腫瘤壞死因子-α在七氟醚后處理減輕體外循環(huán)犬肺損傷中的作用

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基礎(chǔ)醫(yī)學(xué)研究

腫瘤壞死因子-α在七氟醚后處理減輕體外循環(huán)犬肺損傷中的作用

謝菲1，李冬冬2，羅俊麗3，何苗1，張紅1

(1.遵義醫(yī)學(xué)院 貴州省麻醉學(xué)研究生教育創(chuàng)新基地暨貴州省麻醉與器官保護(hù)基礎(chǔ)研究重點(diǎn)實(shí)驗(yàn)室，貴州 遵義563099；2.益都中心醫(yī)院，山東 濰坊261000；3.遵義醫(yī)學(xué)院附屬醫(yī)院，貴州 遵義563099)

[摘要]目的 通過觀察七氟醚后處理對(duì)體外循環(huán)(CPB)犬肺組織腫瘤壞死因子-α(TNF-α)表達(dá)的影響，探討七氟醚后處理減輕CPB肺損傷的機(jī)制。方法 健康雜種犬18只隨機(jī)分為3組，每組6只(n=6)：?jiǎn)渭兪中g(shù)組(A組)、體外循環(huán)缺血再灌注組(B組)、七氟醚后處理組(C組)。3組犬麻醉固定后，行雙腔氣管插管機(jī)械通氣。經(jīng)胸骨正中切口開胸，B、C組建立犬體外循環(huán)單肺缺血再灌注損傷模型。C組在開放左肺動(dòng)脈即刻通過人工膜肺給予呼氣末2%七氟醚后處理30 min，A組只開胸，不做其他處理。B、C組CPB前(T1)、開放左肺動(dòng)脈后(T2)及實(shí)驗(yàn)結(jié)束時(shí)(T3)；A組與B、C組相同時(shí)間的3個(gè)時(shí)間點(diǎn)抽取股動(dòng)脈血行血?dú)夥治鲇?jì)算氧合指數(shù)(OI)和呼吸指數(shù)(RI)；取左肺組織保存，HE染色光鏡觀察形態(tài)學(xué)變化；采用ELISA法檢測(cè)肺組織TNF-α的含量。結(jié)果 ①肺功能指標(biāo)的變化：3組內(nèi)不同時(shí)點(diǎn)比較結(jié)果發(fā)現(xiàn)，A組各時(shí)點(diǎn)OI、RI未見明顯改變(P>0.05)，B、C組T1~T3時(shí)點(diǎn)OI逐漸降低，而RI則逐漸升高(P<0.05)。3組間同時(shí)點(diǎn)比較結(jié)果顯示，T1時(shí)刻，3組犬之間OI、RI無明顯差異(P>0.05)；T2、T3時(shí)刻，與A組比較，B、C組OI顯著減低，RI則明顯升高(P<0.05)，其中C組在T3時(shí)刻OI明顯高于B組，而RI則相反(P<0.05)。②肺組織光鏡形態(tài)學(xué)觀察：各組T1~T3時(shí)點(diǎn)，肺組織結(jié)構(gòu)損傷逐漸加重。T3時(shí)刻A組肺組織損傷程度較B、C組輕，而C組又明顯較B組輕。③肺組織TNF-α含量的變化：3組內(nèi)各時(shí)點(diǎn)比較結(jié)果顯示，A組各時(shí)點(diǎn)肺組織TNF-α含量未見明顯改變(P>0.05)；B、C組T1~T3時(shí)點(diǎn)肺組織TNF-α含量逐漸增加(P<0.05)。3組間同時(shí)點(diǎn)比較結(jié)果顯示，T1時(shí)刻3組犬肺組織TNF-α含量無明顯差異(P>0.05)；T2、T3時(shí)刻，與A組比較，B、C組肺組織TNF-α含量明顯增加(P<0.05)，其中C組在T3時(shí)刻TNF-α含量明顯低于B組(P<0.05)。結(jié)論 CPB后隨著犬肺組織TNF-α含量的增加，肺組織結(jié)構(gòu)破壞嚴(yán)重，肺功能降低，肺組織TNF-α含量的增加可能是加重肺損傷的重要原因。七氟醚后處理后肺功能明顯改善，肺組織破壞程度減輕，對(duì)CPB犬肺損傷有一定保護(hù)作用。七氟醚后處理減輕CPB肺損傷的作用可能與抑制肺組織TNF-α表達(dá)有關(guān)。

[關(guān)鍵詞]TNF-α；七氟醚后處理；體外循環(huán)；肺損傷

體外循環(huán)(cardiopulmonary bypass, CPB)后肺損傷是心臟手術(shù)最常見的并發(fā)癥，尤其是老年人和嬰兒，由此導(dǎo)致的肺功能障礙已成為術(shù)后死亡的主要原因之一[1]。如何減輕CPB肺損傷，降低心臟手術(shù)的死亡率，一直是人們研究的熱點(diǎn)。CPB引發(fā)的全身性炎癥反應(yīng)是肺損傷最重要的因素，CPB過程中肺缺血/再灌注損傷、補(bǔ)體系統(tǒng)的激活、內(nèi)毒素血癥和炎癥因子相互作用都可導(dǎo)致炎癥“瀑布”效應(yīng)的發(fā)生，大量激活中性粒細(xì)胞使其在肺組織內(nèi)聚集活化，產(chǎn)生大量氧自由基直接破壞肺泡上皮細(xì)胞和內(nèi)皮細(xì)胞，破壞肺組織正常結(jié)構(gòu)，其中腫瘤壞死因子-α(tumor necrosis factor α，TNF-α)在炎性反應(yīng)中發(fā)揮著重要作用[2]。七氟醚是一種鹵素類吸入麻醉藥，研究發(fā)現(xiàn)，0.5%~3%濃度七氟醚后處理能改善脂多糖誘導(dǎo)肺損傷的氣體交換功能并減輕肺組織損傷[3]。七氟醚可改善肺功能，對(duì)肺損傷有一定的保護(hù)作用，但具體機(jī)制不清。本研究是通過觀察七氟醚后處理對(duì)CPB犬肺組織TNF-α表達(dá)的影響，來探討其減輕CPB肺損傷的機(jī)制，為CPB中的肺保護(hù)提供新的手段，也為拓展七氟醚的用途提供理論依據(jù)。

1材料與方法

1.1動(dòng)物與分組健康成年雜種犬18只，體重10~15 kg，雌雄不拘，由遵義醫(yī)學(xué)院動(dòng)物實(shí)驗(yàn)中心提供。隨機(jī)分為3組(n=6)，單純手術(shù)組(A組)：犬麻醉固定，雙腔插管，機(jī)械通氣，開胸，分離左肺動(dòng)脈，不做任何處理直至實(shí)驗(yàn)結(jié)束；體外循環(huán)缺血再灌注組(B組)：建立CPB左肺缺血再灌注損傷模型[4]；七氟醚后處理組(C組)：建立CPB左肺缺血再灌注損傷模型，于開放左肺動(dòng)脈即刻通過人工膜肺給予2%七氟醚后處理30 min，其余步驟同B組。

1.2模型制備和分組處理3組犬腹腔內(nèi)注射2.5%戊巴比妥25 mg/kg麻醉后，仰臥位固定，行雙腔氣管插管，連接麻醉機(jī)行機(jī)械通氣，維持潮氣量12~15 mL/kg，呼吸頻率16次/min，I∶E=1∶2，F(xiàn)iO2∶99%。股動(dòng)脈穿刺置管，監(jiān)測(cè)MAP；股靜脈中心靜脈穿刺置管，監(jiān)測(cè)CVP和術(shù)中補(bǔ)液；同時(shí)監(jiān)測(cè)HR、舌黏膜氧飽和度(SpO2)及鼻咽溫度(T)。經(jīng)胸骨正中切口開胸，右心房?jī)?nèi)注入肝素3 mg/kg，肝素化后行升主動(dòng)脈和右心房插管，連接人工心肺機(jī)及CPB管道，建立犬CPB。待ACT大于480 s后開始CPB轉(zhuǎn)流，并行循環(huán)10 min后阻斷左肺動(dòng)脈，行右側(cè)單肺通氣。并行循環(huán)60 min后，開放左肺動(dòng)脈恢復(fù)左肺機(jī)械通氣，繼續(xù)并行循環(huán)30 min后停CPB，停機(jī)時(shí)用魚精蛋白(1∶1.5)中和肝素，充分止血，拔出CPB管道，機(jī)血回收后回輸犬體內(nèi)，維持循環(huán)穩(wěn)定2 h后實(shí)驗(yàn)結(jié)束。CPB期間維持平均動(dòng)脈壓在50~80 mmHg，灌注流量在100~120 mL/kg/min，通過動(dòng)脈血?dú)夥治鰜碚{(diào)整酸堿及電解質(zhì)平衡。C組于開放左肺動(dòng)脈時(shí)通過人工膜肺給予七氟醚處理(呼氣末濃度為2%)30 min，其余步驟同B組。

1.3指標(biāo)的檢測(cè)B、C組CPB前(T1)、開放左肺動(dòng)脈后(T2)及實(shí)驗(yàn)結(jié)束時(shí)(T3)；A組與B、C組相同時(shí)間的3個(gè)時(shí)間點(diǎn)取股動(dòng)脈血行血?dú)夥治觯∽蠓谓M織，并將每個(gè)時(shí)間點(diǎn)的組織分為2份保存，一份用于病理學(xué)觀察，另一份用于TNF-α含量測(cè)定。

1.3.1肺功能測(cè)定根據(jù)動(dòng)脈血?dú)夥治鼋Y(jié)果計(jì)算氧合指數(shù)(OI)、呼吸指數(shù)(RI)：OI(氧合指數(shù))= PaO2/FiO2，RI(呼吸指數(shù))=P(A-a)O2/PaO2。

1.3.2病理學(xué)觀察3組分別于T1、T2、T3時(shí)刻取犬左肺組織10%福爾馬林固定，制作石蠟切片，行HE染色后封片，在100倍光學(xué)顯微鏡下觀察肺組織形態(tài)，并拍攝保存圖片。

1.3.3肺組織TNF-α含量測(cè)定采用ELISA法檢測(cè)。3組分別于T1、T2、T3時(shí)刻取犬左肺組織稱重，取緩沖液，以1/9容量比配置，勻漿，離心后取上清液-80 ℃保存，嚴(yán)格按照TNF-α酶聯(lián)免疫試劑盒(RD公司，美國(guó))說明書的步驟進(jìn)行操作。

2結(jié)果

2.1一般資料3組犬體重、體表面積、并循時(shí)間、左肺動(dòng)脈阻斷時(shí)間、CPB時(shí)間、灌注液體量以及最低鼻咽溫度等比較差異均無統(tǒng)計(jì)學(xué)意義(P>0.05)。

2.2氧合指數(shù)變化3組內(nèi)不同時(shí)點(diǎn)比較：與T1時(shí)點(diǎn)比較，T2、T3時(shí)刻B、C組OI顯著降低(P<0.05)，A組未見明顯改變(P>0.05)；與T2時(shí)刻比較，T3時(shí)刻B、C組OI明顯降低(P<0.05)，A組未見明顯改變(P>0.05)。3組間同時(shí)點(diǎn)比較：T1時(shí)刻，3組犬之間OI無明顯差異(P>0.05)；T2時(shí)刻，與A組比較，B、C組OI顯著減低(P<0.05)；T3時(shí)刻，B、C組OI低于A組(P<0.05)，C組OI明顯高于B組(P<0.05，見表1)。

組別氧合指數(shù)T1T2T3呼吸指數(shù)T1T2T3A464.67±17.56430.75±15.33415.67±15.430.298±0.0160.348±0.0330.363±0.034B457.50±20.71373.33±20.06#◆234.33±22.69#※◆★0.283±0.0241.070±0.040#◆3.257±0.044#※◆★C466.33±18.36376.33±16.10#◆321.67±11.30#※◆0.291±0.0211.115±0.094#◆1.815±0.057#※◆

與T1比，#P<0.05；與T2比，※P<0.05；與A組比，◆P<0.05；與C組比，★P<0.05。

2.3呼吸指數(shù)變化3組內(nèi)不同時(shí)點(diǎn)比較：與T1時(shí)點(diǎn)比較，T2、T3時(shí)刻B、C組RI顯著升高(P<0.05)，A組未見明顯改變(P>0.05)；與T2時(shí)刻比較，T3時(shí)刻B、C組RI明顯升高(P<0.05)，A組未見明顯改變(P>0.05)。3組間同時(shí)點(diǎn)比較：T1時(shí)刻，3組犬之間RI無明顯差異(P>0.05)； T2時(shí)刻，與A組比較，B、C組RI顯著升高(P<0.05)；T3時(shí)刻，B、C組RI高于A組(P<0.05)，C組RI明顯低于B組(P<0.05，見表1)。

2.43組犬肺組織病理學(xué)變化T1時(shí)刻，3組犬肺組織結(jié)構(gòu)完整清晰，肺泡壁光滑、完整，肺泡腔內(nèi)未見明顯出血及炎性滲出。T2時(shí)刻，B、C組犬肺組織結(jié)構(gòu)稍紊亂，可見部分肺泡壁斷裂，少量炎性細(xì)胞滲出及紅細(xì)胞漏出，A組無明顯變化。T3時(shí)刻，A組肺組織結(jié)構(gòu)較清晰，見少量肺泡壁斷裂及炎性滲出；B組肺組織結(jié)構(gòu)紊亂，肺泡壁斷裂較重，部分肺泡腔塌陷，肺泡腔和肺間質(zhì)可見大量炎性細(xì)胞浸潤(rùn)和少量紅細(xì)胞漏出；C組肺組織結(jié)構(gòu)尚清晰，部分肺泡腔塌陷，肺泡壁斷裂，可見少量炎性細(xì)胞滲出及紅細(xì)胞漏出，肺損傷程度明顯較B組輕(見圖1)。

A:A組T1;B:B組T2;C:C組T2；D：A組T3;E:B組T3;F:C組T3。圖1　各時(shí)點(diǎn)3組犬左肺組織病理學(xué)的變化(×100)

2.53組犬肺組織TNF-α含量變化3組犬不同時(shí)間點(diǎn)之間比較：A組各時(shí)點(diǎn)肺組織TNF-α含量未見明顯改變(P>0.05)；與T1比較，T2、T3時(shí)刻B、C組肺組織TNF-α含量明顯增加(P<0.05)；與T2時(shí)刻比較，T3時(shí)刻B、C組犬肺組織TNF-α含量明顯升高(P<0.05)。3組犬同時(shí)間點(diǎn)比較：T1時(shí)刻3組犬肺組織TNF-α含量無明顯差異(P<0.05)；T2時(shí)刻：與A組比較，B、C組肺組織TNF-α含量明顯增加(P<0.05)；T3時(shí)刻，B、C組肺組織TNF-α含量明顯高于A組(P<0.05)，C組TNF-α含量明顯低于B組(P<0.05，見表2)。

組別T1T2T3A19.4967±1.534926.7367±1.663629.4470±2.4512B22.8533±2.319642.9233±4.4244#◆70.3467±5.7087#※◆★C24.0733±3.005143.1200±2.2255#◆55.9233±4.6555#※◆

與T1比，#P<0.05；與T2比，※P<0.05；與A組比，◆P<0.05；與C組比，★P<0.05。

3討論

本次實(shí)驗(yàn)參照游露等[4]已成功建立的實(shí)驗(yàn)?zāi)Ｐ?。結(jié)果顯示：實(shí)驗(yàn)結(jié)束時(shí)，左肺缺血再灌注損傷組(B組)與單純開胸組(A組)比較，B組肺組織結(jié)構(gòu)破壞及損傷程度比A組嚴(yán)重，犬肺功能降低，說明CPB導(dǎo)致了犬肺組織損傷，模型成功建立。

目前多數(shù)研究認(rèn)為，體外循環(huán)誘發(fā)的全身性炎癥反應(yīng)及肺缺血再灌注損傷是CPB肺損傷的主要原因。CPB后血液與人工材料表面的直接接觸、手術(shù)自身創(chuàng)傷、體溫變化、肝素及魚精蛋白的使用等均可產(chǎn)生全身性炎癥反應(yīng)，肺組織內(nèi)皮細(xì)胞損傷，破壞肺組織結(jié)構(gòu)，影響術(shù)后肺功能[5]。當(dāng)心臟復(fù)跳、腔靜脈開放后，肺循環(huán)重新開放，大量高氧合血液再次進(jìn)入肺循環(huán)，產(chǎn)生大量氧自由基直接破壞肺泡上皮細(xì)胞和內(nèi)皮細(xì)胞，引起細(xì)胞過度凋亡，進(jìn)一步加重CPB肺損傷[6]。

七氟醚是一種新型的鹵素類吸入麻醉藥，作用時(shí)間相對(duì)較短，可控性好，肌松藥用量小，術(shù)后蘇醒快，具有穩(wěn)定血流動(dòng)力學(xué)和減少麻醉藥用量的特點(diǎn)，且呼吸抑制作用小，對(duì)器官也有保護(hù)作用，目前已廣泛應(yīng)用于臨床。Cho等[7]研究證明，體外循環(huán)下行瓣膜置換手術(shù)的病人通過七氟醚處理，患者術(shù)后肺部炎癥反應(yīng)明顯輕于芬太尼和舒芬太尼處理組。七氟醚預(yù)處理能夠改善CPB患者的肺換氣功能，并提高其肺順應(yīng)性，明顯縮短患者呼吸支持時(shí)間[8]。同時(shí)臨床研究發(fā)現(xiàn)七氟烷可改善心肺轉(zhuǎn)流冠狀動(dòng)脈旁路移植術(shù)患者肺功能指標(biāo)，可選擇性的抑制中性粒細(xì)胞的活性，降低肺泡毛細(xì)血管的通透性，減輕肺損傷[9]。七氟醚抑制單核巨噬細(xì)胞釋放TNF-α，可減輕肺組織炎癥反應(yīng)及細(xì)胞過度凋亡，對(duì)肺損傷有一定保護(hù)作用[10]。本實(shí)驗(yàn)結(jié)果顯示：在CPB后開放肺動(dòng)脈時(shí)，經(jīng)人工膜肺進(jìn)行七氟醚后處理30 min發(fā)現(xiàn)，與B組相比，C組肺功能得到明顯改善，肺組織病理改變減輕，說明七氟醚后處理可改善肺功能，減輕CPB肺損傷。

TNF- α是一個(gè)強(qiáng)有力的炎癥介質(zhì)，是炎性細(xì)胞因子網(wǎng)絡(luò)中的關(guān)鍵成分。主要由肺泡單核巨噬細(xì)胞產(chǎn)生，它協(xié)同激活核因子-KB(NF-KB)產(chǎn)生細(xì)胞因子，啟動(dòng)炎癥級(jí)聯(lián)反應(yīng)，誘導(dǎo)多形核粒細(xì)胞(PMNs)遷移和聚集，不僅啟動(dòng)早期炎癥反應(yīng)而且維持炎癥[11]。TNF-α在肺組織表達(dá)水平的高低，影響著CPB后肺部炎癥的發(fā)生發(fā)展及肺泡活性因子的含量，決定著肺組織損傷程度[12]。CPB后血液與管道接觸可直接刺激白細(xì)胞生成和聚集，使白細(xì)胞表面TNF- α基因表達(dá)增加；同時(shí)主動(dòng)脈開放后，缺血器官再灌注進(jìn)一步導(dǎo)致補(bǔ)體激活，尤其是 C5a 可以誘導(dǎo)白細(xì)胞聚集產(chǎn)生TNF- α[13]。臨床研究表明，CPB過程中，各種炎性因子(如IL-1、IL-6等)的活化都遠(yuǎn)遠(yuǎn)滯后于TNF-α的激活，TNF-α是各種炎癥因子發(fā)生激活的觸發(fā)點(diǎn)，在CPB導(dǎo)致肺損傷的炎性過程中起著重要作用[2]。通過用腫瘤壞死因子-α抗體進(jìn)行干預(yù)能減輕CPB肺損傷[14]。本研究結(jié)果：隨CPB時(shí)間的延長(zhǎng)，肺組織結(jié)構(gòu)紊亂，肺泡壁斷裂嚴(yán)重，肺泡腔塌陷充滿水腫液，B組RI明顯升高，而OI則顯著降低；同時(shí)B組肺組織TNF-α的含量明顯增加；說明CPB導(dǎo)致的肺損傷可能與肺組織TNF-α的含量增加有關(guān)。通過七氟醚后處理，C組犬肺功能明顯改善，肺組織病理改變明顯比B組輕，同時(shí)肺組織TNF-α的含量低于B組(P<0.05)，說明七氟醚后處理可降低肺組織TNF-α含量，減輕CPB肺損傷。

本實(shí)驗(yàn)表明：CPB導(dǎo)致的肺損傷可能與肺組織TNF-α增加有關(guān)。七氟醚后處理可抑制TNF-α的產(chǎn)生，減輕CPB犬肺損傷。但TNF-α在CPB肺損傷是否占主導(dǎo)地位仍有待進(jìn)一步研究。

[參考文獻(xiàn)]

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收稿2015-10-20;修回2015-11-12]

(編輯：王靜)

The role of tumor necrosis factor-α in sevoflurane postconditioning alleviating cardiopulmonary bypass related lung injury in dogs

XieFei1,LiDongdong2,LuoJunli3,HeMiao1,ZhangHong1

(1.Guizhou Graduate Education Innovation Base of Anesthesiology, Guizhou Key Laboratory of Anesthesia and Organ Protection, Zunyi Medical University, Zunyi Guizhou 563099, China; 2.Yidu Central Hospital, Weifang Shandong 261000, China; 3.The Affiliated Hospital of Zunyi Medical University, Zunyi Guizhou 563099, China)

[Abstract]Objective To observe the effect of sevoflurane postconditioning on expression of TNF-α in lungs of dogs during cardiopulmonary bypass (CPB) and explore mechanisms of sevoflurane postconditioning alleviating CPB-induced lung injury.Methods Eighteen healthy dogs were randomly divided into 3 groups (n=6 each): simple operation group (group A), left lung ischemia-reperfusion injury of CPB group (group B), sevoflurane postconditioning group (group C). All dogs received intraperitoneal injection of 2.5% sodium pentobarbital and double lumen bronchial tube intubation. After cutting the sternum，the left pulmonary artery was separated and blocked to establish lung ischemia reperfusion injury model in B and C group. In C group, 2% sevoflurane was administrated for 30 minutes at the onset of opening left pulmonary artery. In A group, sternotomy was completed without other treatments. Arterial blood was collected before CPB (T1), at the onset of opening artery (T2) and 2 h after CPB (T3) to calculate oxygenation index (OI) and respiration index (RI). Left lung tissues were taken to measure following indexes. Hematoxylin-eosin staining was performed to observe the pathological changes. The expression of TNF-α was measured with enzyme linked immunosorbent assay method (ELISA).Results (1) The changes of lung function index: In A group, there was no significant difference among three time points (P>0.05); The value of OI was gradually decreased at T1~T3in B and C group (P>0.05), while RI showed a contrary trend obviously (P<0.05). There were no obvious changes of OI and RI in every group at T1time (P>0.05). The value of OI in the group B and C were obviously lower than that of group A at T2and T3time, while RI showed a contrary trend (P<0.05). C groups’ OI values were obviously higher than group B (P<0.05), and RI values were obviously lower than group B (P<0.05). (2) Pathological changes under optical microscope: The damage of lung tissue was gradually aggravated at T1~T3in all groups. At T3, the injury of lung tissue in A group was lower than that in B and C group, while the C group was significantly lower than the B group. (3) The expression changes of TNF-α: There were no changes at any time in group A (P>0.05). The changes of TNF-α was gradually decreased at T1~T3in B and C group (P<0.05). The changes at the same time in the three groups: There were no obviously difference in every group at T1time (P>0.05). The expression in the B and C group was obviously higher than that in group A at T2、T3time (P<0.05), while the C group was significantly lower than the B group (P<0.05).Conclusion With the increase of TNF-α during CPB, the damage of lung tissue was severe and the lung function reduced, It indicates that the content of TNF-α could lead to lung injury. During CPB, sevoflurane postconditioning exerts protective effects on the lung indicated by improved lung function, alleviated pulmonary damage. The protective effect of sevoflurane postconditioning against lung injury caused by CPB is related with inhibition of TNF-α.

[Key words]TNF-α; sevoflurane postconditioning; cardiopulmonary bypass; lung injury

[中圖法分類號(hào)]R332

[文獻(xiàn)標(biāo)志碼]A

[文章編號(hào)]1000-2715(2015)06-0576-05

[通信作者]張紅，女，碩士，教授，碩士生導(dǎo)師，研究方向：體外循環(huán)，E-mail:hianzhang@tom.com。

[基金項(xiàng)目]貴州科技廳聯(lián)合基金資助項(xiàng)目(NO: 黔科合J字LKZ[2012]05)。