外泌體microRNA在豬成熟和閉鎖卵泡中的表達差異及功能分析

陳慧芳，黃綺亮, 胡智超, 潘曉婷, 吳志勝, 白銀山

外泌體microRNA在豬成熟和閉鎖卵泡中的表達差異及功能分析

陳慧芳，黃綺亮, 胡智超, 潘曉婷, 吳志勝, 白銀山

佛山科學技術(shù)學院生命科學與工程學院，廣東佛山528231

【】通過分析成熟卵泡液外泌體（mature follicular fiuid Exosomes, mffEXs）和閉鎖卵泡液外泌體（atretic follicular fiuid Exosomes, affEXs）miRNA的表達差異，探索卵泡液外泌體（EXs）miRNA在卵泡發(fā)育和閉鎖過程中的調(diào)控作用。本研究通過抽提4—6 mm豬成熟發(fā)育和閉鎖卵泡的卵泡液分離外泌體，進行粒徑分析及Western Blot檢測對EXs進行鑒定，接下來對特征性EXs攜帶的miRNA測序和功能富集分析，篩選關(guān)鍵信號通路和差異基因。最后，將mffEXs和affEXs作為添加劑進行顆粒細胞培養(yǎng)，利用Q-PCR檢測技術(shù)分析關(guān)鍵基因的表達，驗證兩類卵泡液內(nèi)EXs miRNA在卵泡發(fā)育中的調(diào)控功能。成功分離了mffEXs和affEXs，對比mffEXs測序結(jié)果，affEXs中有90個miRNA上調(diào)表達，220個miRNA下調(diào)表達，表明了卵泡液中的miRNA表達水平與調(diào)控卵泡發(fā)育有關(guān)；KEGG富集分析結(jié)果顯示兩類卵泡的差異信號通路主要集中在Ras、cAMP、P53和MAPK等信號通路，涉及調(diào)控卵母細胞發(fā)育、減數(shù)分裂以及顆粒細胞細胞周期等生物學功能。在閉鎖卵泡中，上調(diào)表達的ssc-let-7a和ssc-miR-133a-3p分別潛在靶向調(diào)控細胞周期蛋白依賴性激酶（）和胰島素生長因子（），抑制了G1和G2/M期的運轉(zhuǎn)和類固醇激素代謝，促使顆粒細胞周期運轉(zhuǎn)受阻和顆粒細胞凋亡，引起卵泡閉鎖的發(fā)生；下調(diào)的ssc-miR-21-5p潛在靶向腫瘤抑癌基因（），抑制細胞周期運轉(zhuǎn)，促使顆粒細胞凋亡。在體外培養(yǎng)的顆粒細胞中分別添加mffEXs和affEXs，Q-PCR結(jié)果顯示在mffEXs中顯著上調(diào)表達，而顯著下調(diào)表達，表明了測序分析結(jié)果的可靠性。這些結(jié)果均顯示了affEXs中miRNA表達水平的變化促使顆粒細胞凋亡和細胞周期阻滯，引起卵泡閉鎖。豬affEXs攜帶miRNA增加了對、和的表達調(diào)控，抑制顆粒細胞細胞周期運轉(zhuǎn)和類固醇激素代謝等信號通路，引起顆粒細胞凋亡，導致卵泡閉鎖。

豬；成熟卵泡液；閉鎖卵泡液；EXs；miRNA

0 引言

【研究意義】卵泡閉鎖是指一些卵泡發(fā)育到一定階段后停止發(fā)育并發(fā)生退化的一種普遍存在的現(xiàn)象。在很多雌性動物卵泡發(fā)育過程中，只有極少數(shù)卵泡能夠發(fā)育成熟并且排卵，絕大多數(shù)卵泡都會發(fā)生閉鎖[1-2]，這可能與卵泡液所含營養(yǎng)物質(zhì)的差異[3]、激素[4]、氧化應激[5]和各種細胞因子[6]等相關(guān)，它們能夠通過不同途徑將刺激信號級聯(lián)放大，引起通路中相關(guān)作用基因或轉(zhuǎn)錄因子活性增加或減少進而調(diào)控細胞凋亡[7-8]。以往研究顯示激素和營養(yǎng)物質(zhì)均能參與調(diào)控卵泡閉鎖，而關(guān)于外泌體（exosomes, EXs）的調(diào)控作用，目前還知之甚少[9]。EXs是一類由細胞分泌的直徑大小約30—150 nm的細胞外囊泡，因攜帶多種生物活性物質(zhì)，可進行細胞間信息傳遞[10]。已有報道在各種體液中均含有大量EXs[11-12]，且EXs可選擇性地包裹細胞內(nèi)的多種活性調(diào)節(jié)物質(zhì)（miRNA、LncRNA和CircRNA等），其中miRNA含量最豐富，廣泛進行細胞間物質(zhì)轉(zhuǎn)運和信號轉(zhuǎn)導，調(diào)節(jié)功能基因表達[13-14]。近年來，相關(guān)卵泡液EXs miRNA的研究越來越多，但關(guān)于EXs miRNA對卵泡閉鎖的調(diào)控機制仍未見報道。【前人研究進展】適宜的卵泡液微環(huán)境是促使卵泡發(fā)育的主要場所，研究顯示卵泡液EXs中的miRNA廣泛參與調(diào)控顆粒細胞類固醇激素代謝的相關(guān)通路[15]，涉及調(diào)節(jié)顆粒細胞增殖和雌二醇的合成與分泌[16]。卵泡液EXs miRNA的相關(guān)研究已在多種女性生殖系統(tǒng)疾病中作為檢測和治療的關(guān)鍵靶標[17-20]。如高水平表達的EXs-miR-199可以抑制芳香化酶的合成與代謝，促使雄激素升高，引起了多囊卵巢綜合癥的發(fā)生發(fā)展[21]。卵泡閉鎖也可能直接受到卵泡液EXs miRNA的調(diào)控，但目前miRNA在卵泡閉鎖過程中的調(diào)控機制還并不清楚?！颈狙芯壳腥朦c】卵泡液攜帶的EXs miRNA在調(diào)控卵泡閉鎖方面的研究有待解析。本研究對豬EXs miRNA進行測序分析，通過GO和KEGG功能富集分析，探究EXs miRNA對卵泡閉鎖的調(diào)控機制。【擬解決的關(guān)鍵問題】通過對比成熟及閉鎖卵泡液中的EXs miRNA的表達情況，擬探究豬卵泡液EXs攜帶的miRNA在調(diào)控成熟卵泡發(fā)育和閉鎖卵泡發(fā)生的潛在分子機制。

1 材料與方法

試驗于2018年10月至2019年12月于廣東省佛山科學技術(shù)學院生命科學與工程學院和華南農(nóng)業(yè)大學動物科學學院進行。

1.1 卵泡液的收集

從廣州市嘉禾屠宰場采集豬卵巢，裝入38.5℃含有青霉素和鏈霉素生理鹽水中的保溫瓶內(nèi)，2 h內(nèi)運回實驗室。用37℃添加青鏈霉素的生理鹽水清洗3遍，隨后使用10 mL注射器吸取直徑為4—6 mm卵泡中的液體，4℃，12 000 r/min離心3 min去除細胞碎片；然后使用0.22mm過濾器過濾，進行EXs提取。

1.2 外泌體的分離與鑒定

使用ExoQuick Exosome Isolation Kit（System Bioscience, SBI）分離mffEXs和affEXs，詳細步驟均參照試劑盒說明書。分離完成后，一部分用于檢測鑒定，另一部分置于-80℃冰箱中保存?zhèn)溆谩?/p>

1.3 外泌體粒徑分析（NTA）

取一部分mffEXs和affEXs，用PBS重懸，隨后將液體添加到Nanosight（NS300）納米顆粒跟蹤分析儀（NTA）中，利用NTA對分離出的EXs的大小和形態(tài)進行分析。

1.4 Western Blot技術(shù)檢測

將RIPA裂解液混合加入mffEXs和affEXs中，進行裂解和變性。根據(jù)Western-Blot步驟檢測外泌體表面標志蛋白CD9、CD63和CD81（Rabbit anti-CD9, 1﹕2000 Dilution, ab92726 Abcam; Rabbit anti-CD63, 1﹕2000 Dilution, ab231975, Abcam和Rabbit anti-CD81, 1﹕2000 Dilution, ab109201, Abcam）的表達，4℃過夜孵育，1×TBST洗脫3次后，添加二抗（sc-2030, Santa Cruz, 1﹕2000 Dilution），室溫孵育2 h，1×TBST洗脫3次，添加顯色劑顯色后使用凝膠成像系統(tǒng)拍照[22-23]。

1.5 外泌體總RNA提取、cDNA文庫構(gòu)建和RNA測序

采用Trizol（Life teachnologies）傳統(tǒng)法提取mffEXs和affEXs總RNA，通過NanoDrop（Nanodrop 2000，Thermo，USA）以及Agilent 2100（Agilent Technologies, Palo Alto, CA, USA）對提取的總RNA的濃度和完整性進行檢測，并構(gòu)建小RNA文庫，使用Illumina HiSeqTM2000（Illumina, San Diego, CA, USA）進行測序，該部分由廣州基迪奧生物科技有限公司完成。

1.6 miRNA的差異分析與靶基因預測

評估m(xù)iRNA測序質(zhì)量，計算RNA的長度分布，過濾掉原始數(shù)據(jù)中低質(zhì)量序列（質(zhì)量值＜20的堿基數(shù)超過1個或含N的序列），獲得高質(zhì)量的Reads。通過計算TPM（Tags per million）的表達量[24]，篩選出mffEXs和affEXs中差異表達的miRNA。使用RNAhybrid（v2.1.2）、Miranda（v3.3a）和TargetScan（Version：7.0）進行miRNA靶基因預測和功能分析，并將獲得的靶基因用Cytoscape軟件繪制成可視化互作網(wǎng)絡圖。

1.7 GO和KEGG通路富集分析

篩選出卵泡液EXs中差異表達miRNA對應的靶基因，進行GO（http://www.geneontology.org/）和KEGG（kyoto encyclopedia of genesand genomes; http: //www.kegg.jp/kegg）顯著性富集分析[25]，分別描述GO的分子功能（molecular function）、細胞組分（cellular component）和生物進程（biological process），并選擇與卵母細胞發(fā)育相關(guān)的主要調(diào)節(jié)基因和信號通路進行分析。

1.8 顆粒細胞試驗

將配制20 μg·mL-1的mffEXs和affEXs作為添加劑加到顆粒細胞培養(yǎng)基中進行培養(yǎng)，48 h后提取顆粒細胞的RNA，反轉(zhuǎn)錄合成cDNA，通過Q-PCR分析和的表達情況，具體反應條件為95℃預變性4 min；95℃變性30 s；59℃退火30 s；72℃延伸15 s，共33個循環(huán)。其中引物（上游AGAGTCGCTGGGGATCTACC；下游：TCATGGC TACCACTTGACCTG，擴增產(chǎn)物134 bp；XM_ 005671013）、引物（上游AAGGGAATTTACGGGC CGAG；下游CACGCACCTCAAAGCTGTTC，擴增產(chǎn)物228 bp；NM_213824）和內(nèi)參引物（上游TCGGAGTGAACGGATTTGGC；下游TGACAAG CTTCCCGTTCTCC，擴增產(chǎn)物189 bp；NM_001206359）。

2 結(jié)果

2.1 成熟及閉鎖卵泡液EXs的分離鑒定與miRNA測序分析

將分離的成熟卵泡和閉鎖卵泡（圖1-A，B）EXs進行粒徑分析和蛋白檢測，結(jié)果顯示粒徑峰值約100 nm（圖1-C，D）；Western-Blot結(jié)果顯示，CD9、CD63和CD81（圖1-E）這些標志性蛋白均在分離的mffEXs和affEXs中表達。測序結(jié)果顯示，mffEXs和affEXs攜帶的miRNA序列長度主要分布在20—22 nt范圍內(nèi)（圖1-F）；miRNA表達譜比較分析顯示，90個miRNA在affEXs中顯著上調(diào)表達，220個miRNA顯著下調(diào)表達（圖1-G）。

2.2 mffEXs和affEXs中主要差異的miRNA

根據(jù)mffEXs和affEXs攜帶的miRNA測序結(jié)果，以差異倍數(shù)|log2（F/C）|≥1.2，且＜0.05為閾值進行篩選，發(fā)現(xiàn)這些miRNA在mffEXs和affEXs中存在顯著差異，且可能直接參與調(diào)控卵泡的成熟發(fā)育及閉鎖（表1）。

2.3 GO功能注釋分析

根據(jù)差異表達miRNA的靶基因進行GO功能注釋分析，結(jié)果發(fā)現(xiàn)它們主要富集于11個生物過程條目（細胞周期進程、蛋白絲氨酸/蘇氨酸激酶活性、MAPK級聯(lián)和有絲分裂細胞周期的G2/M轉(zhuǎn)變等）、11個細胞組分條目（紡錘、周期蛋白依賴的蛋白激酶全酶復合物、蛋白激酶復合物和泛素連接酶復合物等）和13個分子功能條目（磷酸酶結(jié)合、鈣調(diào)素結(jié)合、蛋白磷酸酶結(jié)合和腺苷酸環(huán)化酶活性等）。進一步研究發(fā)現(xiàn)，這些差異基因被劃分為多種生物功能類別，包括細胞增殖、代謝和凋亡等。差異表達基因的分布及生物學過程類別如圖2所示。

A：閉鎖卵泡的形態(tài)觀察結(jié)果；B：成熟卵泡的形態(tài)觀察結(jié)果；C：mffEXs粒徑分析結(jié)果；D：affEXs粒徑分析結(jié)果；E：Westem blot檢測結(jié)果；F：miRNA的序列長度分布；G：mffEXs和affEXs中miRNA的表達差異結(jié)果

2.4 KEGG富集分析

KEGG通路分析顯示差異表達的miRNA涉及調(diào)控169個靶基因，主要富集于Ras信號通路（ssc04014）、cAMP信號通路（ssc04024）、P53信號通路（ssc04115）、MAPK信號通路（ssc04010）、卵母細胞減數(shù)分裂（ssc04114）、泛素介導的蛋白水解作用（ssc04120）和細胞周期（ssc04110）等通路（圖3），可能參與調(diào)控卵泡的成熟與閉鎖。

2.5 富集關(guān)鍵信號通路中miRNA潛在的靶基因

差異表達的miRNA靶標富集的主要信號通路顯示了靶基因的調(diào)控作用（表2）。通過富集通路篩選出一些參與顆粒細胞凋亡和卵母細胞發(fā)育的關(guān)鍵基因，結(jié)合信號傳導通路深入研究豬卵泡閉鎖調(diào)控機制，發(fā)現(xiàn)介導卵泡閉鎖的途徑顯著富集于7條信號通路，且這些通路可能直接參與調(diào)控卵泡閉鎖。

表1 卵泡液EXs中主要差異表達的miRNA

2.6 構(gòu)建miRNA-靶基因互作網(wǎng)絡

將篩選獲得的與卵泡成熟發(fā)育和閉鎖相關(guān)且差異表達顯著的miRNA及靶基因進行了共表達網(wǎng)絡分析。在互作網(wǎng)絡中，我們檢測了3個特異性miRNA（ssc-miR-21-5p、ssc-miR-133a-3p和ssc-let-7a），發(fā)現(xiàn)它們分別作用于和等基因（圖4）。其中，是一種調(diào)節(jié)細胞周期運轉(zhuǎn)的重要激酶，它能在特定時間被激活，通過底物磷酸化結(jié)合cyclin B驅(qū)動細胞周期運轉(zhuǎn)，而ssc-let-7a潛在靶向，抑制了細胞G2/M期的運轉(zhuǎn)，抑制其增殖，誘導顆粒細胞凋亡；是調(diào)控細胞凋亡的關(guān)鍵因子，進一步分析發(fā)現(xiàn)affEXs中ssc-miR-21-5p下調(diào)潛在靶向調(diào)控表達，調(diào)控顆粒細胞凋亡；胰島素生長因子（）作為ssc-miR-133a-3p的潛在靶基因，在IGFBP-1作用下參與調(diào)控，增強了卵巢顆粒細胞芳香化酶的活性，抑制了類固醇激素代謝，誘導顆粒細胞凋亡，揭示了這些miRNA的靶標可能潛在調(diào)控卵泡的成熟發(fā)育和閉鎖。

2.7 卵泡液EXs添加顆粒細胞的驗證

根據(jù)以上mffEXs和affEXs的差異表達及功能富集分析結(jié)果，我們將mffEXs和affEXs分別添加到顆粒細胞中培養(yǎng)48 h。鏡檢結(jié)果顯示，mffEXs添加的顆粒細胞顯示出增殖狀態(tài)，長勢較好；affEXs添加顆粒細胞，形態(tài)發(fā)生拉長，部分出現(xiàn)凋亡現(xiàn)象（圖5-A、B）。Q-PCR結(jié)果顯示在mffEXs中顯著上調(diào)表達，而在affEXs中顯著上調(diào)（圖5-C、D），表明affEXs可能潛在促進了顆粒細胞凋亡的發(fā)生。

圖2 卵泡液EXs中差異miRNA靶基因的GO富集分析

圖3 KEGG功能富集分析

表2 主要信號通路中miRNA調(diào)控靶基因信息表

圖4 差異性表達miRNA的靶基因互作網(wǎng)絡

A：添加mffEXs培養(yǎng)的顆粒細胞結(jié)果；B：添加affEXs培養(yǎng)顆粒細胞的結(jié)果；C和D：Q-PCR檢測CDK1和P53表達結(jié)果

3 討論

卵泡閉鎖是一種通過凋亡機制產(chǎn)生的生理性程序性細胞死亡，最終引起卵母細胞發(fā)育停滯和顆粒細胞凋亡，其中顆粒細胞凋亡是引起閉鎖卵泡發(fā)生的主要原因[26]。大量研究報道EXs介導細胞間信號轉(zhuǎn)導，涉及多種生理功能調(diào)控。已有研究顯示，卵泡液中的EXs攜帶大量miRNA[27-28]，參與卵泡發(fā)育調(diào)控。但EXs miRNA參與調(diào)控卵泡閉鎖的調(diào)控機制還不清楚。本研究通過抽提卵泡液，發(fā)現(xiàn)豬成熟及閉鎖卵泡中均含有粒徑峰值約100 nm，且表面表達特異性標志蛋白CD9、CD63和CD81的EXs，這與先前的研究結(jié)果一致[29]。EXs是細胞向胞外分泌的一種囊泡小體[30]，在卵泡液[31]、輸卵管[32]和子宮液[33]中均存在。卵泡液中EXs攜帶大量調(diào)控分子可以直接調(diào)控卵母細胞和顆粒細胞，構(gòu)成卵母細胞和顆粒細胞生存的微環(huán)境，促使卵泡發(fā)育[34]。miRNA是一類EXs中含量最豐富的RNA小分子物質(zhì)，廣泛參與基因表達調(diào)控[35-36]，促使完成動物機體的各項生命活動[37]。

在卵泡發(fā)育過程中，EXs介導的信號傳遞發(fā)揮重要的調(diào)控作用，不僅影響卵母細胞發(fā)育，而且還會影響卵母細胞受精和胚胎質(zhì)量等[20,38-40]。本文通過對豬成熟和閉鎖卵泡液中的EXs miRNA進行測序，發(fā)現(xiàn)許多miRNA呈現(xiàn)顯著差異表達，且參與介導關(guān)鍵基因和信號轉(zhuǎn)導調(diào)控，顯示了在卵泡發(fā)育過程中的重要調(diào)控作用。兩類差異表達的EXs miRNA功能富集分析發(fā)現(xiàn)這些miRNA對顆粒細胞凋亡、細胞周期運轉(zhuǎn)和類固醇代謝等方面具有潛在的調(diào)控作用，表明了EXs中的miRNA可能直接參與調(diào)控卵泡的成熟發(fā)育和閉鎖[38,41]。

卵泡閉鎖是從顆粒細胞凋亡開始的[42]，屬于一種促細胞凋亡基因，其功能主要涉及調(diào)控G1和G2/M期的運轉(zhuǎn)，促進細胞凋亡[40,43]。研究通過對豬[44]和牛[45-46]進行miRNA表達譜分析，發(fā)現(xiàn)miR-21-5p在卵泡發(fā)育過程中呈現(xiàn)不同表達[45]；在培養(yǎng)小鼠顆粒細胞時發(fā)現(xiàn)miR-21-5p屬于一種抗凋亡因子，可以直接靶向同源基因，促進細胞增殖[47]，證明了在顆粒細胞凋亡發(fā)生過程中起到重要調(diào)控作用[48]；本研究顯示affEXs中下調(diào)表達的ssc-miR-21-5p，潛在靶向，促使表達增加，引起顆粒細胞凋亡；在體外培養(yǎng)顆粒細胞時添加affEXs可增加的表達，引起卵巢顆粒細胞的凋亡，這說明了affEXs中ssc-miR-21-5p下調(diào)可以引起卵母細胞凋亡；也有研究顯示miR-21高表達受到抑制時，體外胚胎發(fā)育停滯[49-50]。

let-7家族（let-7a、let-7b和let-7c等）在豬早期卵泡閉鎖和逐步閉鎖過程中特異性表達，且卵巢卵泡的數(shù)量也會隨卵泡閉鎖的發(fā)生逐漸減少[51]。隨著研究的深入，let-7a在動物生長發(fā)育、細胞增殖、分化和凋亡等方面發(fā)揮著重要調(diào)控作用[52]。相關(guān)報道顯示let-7a通過下調(diào)抑制哮喘氣道平滑肌細胞的增殖，促進其凋亡[53]；還可以在細胞周期蛋白-D1的調(diào)控作用下，抑制肺腺癌細胞增殖[54]；在骨肉瘤細胞中l(wèi)et-7a潛在靶向，促使細胞生長和肺轉(zhuǎn)移受到抑制[55-56]；let-7a還可以通過抑制和表達誘導滋養(yǎng)層細胞凋亡[57]；還有研究顯示let-7a靶向凋亡基因、和發(fā)揮調(diào)控作用[58-59]。miR-133a-3p在多種細胞中都起到了抑制增殖、侵襲，阻滯細胞周期等作用，促進細胞凋亡[60-61]，如在口腔鱗狀細胞癌研究中發(fā)現(xiàn)miR-133a-3p下調(diào)表達，抑制癌細胞的增殖、侵襲和轉(zhuǎn)移[62-63]；它還可以通過阻斷自噬介導的谷氨酰胺分解，抑制胃癌細胞的生長和轉(zhuǎn)移等[64]；在視網(wǎng)膜母細胞瘤中通過靶向，促進細胞凋亡，阻滯細胞周期[65]；還有研究顯示胰島素生長因子（）被認為是卵泡發(fā)育所必須的生長因子[66]，顯然缺失會對卵泡閉鎖的發(fā)生產(chǎn)生一定的影響。我們發(fā)現(xiàn)與mffEXs相比，affEXs中上調(diào)表達的ssc-let-7a和ssc-miR-133a-3p潛在靶向和，催化底物磷酸化，抑制了G2/M期的運轉(zhuǎn)，降低了細胞的增殖能力，并使卵泡細胞與LH作用，誘導雄激素釋放，促使卵母細胞降解和顆粒細胞凋亡或其受IGFBP-1調(diào)控，增強卵巢顆粒細胞芳香化酶的活性，抑制類固醇激素代謝，解釋了EXs攜帶的miRNA上調(diào)表達可以促使顆粒細胞凋亡，進而誘導卵泡閉鎖的發(fā)生，并且有望成為解析卵泡發(fā)育規(guī)律的分子機制[67]。

4 結(jié)論

近年來EXs已成為動物生殖發(fā)育領域的研究熱點，許多潛在的功能機制逐漸被人們揭曉，然而卵泡液EXs攜帶的miRNA對豬卵泡發(fā)育調(diào)控作用仍然不清楚。本文研究顯示卵泡液EXs攜帶的miRNA（ssc-miR-133-3p、ssc-miR-21-5p和ssc-let-7a）在顆粒細胞凋亡、細胞周期調(diào)控和類固醇激素代謝等過程中具有重要的調(diào)控作用，這將為豬卵泡發(fā)育和閉鎖相關(guān)的主要調(diào)控機制提供重要數(shù)據(jù)。

[1] JONG E D. Weaning practices and culling policy: Critical steps for optimal reproductive performance of female breeding pigs. 2014.

[2] FORTUNE J E. Ovarian follicular growth and development in mammals. Biology of Reproduction, 1994, 50(2): 225-232.

[3] DISKIN M G, MACKEY D R, ROCHE J F, SREENAN J M. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Animal Reproduction Science, 2003, 78(3/4): 345-370. doi:10.1016/s0378-4320(03)00099-x.

[4] HSUEH A J, BILLIG H, TSAFRIRI A. Ovarian follicle atresia: a hormonally controlled apoptotic process. Cancer Management and Research, 1994, 15(6): 707-724. doi:10.1210/edrv-15-6-707.

[5] TILLY J L, TILLY K I. Inhibitors of oxidative stress mimic the ability of follicle-stimulating hormone to suppress apoptosis in cultured rat ovarian follicles. Microorganisms, 1995, 136(1): 242-252. doi:10.1210/endo.136.1.7828537.

[6] PERSANI L, ROSSETTI R, CACCIATORE C, FABRE S. Genetic defects of ovarian TGF-β-like factors and premature ovarian failure. Journal of Endocrinological Investigation, 2011, 34(3): 244-251. doi:10.1007/BF03347073.

[7] SOHEL M M, HOELKER M, NOFERESTI S S, SALILEW- WONDIM D, THOLEN E, LOOFT C, RINGS F, UDDIN M J, SPENCER T E, SCHELLANDER K, TESFAYE D. Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: Implications for bovine oocyte developmental competence. PLoS One, 2013, 8(11): e78505. doi:10.1371/journal.pone.0078505.

[8] LUO F, JIA R, YING S, WANG Z, WANG F. Analysis of genes that influence sheep follicular development by different nutrition levels during the luteal phase using expression profiling. Animal Genetics, 2016, 47(3): 354-364. doi:10.1111/age.12427.

[9] 胡軍和, 唐濤, 譚顯勝, 曾智, 吳娟. 外泌體調(diào)控卵泡中母細胞發(fā)育的研究進展. 中國農(nóng)學通報, 2019(27): 153-157.

HU J H, TANG T, TAN X S, ZENG Z, WU J. The advance of exosomes regulating oocyte development in ovarian follicle. Chinese Agricultural Science Bulletin, 2019(27): 153-157. (in Chinese)

[10] THéRY C, OSTROWSKI M, SEGURA E. Membrane vesicles as conveyors of immune responses. Nature Reviews Immunology, 2009, 9(8): 581-593. doi:10.1038/nri2567.

[11] VLASSOV A V, MAGDALENO S, SETTERQUIST R, CONRAD R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochimica et Biophysica Acta, 2012, 1820(7): 940-948. doi:10.1016/j.bbagen.2012. 03.017.

[12] KELLER S, RUPP C, STOECK A, RUNZ S, FOGEL M, LUGERT S, HAGER H D, ABDEL B MS，GUTWEIN P, ALTEVOGT P. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney International, 2007, 72(9): 1095-1102.

[13] DE LA TORRE GOMEZ C, GOREHAM R V, BECH SERRA J J, NANN T, KUSSMANN M. “exosomics”-A review of biophysics, biology and biochemistry of exosomes with a focus on human breast milk. Frontiers in Genetics, 2018, 9: 92. doi:10.3389/fgene.2018. 00092.

[14] BORIACHEK K, UMER M, ISLAM M N, GOPALAN V, LAM A K, NGUYEN N T, SHIDDIKY M J A. An amplification-free electrochemical detection of exosomal miRNA-21 in serum samples. The Analyst, 2018, 143(7): 1662-1669. doi:10.1039/c7an01843f.

[15] TAO W, SUN L, SHI H, CHENG Y, JIANG D, FU B, CONTE M A, GAMMERDINGER W J, KOCHER T D, WANG D. Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation. BMC Genomics, 2016, 17(1): 328.

[16] SANG Q, YAO Z, WANG H, FENG R, WANG H, ZHAO X, XING Q, JIN L, HE L, WU L, WANG L. Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo. The Journal of Clinical Endocrinology and Metabolism, 2013, 98(7): 3068-3079. doi:10.1210/jc.2013-1715.

[17] WILLIS G R, CONNOLLY K, LADELL K, DAVIES T S, GUSCHINA I A, RAMJI D, MINERS K, PRICE D A, CLAYTON A, JAMES P E, REES D A. Young women with polycystic ovary syndrome have raised levels of circulating annexin v-positive platelet microparticles. Human Reproduction, 2014, 29(12): 2756-2763.

[18] S?RENSEN A E, WISSING M L, ENGLUND A L M, DALGAARD L T. MicroRNA species in follicular fluid associating with polycystic ovary syndrome and related intermediary phenotypes. The Journal of Clinical Endocrinology & Metabolism, 2016, 101(4): 1579-1589.

[19] 楊倩, 劉蘭心, 黃荷鳳. 多囊卵巢綜合征患者卵泡液外泌體的提取鑒定及其miRNAs的提取和檢測. 上海交通大學學報(醫(yī)學版), 2017, 37(8): 1085-1089. doi:10.3969/j.issn.1674-8115.2017. 08.007.

YANG Q, LIU L X, HUANG H F. Extraction and identification of exosomes in follicular fluid from patients with polycystic ovary syndrome and isolation and detection of miRNAs in exosomes. Journal of Shanghai Jiao Tong University (Medical Science), 2017, 37(8): 1085-1089. doi:10.3969/j.issn.1674-8115.2017.08.007. (in Chinese)

[20] COTICCHIO G, DAL CANTO M, MIGNINI RENZINI M, GUGLIELMO M C, BRAMBILLASCA F, TURCHI D, NOVARA P V, FADINI R. Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization. Human Reproduction Update, 2015, 21(4): 427-454. doi:10.1093/humupd/dmv011.

[21] 劉凱魯, 胡夢婷, 蔡令波, 李涵, 楊瑋杰, 劉嘉茵, 崔毓桂, 千日成. 多囊卵巢綜合征患者卵泡液中6種miRNAs表達的檢測. 國際生殖健康/計劃生育雜志, 2018, 37(1): 5-10. doi:10.3969/j.issn.1674-1889. 2018.01.001.

LIU K L, HU M T, CAI L B, LI H, YANG W J, LIU J Y, CUI Y G, QIAN R C. Expressions of six MiRNAs in follicular fluid of patients with polycystic ovary syndrome. International Journal of Reproductive Health/Family Planning, 2018, 37(1): 5-10. doi:10.3969/j.issn.1674- 1889.2018.01.001. (in Chinese)

[22] 詹小舒, 羅惠娜, 羅冬章, 陳勝鋒, 王丙云, 白銀山, 陳志勝, 劉璨穎, 計慧琴. 犬臍帶間充質(zhì)干細胞來源外泌體對血管內(nèi)皮細胞增殖、遷移和凋亡的調(diào)控作用. 中國組織工程研究, 2019, 23(29): 4637-4643. doi:10.3969/j.issn.2095-4344.1808.

ZHAN X S, LUO H N, LUO D Z, CHEN S F, WANG B Y, BAI Y S, CHEN Z S, LIU C Y, JI H Q. Effects of exosomes derived from canine umbilical cord mesenchymal stem cells on proliferation, migration and apoptosis of vascular endothelial cells. Journal of Clinical Rehabilitative Tissue Engineering Research, 2019, 23(29): 4637-4643. doi:10.3969/j.issn.2095-4344.1808. (in Chinese)

[23] ZHANG H, XU S, LIU X. microRNA profiling of plasma exosomes from patients with ovarian cancer using high-throughput sequencing. Oncology Letters, 2019, 17(6): 5601-5607. doi:10.3892/ol.2019. 10220.

[24] WAGNER G P, KIN K, LYNCH V J. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory in Biosciences, 2012, 131(4): 281-285. doi:10.1007/s12064-012-0162-3.

[25] YOUNG M D, WAKEFIELD M J, SMYTH G K, OSHLACK A. Gene ontology analysis for RNA-seq: Accounting for selection bias. Proceedings Biological Sciences, 2010, 11(2): R14. doi:10.1186/gb- 2010-11-2-r14.

[26] WANG C L, FAN Y C, CHUN-HSIEN TSENG, CHIU C H, TSAI H J, CHOU C H. Salmonella Enteritidis infection slows steroidogenesis and impedes cell growth in hen granulosa cells. Avian Diseases, 2014, 58(4): 511-517. doi:10.1637/10846-041414-reg.1.

[27] DA SILVEIRA J, ANDRADE G M, PERECIN F, MEIRELES F V, WINGER Q A, BOUMA G J. Isolation and analysis of exosomal microRNAs from ovarian follicular fluid. Methods in Molecular Biology (Clifton, N J), 2018, 1733: 53-63. doi:10.1007/978-1-4939- 7601-0_4.

[28] HU J, TANG T, ZENG Z, WU J, TAN X S, YAN J. The expression of small RNAs in exosomes of follicular fluid altered in human polycystic ovarian syndrome. PeerJ, 2020, 8: e8640.

[29] VICTOR N T, YONATHAN G, GIOVANNI C, MARIA F B. Extracellular vesicles: new players in lymphomas. International Journal of Molecular Sciences, 2018, 20(1). doi：10.3390/ ijms20010041.

[30] L?TVALL J, HILL A F, HOCHBERG F, BUZáS E I, VIZIO D D, GARDINER C, GHO Y S, KUROCHKIN I V, MATHIVANAN S, QUESENBERRY P. Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles, 2014, 3(1): 26913.

[31] HUNG W T, NAVAKANITWORAKUL R, KHAN T, ZHANG P, DAVIS J S, MCGINNIS L K, CHRISTENSON L K. Stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation. Biology of Reproduction, 2017, 97(4): 644-655. doi:10.1093/biolre/iox106.

[32] HUNG W T, HONG X, CHRISTENSON L K, MCGINNIS L K. Extracellular vesicles from bovine follicular fluid support cumulus expansion. Biology of Reproduction, 2015, 93(5): 117. doi:10.1095/ biolreprod.115.132977.

[33] AL-DOSSARY A A，STREHLER E E，MARTIN-DELEON P A. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine Estrus: Association with oviductal exosomes and uptake in sperm. PLoS ONE, 2013, 8(11): e80181.

[34] FIELD S L, DASGUPTA T, CUMMINGS M, ORSI N M. Cytokines in ovarian folliculogenesis, oocyte maturation and luteinisation. Molecular Reproduction and Development, 2014, 81(4): 284-314. doi:10.1002/mrd.22285.

[35] BENTWICH I, AVNIEL A, KAROV Y, AHARONOV R, GILAD S, BARAD O, BARZILAI A, EINAT P, EINAV U, MEIRI E, SHARON E, SPECTOR Y, BENTWICH Z. Identification of hundreds of conserved and nonconserved human microRNAs. Nature Genetics, 2005, 37(7): 766-770. doi:10.1038/ng1590.

[36] KROL J, LOEDIGE I, FILIPOWICZ W. The widespread regulation of microRNA biogenesis, function and decay. Nature Reviews Genetics, 2010, 11(9): 597-610. doi:10.1038/nrg2843.

[37] MASOUMI-DEHGHI S, BABASHAH S, SADEGHIZADEH M. microRNA-141-3p-containing small extracellular vesicles derived from epithelial ovarian cancer cells promote endothelial cell angiogenesis through activating the JAK/STAT3 and NF-κB signaling pathways. Journal of Cell Communication and Signaling, 2020, 14(2): 233-244. doi:10.1007/s12079-020-00548-5.

[38] MACHTINGER R, RODOSTHENOUS R S, ADIR M, MANSOUR A, RACOWSKY C, BACCARELLI A A, HAUSER R. Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: An exploratory study. Journal of Assisted Reproduction and Genetics, 2017, 34(4): 525-533. doi:10.1007/s10815-017-0876-8.

[39] LIANG M, YAO G, YIN M, Lü M, TIAN H, LIU L, LIAN J, HUANG X, SUN F. Transcriptional cooperation between p53 and NF-κB p65 regulates microRNA-224 transcription in mouse ovarian granulosa cells. Molecular and Cellular Endocrinology, 2013, 370(1/2): 119-129. doi:10.1016/j.mce.2013.02.014.

[40] DA SILVEIRA J C, VEERAMACHANENI D N, WINGER Q A, CARNEVALE E M, BOUMA G J. Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: A possible new form of cell communication within the ovarian follicle. Biology of Reproduction, 2012, 86(3): 71. doi:10.1095/biolreprod.111.093252.

[41] PIETRO C D. Exosome-mediated communication in the ovarian follicle. Journal of Assisted Reproduction and Genetics, 2016, 33(3): 303-311.

[42] KRYSKO D V, DIEZ-FRAILE A, CRIEL G, SVISTUNOV A A, VANDENABEELE P, D’HERDE K. Life and death of female gametes during oogenesis and folliculogenesis. Apoptosis, 2008, 13(9): 1065-1087. doi:10.1007/s10495-008-0238-1.

[43] TILLY J L, KOWALSKI K I, JOHNSON A L, HSUEH A J. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Thrombosis and Haemostasis, 1991, 129(5): 2799-2801. doi:10.1210/endo-129-5-2799.

[44] LIN F, LI R, PAN Z X, ZHOU B, YU D B, WANG X G, MA X S, HAN J, SHEN M, LIU H L. miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary. PLoS ONE, 2012, 7(6): e38640. doi:10.1371/journal.pone. 0038640.

[45] SALILEW-WONDIM D, AHMAD I, GEBREMEDHN S, SAHADEVAN S, HOSSAIN M D, RINGS F, HOELKER M, THOLEN E, NEUHOFF C, LOOFT C, SCHELLANDER K, TESFAYE D. The expression pattern of microRNAs in granulosa cells of subordinate and dominant follicles during the early luteal phase of the bovine estrous cycle. PLoS ONE, 2014, 9(9): e106795. doi:10.1371/journal. pone.0106795.

[46] SONTAKKE S D, MOHAMMED B T, MCNEILLY A S, DONADEU F X. Characterization of microRNAs differentially expressed during bovine follicle development. Reproduction (Cambridge, England), 2014, 148(3): 271-283. doi:10.1530/rep-14-0140.

[47] CARLETTI M Z, FIEDLER S D, CHRISTENSON L K. microRNA 21 blocks apoptosis in mouse periovulatory granulosa cells. Molecules and Cells, 2010, 83(2): 286-295. doi:10.1095/biolreprod.109.081448.

[48] 周桔, 羅榮保, 湯長發(fā), 瞿樹林. Bcl-2蛋白家族和p53基因在細胞凋亡中的調(diào)控效應. 中國組織工程研究與臨床康復, 2007, 11(10): 1950-1952. doi:10.3321/j.issn: 1673-8225.2007.10.048.

ZHOU J, LUO R B, TANG C F, QU S L. Effect of Bcl-2 protein family and p53 gene on regulating and controlling cell apoptosis. Journal of Clinical Rehabilitative Tissue Engineering Research, 2007, 11(10): 1950-1952. doi:10.3321/j.issn:1673-8225.2007.10.048. (in Chinese)

[49] LV C, YU W X, WANG Y, YI D J, ZENG M, XIAO H. MiR-21 in extracellular vesicles contributes to the growth of fertilized eggs and embryo development in mice. Bioscience Reports, 2018, 38(4): BSR20180036.

[50] KUSAMA K, NAKAMURA K, BAI R, NAGAOKA K, SAKURAI T, IMAKAWA K. Intrauterine exosomes are required for bovine conceptus implantation. Biochemical and Biophysical Research Communications, 2018, 495(1): 1370-1375. doi:10.1016/j.bbrc.2017. 11.176.

[51] CAO R, WU W J, ZHOU X L, XIAO P, WANG Y, LIU H L. Expression and preliminary functional profiling of the let-7 family during porcine ovary follicle atresia. Molecules and Cells, 2015, 38(4): 304-311. doi:10.14348/molcells.2015.2122.

[52] WANG S, TANG Y, CUI H, ZHAO X, LUO X, PAN W, HUANG X, SHEN N. Let-7/miR-98 regulate Fas and Fas-mediated apoptosis. Genes and Immunity, 2011, 12(2): 149-154. doi:10.1038/gene.2010. 53.

[53] CHEN Y, QIAO L, ZHANG Z, HU G, ZHANG J, LI H. Let-7a inhibits proliferation and promotes apoptosis of human asthmatic airway smooth muscle cells. Experimental and Therapeutic Medicine, 2019, 17(5): 3327-3334. doi:10.3892/etm.2019.7363.

[54] ZHAO W, HU J X, HAO R M, ZHANG Q, GUO J Q, LI Y J, XIE N, LIU L Y, WANG P Y, ZHANG C, XIE S Y. Induction of microRNA?let?7a inhibits lung adenocarcinoma cell growth by regulating cyclin D1. Oncology Reports, 2018, 40(4): 1843-1854. doi:10.3892/or.2018.6593.

[55] YU J J, PI W S, CAO Y, PENG A F, CAO Z Y, LIU J M, HUANG S H, LIU Z L, ZHANG W. Let-7a inhibits osteosarcoma cell growth and lung metastasis by targeting Aurora-B. Cancer Management and Research, 2018, 10: 6305-6315. doi:10.2147/cmar.s185090.

[56] WANG G, WANG J, KHAN M F. Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen. Toxicology Mechanisms and Methods, 2017, 27(7): 511-517. doi:10.1080/ 15376516.2017.1324932.

[57] ZHA W, GUAN S, LIU N, LI Y, TIAN Y, CHEN Y, WANG Y, WU F. Let-7a inhibits Bcl-xl and YAP1 expression to induce apoptosis of trophoblast cells in early-onset severe preeclampsia. The Science of the Total Environment, 2020, 745: 139919. doi:10.1016/j.scitotenv. 2020.139919.

[58] YU Y, LIAO L, SHAO B, SU X, SHUAI Y, WANG H, SHANG F, ZHOU Z, YANG D, JIN Y. Knockdown of microRNA let-7a improves the functionality of bone marrow-derived mesenchymal stem cells in immunotherapy. Molecular Therapy, 2017, 25(2): 480-493. doi:10.1016/j.ymthe.2016.11.015.

[59] TSANG W P, KWOK T T. Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3. Apoptosis, 2008, 13(10): 1215-1222. doi:10.1007/s10495-008-0256-z.

[60] CHANG L L, WANG H C, TSENG K Y, SU M P, WANG J Y, CHUANG Y T, WANG Y H, CHENG K I. Upregulation of miR-133a-3p in the sciatic nerve contributes to neuropathic pain development. Molecular Neurobiology, 2020, 57(9): 3931-3942. doi:10.1007/s12035-020-01999-y.

[61] HUANG Y, WANG Y, LIN L, WANG P, JIANG L, LIU J, WANG X. Overexpression of miR-133a-3p inhibits fibrosis and proliferation of keloid fibroblasts by regulating IRF5 to inhibit the TGF-β/Smad2 pathway. Molecular and Cellular Probes, 2020, 52: 101563. doi:10.1016/j.mcp.2020.101563.

[62] HE B, LIN X, TIAN F, YU W, QIAO B. miR-133a-3p inhibits oral squamous cell carcinoma (OSCC) proliferation and invasion by suppressing COL1A1. Journal of Cellular Biochemistry, 2018, 119(1): 338-346. doi:10.1002/jcb.26182.

[63] YIN Y, DU L, LI X, ZHANG X, GAO Y. miR-133a-3p suppresses cell proliferation, migration, and invasion and promotes apoptosis in esophageal squamous cell carcinoma. Journal of Cellular Physiology, 2019, 234(8): 12757-12770. doi:10.1002/jcp.27896.

[64] ZHANG X, LI Z, XUAN Z, XU P H, WANG W Z, CHEN Z, WANG S, SUN G L, XU J H, XU Z K. Novel role of miR-133a-3p in repressing gastric cancer growth and metastasis via blocking autophagy-mediated glutaminolysis. Journal of Experimental & Clinical Cancer Research, 2018, 37(1)：320.

[65] LI J, LIU X, WANG W, LI C. miR-133a-3p promotes apoptosis and induces cell cycle arrest by targeting CREB1 in retinoblastoma. Archives of Medical Science, 2020, 16(4): 941-956. doi:10.5114/aoms. 2019.86901.

[66] HAN Y, WANG S M, WANG Y Z, ZENG S M. IGF-1 inhibits apoptosis of porcine primary granulosa cell by targeting degradation of BimEL. International Journal of Molecular Sciences, 2019, 20(21): 5356.

[67] SANTONOCITO M, VENTO M, GUGLIELMINO M R, BATTAGLIA R, WAHLGREN J, RAGUSA M, BARBAGALLO D, BORZì P, RIZZARI S, MAUGERI M, SCOLLO P, TATONE C, VALADI H, PURRELLO M, DI PIETRO C. Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: BIoinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation. Fertility and Sterility, 2014, 102(6): 1751-61.e1. doi:10.1016/j. fertnstert.2014.08.005.

Expression Differences and Functional Analysis of Exosomes microRNA in Porcine Mature and Atretic Follicles

CHEN HuiFang, HUANG QiLiang, HU ZhiChao, PAN XiaoTing, WU ZhiSheng, BAI YinShan

School of Life Science and Engineering, Foshan University, Foshan 528231, Guangdong

【】To explore the regulatory role of follicular fluid Exosomes (EXs) miRNA in follicular development and atresia, the difference of miRNA expression between mature follicular fluid Exosomes (mffEXs) and atretic follicular fluid Exosomes (affEXs) were analyzed. 【】In this study, the follicular fluid of 4-6 mm porcine mature development and atresia follicles was extracted. Then EXs were identified by particle size analysis and Western Blot detection, respectively. the sequencing analysis of the characteristic EXs carried miRNA and functional enrichment analysis were carried out, and then the key signal pathways and differential genes were screened. Finally, mffEXs and affEXs were used as additives for granular cell culture, and Q-PCR detection technology was used to analyze the expression of key genes to verify and analyze the regulatory functions of EXs miRNA in the two types of follicular fluid in follicular development. 【】This study successfully separated mffEXs and affEXs. The sequencing results showed that compared with mffEXs, 90 miRNAs in affEXs were up-regulated and 220 miRNAs were down-regulated, indicating that the level of miRNA expression in follicular fluid could directly regulate follicular development. KEGG enrichment analysis showed that the differential signaling pathways of the two types of follicles were mainly concentrated in the signal pathways, such as Ras, cAMP, P53 and MAPK, which involved in the regulation of biological functions, such as oocyte development, meiosis, and granulosa cell cycle. In atretic follicles, the up-regulated expression of ssc-let-7a and ssc-miR-133a-3p potentially targeted and regulated cyclin-dependent kinase () and insulin growth factor (), which inhibited G1 and G2/M Phase operation, and steroid hormone metabolism promoted the obstruction of granular cell cycle and the apoptosis of granular cells, causing follicular atresia; down-regulated ssc-miR-21-5p potentially targeted tumor suppressor gene () and inhibited cell cycle operation to promote the apoptosis of granular cells. mffEXs and affEXs were added to granular cells cultured in vitro, and Q-PCR results showed thatwas significantly up-regulated in mffEXs, whilewas significantly down-regulated, indicating the reliability of the sequencing analysis results. These results all showed that changes in miRNA expression levels in affEXs promoted granular cell apoptosis and cell cycle arrest, causing follicular atresia. 【】 Porcine affEXs carry miRNAs increased the regulation of,andgene expression, and inhibited the cell cycle of granulosa cells and steroid hormone metabolism and other signal pathways, causing granulosa cell apoptosis and follicular atresia.

porcine; mature follicular fluid; atretic follicular fluid; EXs; miRNA

10.3864/j.issn.0578-1752.2021.21.015

2020-09-27；

2021-04-25

廣東省畜禽疫病防治研究重點實驗室基金項目（YDWS1902）、佛山科學技術(shù)學院高層次人才科研啟動項目（gg040969）

陳慧芳，E-mail：chenhuifang07@163.com。通信作者白銀山，E-mail：xuefei200403@163.com

（責任編輯 林鑒非）