潘祉諭， 達靜靜， 董 蓉， 吳 靜， 皮明婧， 俞佳麗， 孫 翼， 聶瑛潔， 查 艷△

(1貴州醫(yī)科大學免疫學教研室， 貴州 貴陽 550004； 貴州省人民醫(yī)院 2腎內(nèi)科， 3中心實驗室， 貴州 貴陽 550002)

Snail1/IGF-1信號通路介導高糖誘導的腎小管上皮細胞EMT*

潘祉諭1， 達靜靜2， 董 蓉2， 吳 靜2， 皮明婧2， 俞佳麗1， 孫 翼1， 聶瑛潔3△， 查 艷2△

(1貴州醫(yī)科大學免疫學教研室， 貴州 貴陽 550004； 貴州省人民醫(yī)院2腎內(nèi)科，3中心實驗室， 貴州 貴陽 550002)

目的： 觀察高糖誘導大鼠腎小管上皮細胞NRK-52E中鋅指轉(zhuǎn)錄因子Snail1和胰島素樣生長因子1(insulin-like growth factor-1，IGF-1)的表達變化，并初步探討Snail1與IGF-1在糖尿病腎臟病(diabetic kidney di-sease，DKD)上皮-間充質(zhì)轉(zhuǎn)化(epithelial to mesenchymal transition，EMT)過程中的關(guān)系。方法: 高糖培養(yǎng)大鼠近端腎小管上皮細胞系NRK-52E 72 h后，給予Snail1 siRNA和IGF-1 siRNA處理，分為高糖組、non-targeting (NT) siRNA組、Snail1 RNAi組和IGF-1 RNAi組，并設(shè)置對照組。于轉(zhuǎn)染后48和72 h兩個時點收獲細胞。分別用實時熒光定量PCR檢測細胞Snail1、IGF-1、 E-鈣黏蛋白(E-cadherin)和纖維連接蛋白(fibronectin，F(xiàn)N)的mRNA表達，用免疫熒光方法檢測各蛋白的表達。結(jié)果: 高糖誘導NRK-52E細胞E-cadherin的mRNA和蛋白表達明顯降低(P<0.01)，F(xiàn)N的mRNA和蛋白表達明顯升高(P<0.01)；同時，Snail1和IGF-1的mRNA和蛋白表達也明顯升高(P<0.01)。Snail1 RNAi組與高糖組比較，細胞中E-cadherin的mRNA和蛋白表達明顯升高(P<0.01)，F(xiàn)N、Snail1和IGF-1的mRNA和蛋白表達明顯降低(P<0.01)，Snail1的mRNA表達減少62.8%。與高糖組比較，IGF-1 RNAi組細胞IGF-1的mRNA表達減少61.1%，E-cadherin 的mRNA和蛋白表達明顯升高(P<0.01)，F(xiàn)N的mRNA和蛋白表達明顯降低(P<0.01)。NT組E-cadherin、FN、Snail1及IGF-1的mRNA和蛋白表達與高糖組比較差異無統(tǒng)計學顯著性。Pearson相關(guān)性分析顯示，NRK-52E細胞中Snail1與IGF-1蛋白的表達呈顯著正相關(guān)(r=0.852,P<0.01)。結(jié)論: Snail1及IGF-1的mRNA和蛋白在高糖誘導的腎小管上皮細胞EMT過程中表達升高，且沉默Snail1基因，IGF-1表達隨之減少，提示Snail1/IGF-1可能促進DKD時腎小管上皮細胞EMT。

糖尿病腎臟??； Snail1蛋白； E-鈣黏蛋白； 纖維連接蛋白

糖尿病腎臟病(diabetic kidney disease，DKD) 是糖尿病(diabetic mellitus，DM)最常見的慢性微血管并發(fā)癥，可發(fā)展至終末期腎功能衰竭，是DM患者的主要死因之一。上皮-間充質(zhì)轉(zhuǎn)化(epithelial to mesenchymal transition，EMT)既是多細胞生物胚胎發(fā)育中的基礎(chǔ)過程，也是腫瘤侵襲和轉(zhuǎn)移的重要機制。近年來，動物模型和腎病患者腎組織活檢結(jié)果均證明了纖維化腎臟存在腎小管上皮-肌成纖維細胞轉(zhuǎn)分化(tubular epithelial-myofibroblast transdifferentiation，TEMT)，在DKD腎小管間質(zhì)纖維化發(fā)生、發(fā)展中也存在TEMT現(xiàn)象[1-3]。新近研究還發(fā)現(xiàn)，阻斷TEMT可有效減輕腎臟纖維化，改善腎功能，表明TEMT在腎臟纖維化過程中發(fā)揮重要作用，但目前對其分子調(diào)節(jié)機制了解甚少[4]。

鋅指轉(zhuǎn)錄因子Snail1為轉(zhuǎn)錄抑制因子，已有大量研究證實Snail1基因在正常胚胎發(fā)育和腫瘤浸潤轉(zhuǎn)移的EMT中起著重要作用[5]。在正常腎臟發(fā)育過程中，Snail1早期表達于腎臟上皮細胞前體細胞中，在上皮細胞分化時處于下調(diào)狀態(tài)，作為上皮細胞可塑性的抑制因子維持著腎臟上皮組織的正常結(jié)構(gòu)及內(nèi)環(huán)境的穩(wěn)態(tài)[6]。研究表明腎皮質(zhì)Snail1與纖維連接蛋白(fibronectin，F(xiàn)N)的mRNA表達水平呈顯著正相關(guān)，與E-鈣粘蛋白(E-cadherin)的表達呈負相關(guān)，并且還發(fā)現(xiàn)Snail1通過與E-cadherin啟動子區(qū)E-盒結(jié)合，抑制其表達，改變了細胞的黏附性，提示Snail1可能通過介導FN生成并抑制E-cadherin轉(zhuǎn)錄而觸發(fā)腎小管上皮細胞EMT，參與了DKD的發(fā)生和發(fā)展[7-8]。

胰島素樣生長因子1(insulin-like growth factor-1，IGF-1)是機體正常生長發(fā)育所必須的因子，具有廣泛的生物學功能。已有研究表明IGF-1參與腫瘤相關(guān)EMT，促進腫瘤浸潤和轉(zhuǎn)移。正常情況下，全身各組織均有IGF-1表達，參與組織生長發(fā)育。最新研究報道，在DKD和多囊性腎病(polycystic kidney disease，PKD)時，腎臟局部IGF-1表達增多[9]。但是Snail1與IGF-1二者是否相關(guān)，檢索文獻國內(nèi)外未見相關(guān)報道。本研究用高糖刺激大鼠腎小管上皮細胞，觀察Snail1和IGF-1的表達變化，而后利用RNA沉默技術(shù)敲低Snail1后，觀察IGF-1的表達變化及EMT相關(guān)標志分子的表達變化，探討Snail1在DKD的EMT過程中是否與IGF-1相關(guān)，明確這一分子機制可為疾病的治療選擇合適的治療靶點提供理論依據(jù)。

材料和方法

1材料

大鼠近端腎小管上皮細胞 NRK-52E由南方醫(yī)科大學侯凡凡院士惠贈； DMEM培養(yǎng)基(Gibco)；胎牛血清(fetal bovine serum，F(xiàn)BS； HyClone)；Snail1 siRNA、non-targeting (NT) siRNA和transfection reagent 2(Dharmacon)；抗IGF-1抗體和抗Snail1抗體(Abcam)；抗E-cadherin抗體和抗FN抗體(北京百奧思科生物技術(shù)有限公司)；AlexaFluor 488和AlexaFluor 594(北京百奧思科生物技術(shù)有限公司)；Trizol(Invitrogen)；逆轉(zhuǎn)錄試劑盒PrimeScriptTMRT reagent Kit(TaKaRa)；PowerUpTMSYBR?Green Master Mix(ThermoFisher)。

2方法

2.1細胞培養(yǎng) NRK-52E細胞分別用含20% FBS和青鏈霉素的DMEM培養(yǎng)基(分別含5 mmol/L和25 mmol/L葡萄糖)培養(yǎng)。培養(yǎng)72 h后，選取融合至80%左右、生命力旺盛的細胞用0.25%胰酶消化，并接種于放或不放蓋玻片的6孔板中，使每孔含5×104個細胞，分別用于制作細胞玻片免疫熒光染色和提取RNA。

2.2分組及siRNA轉(zhuǎn)染 按如下分組：對照組(control group； 含5 mmol/L葡萄糖)、高糖組(high glucose group, HG group； 含25 mmol/L葡萄糖)、NT siRNA組(高糖培養(yǎng)72 h后給予25 nmol/L NT si-RNA處理)、Snail1 RNAi組(高糖培養(yǎng)72 h后給予25 nmol/L Snail1 siRNA處理)和IGF-1 RNAi組(高糖培養(yǎng)72 h后給予25 nmol/L IGF-1 siRNA處理)。將siRNA和轉(zhuǎn)染試劑分別按說明書稀釋，混合后加入相應(yīng)孔內(nèi)，同時設(shè)置陰性對照。于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)48 h和72 h后收獲細胞。

2.3實時熒光定量PCR實驗 (1)提取總RNA： PBS (1×)潤洗細胞3次，Trizol裂解液于冰上裂解細胞10 min，細胞裂解液移至1.5 mL去酶EP管中，加入1/5體積氯仿上下顛倒混勻，冰上沉淀5 min，于4 ℃預(yù)冷的離心機中12 000 r/min離心15 min，取上清并移至新的1.5 mL去酶EP管中，加入等體積的異丙醇上下顛倒混勻，12 000 r/min離心10 min，棄上清，加入75%乙醇(750 μL無水乙醇+25 μL DEPC處理水)洗滌RNA沉淀，7 500 r/min離心5 min，重復洗滌1次，室溫敞口放置5 min，加10 μL RNase-free water溶解RNA沉淀。用核酸蛋白濃度檢測儀檢測RNA吸光度，均在1.8～2.0之間。(2)逆轉(zhuǎn)錄cDNA：按照TaKaRa逆轉(zhuǎn)錄試劑盒說明書，反應(yīng)體系為：5× PrimeScript buffer 2 μL， PrimeScript RT Enzyme Mix 10.5 μL， oligo dT primer 0.5 μL， random 6-mers 0.5 μL， total RNA 6.5 μL。反應(yīng)條件為： 37 ℃逆轉(zhuǎn)錄15 min， 85 ℃滅活逆轉(zhuǎn)錄酶5 s，模板cDNA短期內(nèi)于4 ℃保存。(3)實時熒光定量PCR：用于實時熒光定量PCR的引物序列見表1。反應(yīng)體系中包括 PowerUpTMSYBR? Green Master Mix (2×) 5 μL、上游引物(300～500 nmol/L) 1.5 μL、下游引物(300～500 nmol/L) 1.5 μL、模板cDNA 1 μL和nuclease-free water 3 μL，總體系量為12 μL。反應(yīng)條件為UDG的激活 50 ℃ 2 min，雙脫氧核糖核酸聚合酶活化 95 ℃ 2 min，變性、退火和延伸分別為95 ℃ 15 s、60 ℃ 15 s和72 ℃ 1 min，共40個循環(huán)。每次在退火和延伸階段檢測熒光信號。取循環(huán)閾值(Ct)均值，分別計算各組ΔCt (Ct目的基因-Ct內(nèi)參照基因)，再進行組間比較計算ΔΔCt (ΔCt組1-ΔCt組2)，以2-ΔΔCt表示基因的相對表達水平。

表1 實時熒光定量PCR的引物序列

F: forward; R: reverse.

2.4免疫熒光實驗 PBS潤洗細胞，丙酮于-20 ℃固定細胞15 min，0.1% Triton X-100 4 ℃打孔10 min，1% BSA 4 ℃封閉1 h，下列抗體按1∶50稀釋(IGF-1、Snail1、E-cadherin和FN)于4 ℃孵育過夜。充分洗片后37 ℃孵育熒光 II 抗，DAPI染核37 ℃ 20 min。熒光顯微鏡下隨機選取10個視野，每個視野選取20個細胞。細胞核呈藍色，目的蛋白IGF-1和E-cadherin呈紅色，Snail1和FN呈綠色。IPP軟件檢測陽性細胞平均熒光強度。

3統(tǒng)計學處理

用SPSS 17.0和GraphPad Prism 5軟件進行統(tǒng)計分析。數(shù)據(jù)以均數(shù)±標準差(mean±SD)表示，兩組比較采用t檢驗，多組組間比較采用單因素方差分析，用Pearson相關(guān)性分析法分析Snail1和IGF-1蛋白表達的相關(guān)性。以P<0.05為差異有統(tǒng)計學意義。

結(jié) 果

1E-cadherin和FN的mRNA表達情況

實時熒光定量PCR檢測結(jié)果顯示，與對照組比較，高糖組E-cadherin 的mRNA表達明顯減少(P<0.01)，F(xiàn)N的mRNA表達均明顯增多(P<0.01)。沉默Snail1基因后，Snail1的mRNA表達減少62.8%，E-cadherin 的mRNA表達增多(P<0.01)，F(xiàn)N的mRNA表達減少(P<0.01)。沉默IGF-1基因后，其表達減少約61.1%，E-cadherin的mRNA表達增多(P<0.01)，F(xiàn)N的mRNA表達減少(P<0.01)。NT siRNA組各因子mRNA的表達水平與高糖組間的差異無統(tǒng)計學顯著性,見圖1。

2E-cadherin和FN蛋白的表達情況

免疫熒光結(jié)果顯示，對照組的E-cadherin蛋白高表達，F(xiàn)N蛋白低表達。經(jīng)高糖刺激后E-cadherin蛋白表達明顯減少(P<0.01)，F(xiàn)N蛋白表達均明顯增加(P<0.01)。Snail1 RNAi組的E-cadherin蛋白表達恢復(P<0.01)，F(xiàn)N蛋白表達顯著降低(P<0.01)。沉默IGF-1基因后，E-cadherin的mRNA表達上調(diào)(P<0.01)，F(xiàn)N的mRNA表達減少(P<0.01)。NT siRNA組各因子的蛋白表達與高糖組比較差異無統(tǒng)計學顯著性，見圖2。

3Snail1和IGF-1的mRNA表達情況

實時熒光定量PCR檢測結(jié)果顯示，高糖組較對照組Snail1和IGF-1的mRNA表達均明顯增多(P<0.01)。沉默Snail1基因后，IGF-1的mRNA表達也顯著減少(P<0.01)。NT siRNA組與高糖組相比，Snail1和IGF-1 mRNA表達水平的差異無統(tǒng)計學顯著性,見圖3。

Figure 1. The relative mRNA expression of E-cadherin and FN in the NRK-52E cells of each group detected by real-time PCR. A: the mRNA expression of E-cadherin and FN after high glucose (HG) stimulation； B, C: Snail1 and IGF-1 mRNA expression after RNAi， respectively； D： the mRNA expression of E-cadherin and FN after HG stimulation and Snail1/IGF-1 RNAi. Mean±SD.n=3.**P<0.01vscontrol;##P<0.01vsHG.

圖1實時熒光定量PCR檢測細胞中E-cadherin和FN的mRNA表達情況

4Snail1和IGF-1蛋白的表達情況

免疫熒光觀察結(jié)果顯示，對照組的IGF-1蛋白低表達，Snail1蛋白微弱表達。高糖組Snail1和IGF-1蛋白與對照組比較表達明顯增多(P<0.01)。Snail1 RNAi組Snail1和IGF-1蛋白比高糖組表達顯著減少(P<0.01)。NT siRNA組與高糖組相比，這2種蛋白表達水平的差異無統(tǒng)計學顯著性，見圖4。

5相關(guān)性分析

Pearson相關(guān)性分析結(jié)果顯示，同一細胞中Snail1與IGF-1蛋白的相關(guān)系數(shù)r=0.852，表達呈顯著正相關(guān)，結(jié)果具有統(tǒng)計學意義(P<0.01),見圖5。

討 論

在世界范圍內(nèi)，DKD是糖尿病患者嚴重的并發(fā)癥之一，最終導致腎功能衰竭。2016年最新WHO-DM報告指出，成人DM已超過5 億，DKD患者人數(shù)達2.85億，是導致終末期腎衰竭的首要原因。在中國，DM 患者近1.33億，DKD 患者達0.6億，是導致終末期腎衰竭的第二位原因，且呈逐年增加的趨勢。預(yù)計到2040年，DKD將會成為我國終末期腎病(end stage renal disease，ESRD)患者透析治療的首要病因，這將給社會經(jīng)濟帶來巨大負擔[10]。因此研究DKD的發(fā)病機制是必要的。腎小管間質(zhì)纖維化是導致DKD不可逆性進展的重要病理過程，增多的細胞外基質(zhì)(extracellular matrix，ECM)沉積于腎間質(zhì)是導致腎小管間質(zhì)纖維化的主要原因[11-12]。近年來研究發(fā)現(xiàn)ECM主要由間質(zhì)肌成纖維細胞合成、分泌，肌成纖維細胞主要由腎小管上皮細胞發(fā)生EMT而來，因此EMT是導致腎臟纖維化的重要機制之一[13-14]。發(fā)生EMT的細胞表現(xiàn)為E-cadherin等上皮細胞標志基因表達減少，F(xiàn)N等間質(zhì)細胞標志基因表達增多[15-16]。本研究在體外高糖培養(yǎng)NRK-52E細胞72 h后，觀察到E-cadherin的mRNA和蛋白表達減少，F(xiàn)N的mRNA和蛋白表達增加，表明高糖刺激腎小管上皮細胞發(fā)生了EMT。

Snail1蛋白屬于鋅指轉(zhuǎn)錄因子Snail超家族成員，1984年首次在黑腹果蠅胚胎中發(fā)現(xiàn)，它是EMT的強誘導因子，誘導上皮細胞發(fā)生EMT，并遷移到中胚層，參與中胚層的形成。在胸腺癌的研究中表明，Snail1抑制E-cadherin的轉(zhuǎn)錄而誘導EMT，顯示Snail1是調(diào)控EMT的一個關(guān)鍵因子[17]。另外，Guo 等[18]研究發(fā)現(xiàn)高糖環(huán)境刺激Snail1表達增加，促進永生型小鼠足細胞發(fā)生EMT，表明高糖環(huán)境影響足細胞中Snail1的表達。方開云等[19]和Sato等[20]在糖尿病大鼠模型中的研究表明，高糖環(huán)境影響腎小管上皮細胞中Snail1的表達，促進腎小管間質(zhì)病變的發(fā)生、發(fā)展。本研究經(jīng)高糖培養(yǎng)NRK-52E細胞后，檢測Snail1的mRNA和蛋白表達，結(jié)果顯示其表達明顯升高，表明Snail1參與高糖環(huán)境誘導的腎小管上皮細胞EMT，提示Snail1可能參與DKD的病程進展，這與上述研究結(jié)果一致。

Figure 2. The protein expression of E-cadherin and FN in the NRK-52E cells of each group observed with immunofluorescence staining (×200). Mean±SD.n=3.**P<0.01vscontrol;##P<0.01vsHG.

圖2免疫熒光檢測細胞中E-cadherin和FN蛋白的表達情況

IGF-1的結(jié)構(gòu)和功能與胰島素類似，可通過內(nèi)分泌、自分泌和旁分泌的方式產(chǎn)生。腎臟既是其分泌器官，也是其靶器官。Wada等[21]報道IGF-1與結(jié)締組織因子在高血糖誘導下具有協(xié)同作用刺激系膜細胞合成細胞外基質(zhì)成分，F(xiàn)N是IGF-1刺激合成的最主要基質(zhì)蛋白。IGF-1可使胞內(nèi)E-cadherin的表達降低，促進EMT[22]。本研究同時檢測IGF-1的mRNA和蛋白表達，結(jié)果顯示其亦明顯升高，提示Snail1及IGF-1均參與DKD的EMT機制。近期研究表明，Snail1和 IGF-1均可通過不同的途徑參與EMT的發(fā)生、發(fā)展。然而尚不清楚Snail1與IGF-1是否為同一信號通路中的上下游分子，或者它們所在的不同信號通路是否存在交叉信號。為了進一步探討二者的關(guān)系，本實驗利用siRNA，特異性沉默Snail1基因后發(fā)現(xiàn)，IGF-1的mRNA和蛋白表達隨之下調(diào)，且E-cadherin表達恢復，F(xiàn)N表達下調(diào)，提示沉默Snail1可能通過下調(diào)IGF-1的表達，逆轉(zhuǎn)EMT過程。另外，應(yīng)用Pearson相關(guān)性分析發(fā)現(xiàn)Snail1與IGF-1蛋白表達呈顯著正相關(guān)(r=0.852)。綜合上述實驗結(jié)果，我們認為在高糖環(huán)境下，IGF-1介導了Snail1誘導的EMT事件的發(fā)生。

Figure 3. The relative mRNA expression of IGF-1 and Snail1 in the NRK-52E cells of each group detected by real-time PCR. A: IGF-1 and Snail1 mRNA expression after HG stimulation； B: IGF-1 and Snail1 mRNA expression after Snail1 RNAi. Mean±SD.n=3.**P<0.01vscontrol；##P<0.01vsHG.

圖3實時熒光定量PCR檢測細胞中IGF-1和Snail1的mRNA表達情況

Figure 4. The protein expression of Snail1 and IGF-1 in the NRK-52E cells of each group (×200). Mean±SD.n=3.**P<0.01vscontrol;##P<0.01vsHG.

圖4免疫熒光檢測細胞中IGF-1和Snail1蛋白的表達情況

Figure 5. Correlation between Snail1 and IGF-1 protein expression determined by Pearson correlation analysis.r=0.852,P<0.01.

圖5Snail1和IGF-1蛋白表達的相關(guān)性

綜上所述，本實驗結(jié)果驗證了Snail1和IGF-1均參與DKD的EMT機制，并首次證明了Snail1可能通過調(diào)節(jié)IGF-1的表達，進而調(diào)節(jié)EMT，在細胞水平做了初步探討，但Snail1調(diào)節(jié)IGF-1的具體機制有待進一步探究。EMT是導致DKD腎纖維化進展的重要機制，明確參與調(diào)節(jié)EMT的細胞因子之間的相關(guān)性是十分必要的，可能為治療DKD提供新的治療靶點。

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(責任編輯： 盧 萍， 羅 森)

Snail1/IGF-1 pathway mediates high glucose-induced EMT in renal tubular epithelial cells

PAN Zhi-yu1, DA Jing-jing2, DONG Rong2, WU Jing2, PI Ming-jing2, YU Jia-li1, SUN Yi1, NIE Ying-jie3, ZHA Yan2

(1Department of Immunology, Guizhou Medical University, Guiyang 550004, China;2Department of Nephrology,3Department of Central Laboratory, Guizhou Provincial People’s Hospital, Guiyang 550002, China. E-mail: zhayan72@126.com)

AIM: To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose, and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS: The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h, and divided into control group, high glucose group, non-targeting (NT) siRNA group, Snail1 RNAi group and IGF-1 RNAi group. The cells were harvested at 48 h and 72 h. Real-time PCR was used to detect the mRNA expression of Snail1, IGF-1, E-cadherin and fibronectin (FN), and the protein levels were determined by immunofluorescence staining.RESULTS: Compared with control group, the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P<0.01), while that of FN was elevated (P<0.01). Meanwhile, the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P<0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group(P<0.01), while that of FN, IGF-1 and Snail1 was significantly down-regulated (P<0.01). The same changes were observed in IGF-1 RNAi group (P<0.01). The protein expression of each factor in NT group had no significant change as compared with high glucose group (P>0.05). Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r=0.852,P<0.01).CONCLUSION: Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.

Diabetic kidney disease; Snail1 protein; E-cadherin; Fibronectin

1000- 4718(2017)09- 1662- 07

2016- 12- 27 [

] 2017- 06- 26

貴州省科技廳-貴州省人民醫(yī)院聯(lián)合基金資助項目(黔科合LH字[2014]7002號; 黔科合LH字[2015]7155號; 黔科合LH字[2015]7154號; 黔科合LH字[2016]7169號); 貴州省衛(wèi)計委科學技術(shù)基金資助項目(No.gzwkj2014-1-046)

R363; R587.1

A

10.3969/j.issn.1000- 4718.2017.09.021

△通訊作者 聶瑛潔 Tel: 13639043101; E-mail: nienyj@hotmail.com； 查 艷 Tel: 13639106123; E-mail: zhayan72@126.com