 2009, 36(8): 756?763.

[8] Staat P, Rioufol G, Piot C,. Postconditioning the human heart[J]. Circulation, 2005, 112(14): 2143?2148.

[9] Ovize M, Baxter GF, Di Lisa F,. Postconditioning and protection from reperfusion injury: where do we stand? Position paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology[J]. Cardiovasc Res, 2010, 87(3): 406?423.

[10] Tawa M, Fukumoto T, Yamashita N,. Postconditioning improves postischemic cardiac dysfunction independently of norepinephrine overflow after reperfusion in rat hearts: comparison with preconditioning[J]. J Cardiovasc Pharmacol, 2010, 55(1): 6?13.

[11] Wang G, Zhang S, Joggerst SJ,. Effects of the number and interval of balloon inflations during primary PCI on the extent of myocardial injury in patients with STEMI: does postconditioning exist in real-world practice[J]? J Invasive Cardiol, 2009, 21(9): 451?455.

[12] Galagudza M, Kurapeev D, Minasian S,. Ischemic postconditioning: brief ischemia during reperfusion converts persistent ventricular fibrillation into regular rhythm[J]. Eur J Cardiothorac Surg, 2004, 25(6): 1006?1010.

[13] Kloner RA, Dow J, Bhandari A. Postconditioning markedly attenuates ventricular arrhythmias after ischemia- reperfusion[J]. J Cardiovasc Pharmacol Ther, 2006, 11(1): 55?63.

[14] Wang XF, Zhang CT, Xu RD,. Effect of aging-related changes in gap junction protein on arrhythmia in rats[J]. Chin J Geriatr, 2011, 30(5): 427?430. [王杏芬, 張存泰, 徐仁德, 等. 大鼠心室肌縫隙連接蛋白增齡性變化對(duì)心律失常發(fā)生率的影響[J]. 中華老年醫(yī)學(xué)雜志, 2011, 30(5): 427?430.]

[15] Dow J, Bhandari A, Kloner RA. The mechanism by which ischemic postconditioning reduces reperfusion arrhythmias in rats remains elusive[J]. J Cardiovasc Pharmacol Ther, 2009, 14(2): 99?103.

[16] Kolettis TM, Vilaeti AD, Tsalikakis DG,Effects of pre- and postconditioning on arrhythmogenesis in therat model[J]. J Cardiovasc Pharmacol Ther, 2013, 18(4): 376?385.

（編輯: 李菁竹）

Ischemic post-conditioning protects aged rat hearts against ischemia/reperfusion injury

YANG Yi, ZHANG Cun-Tai, LI Cai-Ping*, QUAN Xiao-Qing, RUAN Lei

(Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China)

To investigate whether ischemic post-conditioning (IPost) can ameliorate acute myocardial ischemia/reperfusion (I/R) injury in senescent rats and to compare the cardioprotective effects of ischemic post-conditioning between aged and adult rats.A total of 32 male F344 rats were divided into adult (6 to 8 months) group and aged (18 to 20 months) group. After the rats were subjected to 30min ischemia by ligating the left anterior descending coronary artery (LAD), their hearts from each group were further randomized to receive standard, abrupt reperfusion (I/R group), or were post-conditioned with 4 cycles of 10s ischemia interspersed by 10s reperfusion immediately at the end of the ischemia (IPost group). Hemodynamic changes, such as mean arterial pressure (MAP) and rate-pressure product (RPP), and electrocardiogram (ECG) were recorded. Infarct size was measured by Evans blue and triphenyltetrazolium chloride (TTC) staining. At 120min of reperfusion, serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) were assayed.After 2 hours reperfusion, MAP and RPP were significantly higher in IPost group than in the corresponding I/R group for adult rats (＜0.05). However, no significance was achieved in aged rats (＞0.05). During the first 30min reperfusion, in adult rats, the arrhythmia scores were 1.0 and 3.5 respectively for the IPost group and I/R group with significant difference (＜0.05). However, in aged rats, the arrhythmia scores were 2.0 and 3.0 for the IPost group and I/R group, but without significant difference (＞0.05). Post obviously decreased the infarct size in the IPost group than in corresponding I/R group in both adult (by 52%) and aged cohorts (by 44%;＜0.05 for both). The serum levels of CK and LDH were markedly lower in IPost group than in I/R group from both adult and aged rats (＜0.05 for both).IPost, as same powerful as to adult rats, reduces infarct size and decreases the release of myocardial enzymes in aged rats. What’s more, the post-conditioning attenuates myocardial stunning and reperfusion arrhythmia in adult rats, but these effects are not seen in the aged rats.

rats; reperfusion injury; ischemic post-conditioning; cardioprotection

(81300141).

R543

A

10.3724/SP.J.1264.2014.000141

2014?05?08;

2014?06?02

國(guó)家自然科學(xué)基金青年基金（81300141）

李彩萍, E-mail: yangyigracy@gmail.com