沈黃冕 彭祥和 林仕梅* 陳擁軍 黃先智

(1.西南大學(xué)動(dòng)物科技學(xué)院，淡水魚類資源與生殖發(fā)育教育部重點(diǎn)實(shí)驗(yàn)室，重慶400716；2.西南大學(xué)蠶學(xué)與系統(tǒng)生物學(xué)研究所，重慶400716)

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發(fā)酵桑葉對(duì)高脂血癥羅非魚血脂、血糖水平的調(diào)節(jié)作用

沈黃冕1彭祥和1林仕梅1*陳擁軍1黃先智2

(1.西南大學(xué)動(dòng)物科技學(xué)院，淡水魚類資源與生殖發(fā)育教育部重點(diǎn)實(shí)驗(yàn)室，重慶400716；2.西南大學(xué)蠶學(xué)與系統(tǒng)生物學(xué)研究所，重慶400716)

本試驗(yàn)采用高脂飼料誘導(dǎo)建立高脂血癥羅非魚模型，以研究發(fā)酵桑葉對(duì)高脂血癥羅非魚血脂、血糖水平的調(diào)節(jié)作用。將初始均重為45g的450尾雄性吉富羅非魚隨機(jī)分成5組，每組設(shè)3個(gè)重復(fù)，每個(gè)重復(fù)30尾魚。正常對(duì)照組羅非魚飼喂基礎(chǔ)飼料，其他4組羅非魚在高脂血癥模型建成后分別飼喂基礎(chǔ)飼料(模型對(duì)照組)，含7.5%和15.0%發(fā)酵桑葉的飼料(分別為發(fā)酵桑葉低劑量組和發(fā)酵桑葉高劑量組)以及含0.5%銀杏黃酮的飼料(銀杏黃酮組)。試驗(yàn)期為8周。結(jié)果表明：飼養(yǎng)5和8周后，模型對(duì)照組羅非魚體重顯著低于正常對(duì)照組、發(fā)酵桑葉低劑量組和發(fā)酵桑葉高劑量組以及銀杏黃酮組(P<0.05)，而發(fā)酵桑葉高劑量組羅非魚的體重與正常對(duì)照組無(wú)顯著差異(P>0.05)。發(fā)酵桑葉低劑量組、發(fā)酵桑葉高劑量組和銀杏黃酮組羅非魚血清總膽固醇(TCHO)、低密度脂蛋白膽固醇(LDL-C)含量和TCHO/高密度脂蛋白膽固醇(HDL-C)顯著低于模型對(duì)照組(P<0.05)，而發(fā)酵桑葉高劑量組羅非魚血清TCHO、甘油三酯(TG)、LDL-C含量和TCHO/HDL-C與正常對(duì)照組無(wú)顯著差異(P>0.05)。與模型對(duì)照組相比，發(fā)酵桑葉高劑量組和銀杏黃酮組羅非魚血清HDL-C含量顯著升高(P<0.05)。與模型對(duì)照組相比，正常對(duì)照組、發(fā)酵桑葉低劑量組和發(fā)酵桑葉高劑量組以及銀杏黃酮組羅非魚血清超氧化物歧化酶(SOD)、過(guò)氧化氫酶(CAT)活性以及CAT/SOD顯著升高(P<0.05)，而血清丙二醛(MDA)含量和肝脂含量以及肝體指數(shù)(HSI)顯著降低(P<0.05)；而上述這些指標(biāo)在發(fā)酵桑葉高劑量組與正常對(duì)照組之間無(wú)顯著差異(P>0.05)。高脂飼料使模型對(duì)照組、發(fā)酵桑葉低劑量組、發(fā)酵桑葉高劑量組以及銀杏黃酮組羅非魚的血糖水平顯著升高(0周，P<0.05)；飼養(yǎng)8周后，發(fā)酵桑葉高劑量組和銀杏黃酮組羅非魚血糖水平顯著低于模型對(duì)照組(P<0.05)，而與正常對(duì)照組差異不顯著(P>0.05)。由上可知，發(fā)酵桑葉能劑量依賴性地降低高脂血癥羅非魚的血脂、血糖水平，提高機(jī)體的抗氧化能力。

發(fā)酵桑葉；羅非魚；高脂血癥；抗氧化

高脂血癥是導(dǎo)致魚類脂肪肝的重要因素，已引起廣泛的關(guān)注。研究發(fā)現(xiàn)，飼料或營(yíng)養(yǎng)因子同樣會(huì)引起養(yǎng)殖魚類脂質(zhì)代謝紊亂[1-2]，誘發(fā)出現(xiàn)營(yíng)養(yǎng)性脂肪肝，繼而影響?zhàn)B殖魚類生存、生長(zhǎng)和品質(zhì)。桑葉藥食兼佳，在我國(guó)資源豐富，其因富含降糖降脂功能活性物質(zhì)(多糖、黃酮、生物堿)而受人們的關(guān)注[3-4]。動(dòng)物學(xué)試驗(yàn)表明，飼料中添加桑葉或其提取物可以通過(guò)多種途徑實(shí)現(xiàn)降糖、降脂功效[5-6]。本課題組前期研究也證實(shí)，桑葉可有效調(diào)節(jié)魚體的脂質(zhì)代謝[7]。為進(jìn)一步證實(shí)桑葉降脂的功效，本試驗(yàn)通過(guò)高脂飼料誘導(dǎo)羅非魚(Oreochromisniloticus)出現(xiàn)高脂血癥，然后觀察發(fā)酵桑葉對(duì)羅非魚糖脂代謝的影響，為桑葉在水產(chǎn)飼料中的開(kāi)發(fā)利用提供基礎(chǔ)資料。

1 材料與方法

1.1 試驗(yàn)飼料

以魚粉、豆粕、菜籽粕和棉籽粕為蛋白質(zhì)源，以豆油為脂肪源，配制基礎(chǔ)飼料。在基礎(chǔ)飼料中分別用7.5%、15.0%的發(fā)酵桑葉(采用乳酸菌、酵母菌、芽孢桿菌和固氮菌進(jìn)行固態(tài)發(fā)酵而成，其干物質(zhì)含量為32.5%，粗蛋白質(zhì)含量為24.7%，粗脂肪含量為10.3%，粗灰分含量為8.7%)及0.5%的銀杏黃酮(深圳海王藥業(yè)有限公司產(chǎn)品，批準(zhǔn)文號(hào)：國(guó)藥準(zhǔn)字Z20050178，規(guī)格：每片含總黃酮醇苷9.6mg、萜類內(nèi)酯2.4mg，每片重0.14g)替代等量的面粉，配制成3種等氮等能的飼料。試驗(yàn)飼料組成及營(yíng)養(yǎng)水平見(jiàn)表1。飼料原料過(guò)245μm篩網(wǎng)，采取逐級(jí)擴(kuò)大法混合均勻，制成粒徑分別為2.5和4.0mm的顆粒飼料，自然風(fēng)干后于4℃冰柜中保存?zhèn)溆谩?/p>

表1 試驗(yàn)飼料組成及營(yíng)養(yǎng)水平(風(fēng)干基礎(chǔ))Table 1 Composition and nutrient levels of experimental diets (air-dry basis) %

1.2 高脂血癥羅非魚模型的建立

將初始均重為45g的450尾雄性吉富羅非魚隨機(jī)分成5組(正常對(duì)照組和4個(gè)模型組)，每組設(shè)3個(gè)重復(fù)，每個(gè)重復(fù)30尾魚。正常對(duì)照組飼喂基礎(chǔ)飼料；模型組飼喂高脂飼料(高脂飼料組成：豬油10%、膽固醇2%及基礎(chǔ)飼料88%)2周，羅非魚出現(xiàn)高脂血癥后進(jìn)入正式試驗(yàn)。

在羅非魚高脂血癥模型建成后，其中2個(gè)模型組分別飼喂含7.5%和15.0%發(fā)酵桑葉的試驗(yàn)飼料(分別為發(fā)酵桑葉低劑量組和發(fā)酵桑葉高劑量組)；1個(gè)模型組飼喂基礎(chǔ)飼料(模型對(duì)照組)；1個(gè)模型組飼喂含0.5%銀杏黃酮的試驗(yàn)飼料(銀杏黃酮組)。在室內(nèi)淡水循環(huán)水族缸(有效體積為300L)中飼養(yǎng)羅非魚8周，日投飼率為體重的4%～6%，每天08:30、13:30、18:30各投喂1次。水源為曝氣自來(lái)水，試驗(yàn)期間水溫為(26.7±2.1) ℃，溶解氧濃度>6.7mg/L，pH為7.3±0.5，氨氮濃度<0.57mg/L，亞硝酸鹽氮濃度<0.09mg/L。

1.3 樣品制備與分析

試驗(yàn)開(kāi)始前(0周)及試驗(yàn)開(kāi)始后的2、5、8周分別測(cè)定各組試驗(yàn)魚的體重。然后每飼喂10d，取血樣檢測(cè)全血中葡萄糖(血糖)水平。飼養(yǎng)試驗(yàn)結(jié)束后，禁食24h后稱重，每個(gè)重復(fù)隨機(jī)取4尾魚，用MS-222進(jìn)行麻醉，剝離出內(nèi)臟、肝胰臟，用于肝體指數(shù)(HSI)和肝脂含量的測(cè)定；每個(gè)重復(fù)另隨機(jī)取3尾魚于尾靜脈取血，于4000×g4℃條件下離心10min，收集血清，-20℃保存?zhèn)溆谩?/p>

血脂指標(biāo)均采用全自動(dòng)生化分析儀(日立7100)測(cè)定，包括血清總膽固醇(TCHO)、甘油三酯(TG)、高密度脂蛋白膽固醇(HDL-C)和低密度脂蛋白膽固醇(LDL-C)含量。血清超氧化物歧化酶(SOD)和過(guò)氧化氫酶(CAT)活性以及丙二醛(MDA)含量采用試劑盒(南京建成生物工程研究所生產(chǎn))進(jìn)行測(cè)定。血糖水平采用上海強(qiáng)生血糖儀測(cè)定。蛋白質(zhì)含量采用考馬斯亮藍(lán)法測(cè)定。

1.4 數(shù)據(jù)處理與分析

試驗(yàn)數(shù)據(jù)以平均值±標(biāo)準(zhǔn)誤表示，采用SPSS 22.0對(duì)所得數(shù)據(jù)進(jìn)行單因素方差分析(one-way ANOVA)，若差異顯著，再進(jìn)行Tukey’s多重比較。

2 結(jié)果與分析

2.1 發(fā)酵桑葉對(duì)高脂血癥羅非魚體重的影響

由表2可知，試驗(yàn)開(kāi)始時(shí)及飼養(yǎng)2周后，各組羅非魚體重?zé)o顯著差異(P>0.05)。飼養(yǎng)5和8周后，模型對(duì)照組羅非魚體重顯著低于正常對(duì)照組、發(fā)酵桑葉低劑量組和發(fā)酵桑葉高劑量組以及銀杏黃酮組(P<0.05)，而發(fā)酵桑葉高劑量組羅非魚的體重與正常對(duì)照組無(wú)顯著差異(P>0.05)。養(yǎng)殖試驗(yàn)結(jié)束后，各組羅非魚體重均有不同程度的增長(zhǎng)。

表2 發(fā)酵桑葉對(duì)高脂血癥羅非魚體重的影響Table 2 Effects of fermented mulberry leaves on body weight of hyperlipidemia tilapia

2.2 發(fā)酵桑葉對(duì)高脂血癥羅非魚血脂指標(biāo)的影響

由表3可知，飼養(yǎng)8周后，發(fā)酵桑葉低劑量組、發(fā)酵桑葉高劑量組和銀杏黃酮組羅非魚血清TCHO、LDL-C含量和TCHO/HDL-C顯著低于模型對(duì)照組(P<0.05)，而發(fā)酵桑葉高劑量組羅非魚血清TCHO、TG、LDL-C含量和TCHO/HDL-C與正常對(duì)照組無(wú)顯著差異(P>0.05)。與模型對(duì)照組相比，發(fā)酵桑葉高劑量組和銀杏黃酮組羅非魚血清HDL-C含量顯著升高(P<0.05)。

表3 發(fā)酵桑葉對(duì)高脂血癥羅非魚血脂指標(biāo)的影響Table 3 Effects of fermented mulberry leaves on serum lipid indices of hyperlipidemia tilapia

2.3 發(fā)酵桑葉對(duì)高脂血癥羅非魚血清抗氧化指標(biāo)、肝脂含量以及HSI的影響

由表4可知，與模型對(duì)照組相比，正常對(duì)照組、發(fā)酵桑葉低劑量組和發(fā)酵桑葉高劑量組以及銀杏黃酮組羅非魚血清SOD、CAT活性以及CAT/SOD顯著升高(P<0.05)，而血清MDA含量和肝脂含量以及HSI顯著降低(P<0.05)。而上述這些指標(biāo)在發(fā)酵桑葉高劑量組與正常對(duì)照組之間無(wú)顯著差異(P>0.05)。

表4 發(fā)酵桑葉對(duì)高脂血癥羅非魚血清抗氧化指標(biāo)、肝脂含量以及肝體指數(shù)的影響Table 4 Effects of fermented mulberry leaves on serum antioxidant indices, hepatopancreas lipid content and HSI of hyperlipidemia tilapia

2.4 發(fā)酵桑葉對(duì)高脂血癥羅非魚血糖水平的影響

由表5可見(jiàn)，相比正常對(duì)照組，高脂飼料造模使模型對(duì)照組、發(fā)酵桑葉低劑量組、發(fā)酵桑葉高劑量組以及銀杏黃酮組羅非魚的血糖水平顯著升高(0周，P<0.05)。飼養(yǎng)2和4周后，模型對(duì)照組、發(fā)酵桑葉低劑量組、發(fā)酵桑葉高劑量組以及銀杏黃酮組羅非魚的血糖水平仍顯著低于正常對(duì)照組(P<0.05)。飼養(yǎng)8周后，發(fā)酵桑葉高劑量組和銀杏黃酮組羅非魚血糖水平顯著低于模型對(duì)照組(P<0.05)，而與正常對(duì)照組差異不顯著(P>0.05)。

表5 發(fā)酵桑葉對(duì)高脂血癥羅非魚血糖水平的影響Table 5 Effects of fermented mulberry leaves on blood glucose level of hyperlipidemia tilapia

3 討 論

脂質(zhì)的異常，尤其是血清TCHO、TG和LDL-C含量升高以及HDL-C含量的降低，在魚類脂肪肝的發(fā)病中具有重要意義[8]。本試驗(yàn)結(jié)果顯示，發(fā)酵桑葉可降低高脂血癥羅非魚血清TCHO和TG含量，同時(shí)增加血清HDL-C含量，降低血清LDL-C含量和CHO/HDL-C，并呈現(xiàn)劑量依賴性。這表明發(fā)酵桑葉具有降低高脂血癥羅非魚血脂水平的活性，其可能是通過(guò)增加膽固醇的逆向轉(zhuǎn)運(yùn)來(lái)實(shí)現(xiàn)的。已有研究表明，銀杏黃酮通過(guò)提高血漿HDL-C含量、降低血漿膽固醇含量來(lái)起到調(diào)節(jié)血脂水平的作用[9]。本試驗(yàn)結(jié)果也同樣證實(shí)，銀杏黃酮能夠降低高脂血癥羅非魚血脂水平。這提示發(fā)酵桑葉調(diào)節(jié)血脂水平可能是通過(guò)影響血脂相關(guān)指標(biāo)來(lái)實(shí)現(xiàn)的，關(guān)于桑葉調(diào)節(jié)魚類血脂水平的機(jī)制尚需進(jìn)一步探究。

正常情況下，SOD主要清除氧自由基，減輕或避免自由基和脂質(zhì)過(guò)氧化物對(duì)動(dòng)物機(jī)體造成的損傷。而CAT將SOD清除活性氧時(shí)產(chǎn)生的過(guò)氧化氫(H2O2)轉(zhuǎn)化為水(H2O)，保護(hù)機(jī)體免受損傷。本研究發(fā)現(xiàn)，預(yù)防性給予發(fā)酵桑葉能夠增加高脂血癥羅非魚血清SOD和CAT活性以及CAT/SOD，降低血清MDA含量。已有研究指出，桑葉中的黃酮是通過(guò)提高機(jī)體抗氧化能力，促進(jìn)胰島素分泌，加快葡萄糖氧化分解這種途徑達(dá)到降低血糖水平的[10]。本試驗(yàn)結(jié)果也進(jìn)一步證實(shí)這種推斷，即飼料中添加發(fā)酵桑葉可能通過(guò)提高機(jī)體抗氧化能力及抑制脂質(zhì)過(guò)氧化反應(yīng)，改善脂質(zhì)代謝功能，從而起到降低血糖水平、調(diào)節(jié)血脂水平的作用。桑葉中含有生物堿1-脫氧野尻霉素(1-deoxynojirimycin，DNJ)和黃酮類物質(zhì)，能夠抑制動(dòng)物腸道刷狀緣膜上二糖酶的活性，減緩腸道對(duì)碳水化合物的消化和吸收，同時(shí)具有調(diào)控肝臟中糖代謝過(guò)程關(guān)鍵酶活性的作用，從而調(diào)控機(jī)體三大營(yíng)養(yǎng)物質(zhì)的代謝與轉(zhuǎn)化[6]。由此可見(jiàn)，桑葉中不同有效成分降血糖的機(jī)制不一，但其作用機(jī)制已涉及到c-Jun氨基末端激酶(JNK)信號(hào)通路[11]。眾所周知，魚類對(duì)碳水化合物的利用能力較低，提升魚類對(duì)飼料糖的耐受能力具有重要的實(shí)踐意義。在水產(chǎn)動(dòng)物上桑葉究竟通過(guò)何種機(jī)制實(shí)現(xiàn)降糖，有待于深入研究。

4 結(jié) 論

發(fā)酵桑葉能劑量依賴性地降低高脂血癥羅非魚的血脂、血糖水平，提高機(jī)體的抗氧化能力。

[1] 宋理平,冒樹(shù)泉,馬國(guó)紅,等.飼料脂肪水平對(duì)許氏平鲉脂肪沉積、血液生化指標(biāo)及脂肪代謝酶活性的影響[J].水產(chǎn)學(xué)報(bào),2014,38(11):1879-1888.

[2] LIN S M,LUO L.Effects of different levels of soybean meal inclusion in replacement for fish meal on growth,digestive enzymes and transaminase activities in practical diets for juvenile tilapia,Oreochromisniloticus×O.aureus[J].Animal Feed Science and Technology,2011,168:80-87.

[3] PARK M Y,LEE K S,SUNG M K.Effects of dietary mulberry,Korean red ginseng,and banaba on glucose homeostasis in relation toPPAR-α,PPAR-γ,andLPLmRNA expressions[J].Life Sciences,2005,77:3344-3354.

[4] KATSUBE T,IMAWAKA N,KAWANO Y,et al.Antioxidant flavonol glycosides in mulberry (M.albaL.) leaves isolated based on LDL antioxidant activity[J].Food Chemistry,2006,97:25-31.

[5] 陳建國(guó),步文磊,來(lái)偉旗,等.桑葉多糖降血糖作用及其機(jī)制研究[J].中草藥,2011,42(3):515-520.

[6] 李有貴,鐘石,呂志強(qiáng),等.飼料中添加桑葉粉對(duì)單胃哺乳動(dòng)物小鼠的脂肪代謝影響[J].蠶業(yè)科學(xué),2012,38(3):0401-0411.

[7] 李法見(jiàn),楊陽(yáng),陳文燕,等.桑葉粉對(duì)羅非魚生長(zhǎng)性能、脂質(zhì)代謝和肌肉品質(zhì)的影響[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào),2014,26(11):3485-3492.

[8] 林鑫,李法見(jiàn),林仕梅,等.不同碳鏈長(zhǎng)度n-3脂肪酸及脂肪水平對(duì)羅非魚生長(zhǎng)、肝功能和餐后血液指標(biāo)的影響[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào),2015,27(3):775-784.

[9] WANG D W,ZHU H,GAO E.Pharmacological action and clinical application on total ginkgo flavone glycosides[J].Food Drug,2006,8(6):7-9.

[10] 陳玲玲,劉煒,陳建國(guó),等.桑葉黃酮對(duì)糖尿病小鼠調(diào)節(jié)血糖的作用機(jī)制研究[J].中國(guó)臨床藥理學(xué)雜志,2010,26(11):835-838.

[11] 羅明琍.桑葉有效部位降血糖作用與JNK信號(hào)通路的關(guān)系[D].博士學(xué)位論文.廣州:廣州中醫(yī)藥大學(xué),2013.

(責(zé)任編輯 菅景穎)

Regulation of Fermented Mulberry Leaves on Serum Lipid and Blood Glucose Levels of Hyperlipidemia Tilapia (Oreochromisniloticus)

SHEN Huangmian1PENG Xianghe1LIN Shimei1*CHEN Yongjun1HUANG Xianzhi2

(1.KeyLaboratoryofFreshwaterFishReproductionandDevelopment,MinistryofEducation,CollegeofAnimalScienceandTechnology,SouthwestUniversity,Chongqing400716,China; 2.InstituteofSericultureandSystemsBiology,SouthwestUniversity,Chongqing400716,China)

The models of hyperlipidemia tilapia induced by high-fat diet were established to explore the effects of fermented mulberry leaves on serum lipid and blood glucose levels of hyperlipidemia tilapia (Oreochromisniloticus). A total of 450male GIFT tilapia with the initial average body weight of 45g were randomly divided into 5groups with 3replicates per group, and 30fish in each replicate. The fish in normal control group were fed with a basal diet, and the other 4groups were fed the basal diet (model control group) and diets contained 7.5% and 15.0% fermented mulberry leaves (low level fermented mulberry leaves group and high level fermented mulberry leaves group, respectively), and 0.5% ginkgo flavone (ginkgo flavone group) after the establishment of the hyperlipidemia model, respectively. The experiment lasted for 8weeks. The results showed as follows: after feeding 5and 8weeks, the body weight in model control group was significantly lower than that in normal control group, low level fermented mulberry leaves group, high level fermented mulberry leaves group and ginkgo flavone group (P<0.05), but no significant difference was found between high level fermented mulberry leaves group and normal control group (P>0.05). The serum total cholesterol (TCHO) and low-density lipoprotein cholesterol (LDL-C) contents and TCHO/high-density lipoprotein cholesterol (HDL-C) in low level fermented mulberry leaves group, high level fermented mulberry leaves group and ginkgo flavone group were significantly lower than those in model control group (P<0.05), but no significant differences were found in the serum TCHO, triglyceride (TG), LDL-C contents and TCHO/HDL-C between high level fermented mulberry leaves group and normal control group (P>0.05). Compared with the model control group, the serum HDL-C content in high level fermented mulberry leaves group and ginkgo flavone group was significantly increased (P<0.05). Compared with the model control group, the serum superoxide dismutase (SOD) and catalase (CAT) activities and CAT/SOD were significantly increased (P<0.05) and the serum MDA content, hepatopancreas lipid content and hepatosomatic index (HSI) were significantly decreased (P<0.05) in normal control group, low level fermented mulberry leaves group, high level fermented mulberry leaves group and ginkgo flavone group, but no significant differences were found in above indices between high level fermented mulberry leaves group and normal control group (P>0.05). High-fat diet could significantly increase the blood glucose level in model control group, low level fermented mulberry leaves group, high level fermented mulberry leaves group and ginkgo flavone group (0week,P<0.05); after feeding 8weeks, the blood glucose level in high level fermented mulberry leaves group and ginkgo flavone group was significantly decreased than that in model control group (P<0.05), but had no significant difference compared with normal control group (P>0.05). These results indicate that fermented mulberry leaves can decrease the serum lipid and blood glucose levels, and enhance the antioxidant ability of hyperlipidemia tilapia with a dose-dependent manner.[ChineseJournalofAnimalNutrition, 2016, 28(4)：1250-1256]

fermented mulberry leaves; tilapia (Oreochromisniloticus); hyperlipidemia; antioxidant

10.3969/j.issn.1006-267x.2016.04.037

2015-10-29

公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)(201303053)；重慶市現(xiàn)代特色效益農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系(渝農(nóng)辦發(fā)〔2015〕91號(hào))；中央高校基本科研業(yè)務(wù)費(fèi)(XDJK2015A001)

沈黃冕(1993—)，男，浙江仙居人，碩士研究生，從事水產(chǎn)動(dòng)物營(yíng)養(yǎng)與飼料研究。E-mail: shenhuangmian@hotmail.com

*通信作者：林仕梅，副教授，碩士生導(dǎo)師，E-mail: linsm198@163.com

S963

A

1006-267X(2016)04-1250-07

*Corresponding author, associate professor, E-mail: linsm198@163.com