阿依木古麗·阿不都熱依木，阿爾祖古麗·阿依丁，王家敏，石嘉琛，馬芳芳，蔡勇，喬自林*

大豆異黃酮對牦牛卵巢顆粒細胞增殖和凋亡的影響

阿依木古麗·阿不都熱依木1,2，阿爾祖古麗·阿依丁1,2，王家敏1,3，石嘉琛1,2，馬芳芳1,2，蔡勇4，喬自林1,3*

1西北民族大學生物醫(yī)學研究中心甘肅省動物細胞技術(shù)創(chuàng)新中心，蘭州 730030；2西北民族大學生命科學與工程學院，蘭州 730030；3西北民族大學生物醫(yī)學研究中心生物工程與技術(shù)國家民委重點實驗室，蘭州 730030；4西北民族大學實驗教學部，蘭州 730030

【背景】大豆異黃酮主要包括染料木素和大豆苷元，發(fā)揮類雌激素樣作用，能夠清除自由基，促進細胞增殖。顆粒細胞是卵泡內(nèi)重要的細胞群體，其生理狀態(tài)與卵巢功能直接相關(guān)。顆粒細胞凋亡常引起卵泡閉鎖。牦牛是青藏高原特有物種之一，但其繁殖率低，其具體機制尚不明確。卵巢顆粒細胞是研究雌性動物生殖調(diào)控機制的理想細胞模型?！灸康摹刻接懘蠖巩慄S酮對牦牛卵巢顆粒細胞增殖和凋亡的影響，為大豆異黃酮的作用機制研究提供依據(jù)?！痉椒ā糠蛛x培養(yǎng)牦牛卵巢顆粒細胞，F(xiàn)SHR免疫細胞化學染色鑒定，MTT法測定細胞增殖情況，繪制生長曲線；細胞傳代后添加不同濃度染料木素或大豆苷元（0，1 000，2 000，3 000，4 000和5 000 pg·mL-1），選擇染料木素或大豆苷元最佳作用濃度分別處理二代顆粒細胞48 h，收集細胞培養(yǎng)液，ELISA法檢測培養(yǎng)液中雌二醇（E2）和孕酮（P4）濃度；同時收集細胞，提取總RNA，實時熒光定量PCR法檢測細胞增殖相關(guān)基因和細胞凋亡相關(guān)基因、、、、、和的表達水平?！窘Y(jié)果】牦牛卵巢顆粒細胞呈典型的上皮樣細胞生長特性。接種后2 h開始貼壁，12 h出現(xiàn)聚集生長；24 h細胞體積較大，呈長梭形、星形或多邊形；48 h細胞呈“鋪路石”樣單層排布；卵巢顆粒細胞特異標志蛋白FSHR免疫細胞化學染色陽性，表明，分離培養(yǎng)的是卵巢顆粒細胞；MTT法檢測細胞增殖情況，牦牛卵巢顆粒細胞呈“S”形曲線生長：培養(yǎng)24 h內(nèi)生長緩慢，處于潛伏生長期；24—48 h細胞增殖較快，進入指數(shù)生長期；48—120 h細胞平穩(wěn)增殖，處于平頂期；120 h后活細胞密度開始下降，細胞進入退化衰亡期；第二代細胞生長較快，24 h進入指數(shù)增殖期，故試驗選擇第二代顆粒細胞進行。MTT法檢測不同濃度染料木素或大豆苷元對細胞增殖的影響，結(jié)果，添加3 000 pg·mL-1染料木素或大豆苷元作用48 h后，細胞活力均極顯著增強（＜0.01）；染料木素和大豆苷元均促進顆粒細胞分泌雌二醇，大豆苷元促進效果顯著（＜0.05）；染料木素顯著抑制顆粒細胞分泌孕酮（＜0.05）；qRT-PCR結(jié)果顯示，染料木素和大豆苷元均顯著上調(diào)顆粒細胞增殖調(diào)控相關(guān)基因和凋亡相關(guān)基因、和的表達（＜0.01），顯著下調(diào)凋亡相關(guān)基因和的表達（＜0.01），并顯著上調(diào)比值（＜0.01）抑制細胞凋亡。此外，染料木素抑制表達（＜0.01）而大豆苷元促進表達（＜0.01）?！窘Y(jié)論】分離培養(yǎng)了牦牛卵巢顆粒細胞，為牦牛雌性生殖調(diào)控機制研究提供了有效的細胞模型。試驗結(jié)果表明，染料木素和大豆苷元抑制牦牛顆粒細胞分泌孕酮，促進細胞增殖，且主要通過上調(diào)和，以及下調(diào)和的表達，保護卵巢顆粒細胞免于凋亡。

染料木素；大豆苷元；牦牛；卵巢顆粒細胞；增殖；凋亡

0 引言

【研究意義】牦牛（bos grunniens）是分布在高寒低氧牧區(qū)的珍稀高原動物，是世界上罕見的物種資源之一[1]。受高海拔、寒冷等因素的影響，牦牛的繁殖率較低，大多是兩三年產(chǎn)一胎，故牦牛數(shù)量不斷減少[2]。因此，利用人工授精[3]、體外受精[4]等輔助繁殖技術(shù)來提高牦牛繁殖性能廣受關(guān)注。顆粒細胞是卵巢中最大的細胞群，與卵母細胞間存在信息交流，對卵母細胞發(fā)育和成熟起重要作用，故卵巢顆粒細胞成為研究雌性生殖調(diào)控的重要細胞模型[5-6]。卵巢顆粒細胞作為主要的功能細胞參與原始卵泡發(fā)育、卵泡成熟、排卵及黃體形成等過程[7]。研究表明，顆粒細胞中蛋白表達水平影響卵母細胞的生長發(fā)育[8]，顆粒細胞凋亡直接影響卵巢功能[9-10]?！厩叭搜芯窟M展】大豆異黃酮屬于植物雌激素，主要存在于大豆、鷹嘴豆和扁豆等豆類中，主要成分為染料木素和大豆苷元[11]。大豆異黃酮在抗腫瘤[12]、清除自由基[13]、降低膽固醇[14]及神經(jīng)保護[15]等方面具有廣泛的生物活性，同時能增強機體抗應(yīng)激能力，并對腸絨毛有一定保護作用[16]?！颈狙芯壳腥朦c】雌激素能夠促進體外培養(yǎng)的卵巢顆粒細胞合成和分泌孕酮（P4），P4可以顯著促進顆粒細胞合成和分泌雌激素，而雌激素可抑制體外培養(yǎng)的顆粒細胞自發(fā)凋亡，并具有劑量依賴性[17]。大豆異黃酮具有類雌激素樣作用[18]?！緮M解決的關(guān)鍵問題】為了研究大豆異黃酮（染料木素和大豆苷元）對牦牛卵巢顆粒細胞增殖和凋亡的影響，本研究分離培養(yǎng)了牦牛卵巢顆粒細胞，添加不同濃度染料木素和大豆苷元，檢測細胞增殖和凋亡相關(guān)基因，為體外培養(yǎng)牦牛顆粒細胞增殖和凋亡機制的研究奠定理論基礎(chǔ)，為大豆異黃酮的作用機制研究提供基礎(chǔ)數(shù)據(jù)。

1 材料與方法

1.1 試驗動物

2018年11月，在甘肅省臨夏州八坊清和園屠宰場選取12頭4歲左右健康母牦牛。屠宰，檢查無肉眼可視病變，迅速采集卵巢，75%酒精沖洗2次，滅菌PBS沖洗3次，放入含1%青霉素和鏈霉素的37℃ PBS緩沖液中快速帶回實驗室。

1.2 卵巢顆粒細胞原代培養(yǎng)

從含雙抗的PBS緩沖液中取出卵巢，75%酒精浸泡20 s，移入無菌培養(yǎng)皿中，無菌PBS緩沖液沖洗5—6次。注射器抽吸直徑為5—10 mm卵泡的卵泡液，加入等體積DMEM/F12細胞培養(yǎng)液，1 000 r/min離心5 min，棄上清，細胞培養(yǎng)液吹打沉淀的細胞，1 000 r/min離心5 min，棄上清，細胞培養(yǎng)液重懸細胞，按5×105個/mL接種至T25細胞培養(yǎng)瓶，用含10%胎牛血清和1%雙抗的DMEM/F12培養(yǎng)液置37℃、5% CO2培養(yǎng)箱中培養(yǎng)，細胞貼壁后每48 h換液一次。細胞匯合度達90%以上，傳代并凍存。復(fù)蘇的第二代細胞用于后續(xù)試驗。

1.3 卵巢顆粒細胞生長曲線繪制

細胞長滿后進行消化，以2×104個/mL接種于24孔板，置37℃、5% CO2培養(yǎng)箱中培養(yǎng)，臺盼藍計數(shù)法每24 h計數(shù)1次，設(shè)3個重復(fù)，連續(xù)7 d，未計數(shù)組每48 h換液一次，繪制細胞生長曲線。

1.4 卵巢顆粒細胞FSHR免疫細胞化學染色

參照文獻[19]介紹的方法，采用免疫細胞化學SP法檢測促卵泡激素受體（follicle-stimulating hormone receptor, FSHR）表達情況。

1.5 染料木素和大豆苷元對卵巢顆粒細胞增殖的影響

顆粒細胞長滿瓶底時，消化、計數(shù)，按3.5×103個/mL密度接種于96孔板，置37℃、5% CO2培養(yǎng)箱中培養(yǎng)48 h，細胞貼壁后棄培養(yǎng)液，分別加入含不同濃度染料木素（GEN組）（0，1 000，2 000，3 000，4 000和5 000 pg·mL-1）或大豆苷元（DAI組）（0，1 000，2 000，3 000，4 000和5 000 pg·mL-1）的新培養(yǎng)液，其中濃度為0記為對照組（CON組），繼續(xù)培養(yǎng)48 h，MTT法測定細胞增殖。每組設(shè)6個重復(fù)孔，試驗重復(fù)3次。

1.6 染料木素和大豆苷元對卵巢顆粒細胞分泌雌二醇和孕酮的影響

根據(jù)MTT法檢測結(jié)果，篩選出染料木素和大豆苷元對顆粒細胞增殖影響最佳濃度，該濃度作用顆粒細胞48 h后，收集細胞培養(yǎng)液，ELISA法檢測培養(yǎng)液中雌二醇（E2）和孕酮含量。

1.7 RNA提取及增殖和凋亡相關(guān)基因的檢測

根據(jù)MTT法檢測結(jié)果，篩選出染料木素和大豆苷元對顆粒細胞增殖影響最佳濃度，該濃度作用顆粒細胞48 h后，收集細胞，提取總RNA，用TSINGKE逆轉(zhuǎn)錄試劑盒進行反轉(zhuǎn)錄，采用qRT-PCR法分別檢測細胞增殖相關(guān)基因和細胞凋亡相關(guān)基因、、、、、和的表達情況，以為內(nèi)參基因，引物信息詳見表1。

1.8 數(shù)據(jù)處理

所有試驗均設(shè)置3次重復(fù)，數(shù)據(jù)結(jié)果使用Excel進行初步整理，再通過GraphPad Prism 5進行t檢驗。＞0.05表示差異不顯著；＜0.05表示差異顯著，用不同小寫字母表示；＜0.01表示差異極顯著，用不同大寫字母表示。

2 結(jié)果

2.1 卵巢顆粒細胞形態(tài)觀察

倒置顯微鏡下觀察，牦牛卵巢顆粒細胞呈典型的上皮樣細胞生長特性。接種后2 h開始貼壁，12 h出現(xiàn)聚集生長；24 h細胞體積較大，形態(tài)完整，呈長梭形、星形或多邊形；48 h細胞呈“鋪路石”樣單層排布（圖1- A、B）。顆粒細胞第一代生長速度較緩慢，潛伏生長期較長；第二代細胞生長較快，24 h進入指數(shù)增殖期，故試驗選擇第二代顆粒細胞進行。經(jīng)免疫細胞化學SP染色，細胞呈FSHR陽性，表明分離培養(yǎng)的細胞為卵巢顆粒細胞（圖1-C）。牦牛卵巢顆粒細胞呈“S”形曲線生長：培養(yǎng)24 h內(nèi)生長緩慢，處于潛伏生長期；24—48 h細胞增殖較快，進入指數(shù)生長期；48—120 h細胞平穩(wěn)增殖，處于平頂期；120 h后活細胞密度開始下降，細胞進入退化衰亡期（圖1-D）。

表1 Real-time PCR引物信息

A：分離培養(yǎng)24 h ×40；B：分離培養(yǎng)48 h ×100；C：FSHR免疫細胞化學SP染色 ×400；D：生長曲線

2.2 染料木素和大豆苷元可提高卵巢顆粒細胞活力

牦牛卵巢顆粒細胞分離培養(yǎng)48 h長滿瓶底，棄去細胞培養(yǎng)液，加入含有不同濃度染料木素或大豆苷元的培養(yǎng)液，繼續(xù)培養(yǎng)48 h，MTT法檢測細胞活力，結(jié)果如圖2所示：與對照組相比，添加不同濃度染料木素或大豆苷元，均能顯著（＜0.05）或極顯著（＜0.01）提高細胞活力，因此，選擇3 000 pg·mL-1為最佳作用濃度，進行后續(xù)試驗。

不同小寫字母代表差異顯著（P＜0.05）；不同大寫字母代表差異極顯著（P＜0.01）。下同

2.3 染料木素和大豆苷元對卵巢顆粒細胞分泌功能的影響

以添加3 000 pg·mL-1染料木素或大豆苷元的細胞培養(yǎng)液繼續(xù)培養(yǎng)卵巢顆粒細胞48 h，ELISA法檢測培養(yǎng)液中E2和P4的含量，結(jié)果如圖3所示：與對照組相比，大豆苷元顯著促進顆粒細胞分泌E2（＜0.05）；染料木素和大豆苷元均抑制顆粒細胞P4分泌，且染料木素抑制效果顯著（＜0.05）。

2.4 染料木素和大豆苷元對卵巢顆粒細胞增殖和凋亡相關(guān)基因的影響

添加3 000 pg·mL-1染料木素或大豆苷元繼續(xù)培養(yǎng)48 h，qRT-PCR檢測細胞增殖相關(guān)基因和細胞凋亡相關(guān)基因、、、、、和的表達情況，結(jié)果如圖4所示。與對照組相比，染料木素和大豆苷元均顯著上調(diào)增殖相關(guān)基因的表達；都顯著上調(diào)、、和基因表達及比值，且大豆苷元上調(diào)更明顯；染料木素和大豆苷元都顯著抑制的表達；染料木素顯著下調(diào)表達，而大豆苷元顯著上調(diào)；對于，大豆苷元顯著下調(diào)，而添加染料木素組多次檢測均未檢出基因，故無法統(tǒng)計。

圖3 染料木素和大豆苷元對牦牛卵巢顆粒細胞E2和P4分泌的影響

圖4 染料木素和大豆苷元對牦牛卵巢顆粒細胞增殖和凋亡相關(guān)基因的影響

3 討論

大豆異黃酮結(jié)構(gòu)與雌激素相似，在體內(nèi)能與雌激素受體α（estrogen receptor α, ERα）和雌激素受體β（estrogen receptor β, ERβ）結(jié)合，發(fā)揮雌激素樣作用[20]，臨床上用于治療更年期綜合征。研究發(fā)現(xiàn)，染料木素促進大鼠[21]和牛[22]卵巢顆粒細胞增殖，試驗結(jié)果顯示，3 000 pg·mL-1染料木素和大豆苷元都顯著提高了牦牛卵巢顆粒細胞增殖活力；但也有學者報道染料木素抑制豬卵巢顆粒細胞增殖活力[23]，推測染料木素和大豆苷元對顆粒細胞增殖活力的調(diào)節(jié)可能與其濃度和作用時間有一定聯(lián)系，其具體機制仍需進一步研究。

試驗發(fā)現(xiàn)，染料木素顯著抑制顆粒細胞P4分泌，大豆苷元促進牦牛卵巢顆粒細胞分泌E2，與Nynca等[24-25]研究結(jié)果一致。推測，大豆異黃酮對顆粒細胞增殖活力的影響可能是通過增殖和凋亡相關(guān)基因調(diào)控而實現(xiàn)。

又稱為PKBα，為蛋白激酶B（protein kinase B, PKB）家族成員之一，活化的主要通過磷酸化底物蛋白引起下游信號級聯(lián)反應(yīng)，進而參與調(diào)控細胞生長、增殖、遷移及細胞周期等多種細胞活動[26]。試驗發(fā)現(xiàn)染料木素和大豆苷元均顯著上調(diào)牦牛顆粒細胞的表達，從而促進牦牛顆粒細胞增殖。細胞凋亡存在兩種調(diào)控途徑：死亡受體介導的“外源性凋亡通路”和線粒體介導的“內(nèi)源性凋亡通路”。其中，在卵巢顆粒細胞凋亡過程中由蛋白家族介導的線粒體內(nèi)源凋亡通路占主導[27]。和的比值[28]是細胞的“凋亡開關(guān)”，當表達占優(yōu)勢時，細胞發(fā)生凋亡，反之細胞凋亡活動受到抑制。二者的平衡決定了卵泡發(fā)育或是閉鎖[29]。為促凋亡基因，與或等結(jié)合發(fā)揮作用。大豆異黃酮下調(diào)L02細胞中的比值，抑制的表達，從而抑制細胞凋亡[30]。本研究也發(fā)現(xiàn)，染料木素和大豆苷元都能上調(diào)和表達，并顯著下調(diào)表達，上調(diào)比值，從而抑制顆粒細胞凋亡。

是重要的抑癌基因之一，細胞受到DNA 損傷、氧化應(yīng)激等信號刺激時，轉(zhuǎn)錄活性增強，作為一種高效的轉(zhuǎn)錄因子可通過調(diào)控凋亡途徑中的途徑來介導細胞凋亡[31]。本研究發(fā)現(xiàn)，染料木素和大豆苷元都顯著上調(diào)牦牛卵巢顆粒細胞中的表達，從而參與細胞凋亡調(diào)控。

是腫瘤壞死因子受體家族的膜蛋白分子，是的天然配體，與結(jié)合活化相關(guān)死亡結(jié)構(gòu)域及下游的家族，從而啟動外源性凋亡途徑誘導細胞凋亡[32]。本研究發(fā)現(xiàn)，牦牛顆粒細胞中和都有表達，染料木素和大豆苷元都顯著上調(diào)mRNA的表達，但下調(diào)mRNA的表達，且染料木素作用后沒有檢測到的mRNA，促凋亡作用主要通過與其配體結(jié)合后發(fā)揮。因此，大豆異黃酮對死亡受體家族蛋白的調(diào)控機制仍需進一步研究。是介導細胞凋亡的關(guān)鍵效應(yīng)酶，多種細胞凋亡信號通路均需通過激活來進一步激活下游信號調(diào)節(jié)因子促進細胞凋亡[33]。本研究發(fā)現(xiàn)，染料木素顯著降低的表達，而大豆苷元極顯著上調(diào)的表達，這一不同是否和染料木素與大豆苷元結(jié)構(gòu)差異有關(guān)，仍需進一步研究。

4 結(jié)論

通過分離培養(yǎng)牦牛卵巢顆粒細胞，發(fā)現(xiàn)染料木素和大豆苷元可促進牦牛卵巢顆粒細胞增殖，抑制P4分泌，并上調(diào)的表達及比值，下調(diào)表達，從而起到保護卵巢顆粒細胞免受凋亡的作用。

[1] WANG K, YANG Y, WANG L, MA T, SHANG H, DING L, HAN J, QIU Q.Different gene expressions between cattle and yak provide insights into high-altitude adaptation.Animal Genetics, 2016, 47(1): 28-35.

[2] YU S J, HAUANG Y M, CHEN B X.Reproductive patterns of the yak.I.Reproductive phenomena of the female yak.British Veterinary Journal, 1993, 149(6): 579-583.

[3] OOSTHUIZEN N, FONTES P, SANFIORD C D, CIRIACO F M, HENRY D D, CANAL L B, DILORENZO N, LAMB G C.Estrus synchronization and fixed-time artificial insemination alter calving distribution in Bos indicus influenced beef heifers.Theriogenology, 2018, 15(106): 210-213.

[4] 潘陽陽, 王萌, 芮弦, 王立斌, 何翃閎, 王靖雷, 馬睿, 徐庚全, 崔燕, 樊江峰, 余四九.IGF-1調(diào)控RBM3表達抑制低溫應(yīng)激誘導牦牛卵丘細胞凋亡.中國農(nóng)業(yè)科學, 2020, 53(11): 2285-2296.

PAN Y Y, WANG M, RUI X, WANG L B, HE H H, WANG J L, MA R, XU G Q, CUI Y, FAN J F, YU S J.RNA-binding motif protein 3(RBM3) expression is regulated by insulin-like growth factor (IGF-1) for protecting yak (grunniens) cumulus cells from apoptosis during hypothermia stress.Scientia Agricultura Sinica, 2020, 53(11): 2285-2296.(in Chinese)

[5] 畢錫麟, 王鍇, 李鵬飛, 景炅婕, 韓琦, 呂麗華.牛卵泡顆粒細胞PRSS35表達模式及功能研究.畜牧獸醫(yī)學報, 2018, 49(11): 2394-2401.

BI X L, WANG K, LI P F, JING J J, HAN Q, Lü L H.Expression profile and function analysis of PRSS35 in bovine follicle granulosa cells.Acta Veterinaria et Zootechnica Sinica, 2018, 49(11): 2394-2401.(in Chinese)

[6] MATSUDA F, INOUE N, MANABE N, OHKURA S.Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells.The Journal of Reproduction and Development, 2012, 58(1): 44-50.

[7] HATZIRODOS N, HUMMITZSCH K, IRVING-RODGERS H F, HARLAND M L, MORRIS S E, RODGERS R J.Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia.BMC Genomics, 2014, 15: 40.

[8] 趙園園, 李鵬飛, 許勤智, 安清明, 孟金柱.山羊卵泡發(fā)育相關(guān)基因的篩選及分析.中國農(nóng)業(yè)科學, 2020, 53(17): 3597-3605.

ZHAO Y Y, LI P F, XU Q Z, AN Q M, MENG J Z.Screening and analysis of follicular development related genes in goat.Scientia Agricultura Sinica, 2020, 53(17): 3597-3605.(in Chinese)

[9] REGAN S L P, KNIGHT P G, YOVICH J L, LEUNG Y, ARFUSO F, DHARMARAJAN A.Granulosa cell apoptosis in the ovarian follicle- A changing view.Frontiers in Endocrinology, 2018, 9: 61.

[10] ZEUNER A, MüLLER K, REGUSZYNSKI K, JEWGENOW K.Apoptosis within bovine follicular cells and its effect on oocyte development duringmaturation.Theriogenology, 2003, 59(5/6): 1421-1433.

[11] NURMI T, MAZUR W, HEINONEN S, KOKKONEN J, ADLERCREUTZ H.Isoflavone content of the soy based supplements.Journal of Pharmaceutical and Biomedical Analysis, 2002, 28(1): 1-11.

[12] SATHYAPALAN T, MO A, RIGBY A S, FRASER W D, THATCHER N J, KILPATRICK E S, ATKIN S L.Soy reduces bone turnover markers in women during early menopause: a randomized controlled trial.Journal of Bone and Mineral Research, 2017, 32(1): 157-164.

[13] KIM H J, JUNG C L, JEONG Y S, KIM J S.Soybean-derived glyceollins induce apoptosis through ROS generation.Food & Function, 2014, 5(4): 688-695.

[14] LUO T, MIRANDA-GARCIA O, SASAKI G, WANG J L, SHAY N F.Genistein and daidzein decrease food intake and body weight gain in mice, and alter LXR signalingand.Food & Function, 2018, 9(12): 6257-6267.

[15] QIAN Y S, GUAN T, HUANG M H, CAO L X, LI Y M, CHENG H, JIN H X, YU D Y.Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondria-dependent apoptosis pathways and reactive oxygen induced-NF-κB activation in a cerebral ischemia mouse model.Neurochemistry International, 2012, 60(8): 759-767.

[16] 林廈菁, 陳芳, 蔣守群, 蔣宗勇.大豆異黃酮對早期斷奶仔豬生長性能、抗氧化功能及腸粘膜形態(tài)結(jié)構(gòu)的影響.中國農(nóng)業(yè)科學, 2020, 53(10): 2101-2111.

LIN X J, CHEN F, JIANG S Q, JIANG Z Y.Effects of soybean isoflavones on growth performance, antioxidant performance and intestinal morphology of early-weaned piglets.Scientia Agricultura Sinica, 2020, 53(10): 2101-2111.(in Chinese)

[17] MOTTA-MENA N V, PUTS D A.Endocrinology of human female sexuality, mating, and reproductive behavior.Hormones and Behavior, 2017, 91: 19-35.

[18] ANDRES S, HANSEN U, NIEMANN B, PALAVINSKAS R, LAMPEN A.Determination of the isoflavone composition and estrogenic activity of commercial dietary supplements based on soy or red clover.Food & Function, 2015, 6(6): 2017-2025.

[19] SHARMA G T, DUBEY P K, KUMAR G S.Localization and expression of follicle-stimulating hormone receptor gene in buffalo () pre-antral follicles.Reproduction in Domestic Animals, 2011, 46(1): 114-120.

[20] 董曉莉, 謝俊霞.雌激素和植物雌激素對大鼠杏仁核多巴胺能系統(tǒng)的調(diào)節(jié).生理學報, 2003, 55(5): 589-593.

DONG X L, XIE J X.Regulation of estrogen and phytoestrogen on the dopaminergic systems of amygdala in rats.Acta Physiologica Sinica, 2003, 55(5): 589-593.(in Chinese)

[21] WHITEHEAD S A, LACEY M.Protein tyrosine kinase activity of lavendustin A and the phytoestrogen genistein on progesterone synthesis in cultured rat ovarian cells.Fertil Steril, 2000, 73(3): 613-619.

[22] MAKAREVICH A, SIROTKIN A, TARADAJNIK T, CHRENEK P.Effects of genistein and lavendustin on reproductive processes in domestic animals.Journal of Steroid Biochemistry And Molecular Biology, 1997, 63(4-6): 329-337.

[23] TIEMANN U, SCHNEIDER F, VANSELOW J, TOMEK W.In vitro exposure of porcine granulosa cells to the phytoestrogens genistein and daidzein: effects on the biosynthesis of reproductive steroid hormones.Reprod Toxicology, 2007, 24(3-4): 317-325.

[24] NYNCA A, JABLONSKA O, SLOMCZYNSKA M, PETROFF B K, CIERESZKO R E.Effects of phytoestrogen daidzein and estradiol on steroidogenesis and expression of estrogen receptors in porcine luteinized granulosa cells from large follicles.Journal of Physiology and Pharmacology, 2009, 60(2): 95-105.

[25] SWART A C, JOHANNES I D, SATHYAPALAN T, ATKIN S L.The effect of soy isoflavones on steroid metabolism.Frontiers in Endocrinology, 2019, 10: 229-239.

[26] KIM M Y, PARK J Y, PARK H S.Akt1-mediated phosphorylation of RBP-jk controls Notch1 signaling.Biochemistry (Moscow), 2019, 84(12): 1537-1546.

[27] ZHU C C, ZHANG Y E, DUAN X, HAN J, SUN S C.Toxic effects of HT-2 toxin on mouse oocytes and its possible mechanisms.Archives of Toxicology, 2016, 90(6): 1495-1505.

[28] CHOI J Y, JO M W, LEE E Y, YOON B K, CHOI D S.The role of autophagy in follicular development and atresia in rat granulosa cells.Fertility and Sterility, 2010, 93(8): 2532-2537.

[29] FIGUEROA F, MOTTA A, ACOSTA M, MOHAMED F, OLIVEROS L, FORNERIS M.Role of macrophage secretions on rat polycystic ovary: its effect on apoptosis.Reproduction (Cambridge, England), 2015, 150(5): 437-448.

[30] 鄭峰, 金梅花, 金明, 劉思彤, 張?zhí)? 尹學哲, 全吉淑.大豆異黃酮對過氧化氫致L02細胞凋亡的抑制作用.大豆科學, 2020(4): 621-625.

ZHENG F, JIN M H, JIN M, LIU S T, ZHANG T, YIN X Z, QUAN J S.Inhibitory effect of soy isoflavones on apoptosis of L02 cells induced by hydrogen peroxide.Soybean Science, 2020(4): 621-625.(in Chinese)

[31] VOUSDEN K H, PRIVES C.Blinded by the light: the growing complexity of p53.Cell, 2009, 137(3): 413-431.

[32] MYONG N H.Tissue microarray analysis of Fas and FasL expressions in human non-small cell lung carcinomas; with reference to the p53 and bcl-2 overexpressions.Journal of Korean Medical Science, 2005, 20(5): 770-776.

[33] LI X Y, HE Z C, CHENG B Q, FANG Q, MA D, LU T T, WEI D N, KUANG X Y, TANG S S, XIONG J E, WANG J S.Effect of BCLAF1on HDAC inhibitor LMK-235-mediated apoptosis of diffuse large B cell lymphoma cells and its mechanism.Cancer Biology & Therapy, 2018, 19(9): 825-834.

Effects of Soy Isoflavones on the Proliferation and Apoptosis of Yak Ovarian Granulosa Cells

AYIMUGULI·Abudureyimu1,2, AERZUGULI·Ayiding1,2, WANG JiaMin1,3, SHI JiaChen1,2, MA FangFang1,2, CAI Yong4, QIAO ZiLin1,3*

1Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030;2College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030;3Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030;4Department of Experiment & Teaching, Northwest Minzu University, Lanzhou 730030

【Background】Soybean isoflavones, mainly including genistein and daidzein, could exert estrogen-like effects, scavenge free radicals and promote cell proliferation.Granulosa cells are an important cell population in follicles and its physiological state is directly related to ovarian function, and the apoptosis of granulosa cells causes oocyte atresia.Yak (Bos grunniens) is one of the endemic species in Qinghai-Tibet plateau, but its reproductive rate is so low, however, the mechanism is still unclear.Ovary granulosa cell is an ideal model to study the regulating mechanism of female animal reproduction.【Objective】The aim this study was to investigate the effects of soybean isoflavones on the proliferation and apoptosis of granulosa cells of yak ovary, and to provide evidence for the mechanism of soybean isoflavones.【Method】The yak ovarian granulosa cells were isolated and cultured.Immunohistochemistry staining was used to check FSHR for ovarian granulosa cell authenticating.MTT assay was used to detect cell proliferation, then the growth curve was drawn.Ovarian granulosa cells treated with different concentration of genistein and daidzein (0, 1 000, 2 000, 3 000, 4 000 and 5 000 pg·mL-1), the optimal concentration of genistein or daidzein for was selected to treat the second-generation granulosa cells for 48 h, then, the cell culture medium was collected and used to detect the concentration of estradiol and progesterone secreted by granulosa cells by ELISA.At the same time, the cells were collected to extract total RNA and to research the expression of proliferation-related geneand apoptosis-related genesandby qRT-PCR.【Result】The yak ovarian granulosa cells were typical epithelial-like cells.After inoculated for 2 h, the granulosa cells began to adhere to the wall.After 12 h, the cells appeared aggregation and growth.After 24 h, the cells were larger and showed long fusiform, star-shaped or polygonal.After cultured for 48 h, the cells looked like paving appearance.Immunohistochemistry staining showed FSHR positive indicated that the cultured cells were ovarian granulosa cells.MTT assay showed that the growth curve of the yak ovarian granulosa cells was S-shape: the growth was slow in 24 h and the cells were in the latent growth stage, and then, which was rapidly proliferated at 24-48 h and entered the exponential growth phase.The granulosa cells, in the plateau phase, steadily proliferated during 48-120 h.After 120 h, the density of living cells began to decline, and the cells entered the phase of degeneration.The second-generation of granulosa cells grew faster and the exponential proliferation phase at 24 h, so the second-generation of granulosa cells was selected for this experiment.MTT assay showed that treated with 3 000 pg·mL-1genistein or daidzein for 48h, the cell viability were significantly promoted(＜0.01), the secretion of estradiol were induced treated with daidzein, but the progesterone secretion were markedly inhibited treated with genistein.The results of qRT-PCR showed that 3 000 pg·mL-1genistein or daidzein significantly up-regulated the expression of,,,,and/(＜0.01), down-regulated the expression ofand.In addition, genistein significantly down-regulated the expression ofand daidzein significantly up-regulated it (＜0.01).【Conclusion】The yak ovarian granulosa cells were isolated and cultured to provide an effective cell model for further study on the yak female reproduction regulating mechanism.The results indicated that genistein and daidzein inhibited the progesterone secretion of yak ovarian granulosa cells, promoted cell proliferation, and protected cells from apoptosis by up-regulating,,andand down-regulatingand.

genistein; daidzein; yak; ovarian granulosa cells; proliferation; apoptosis

甘肅省自然科學基金（20JR10RA122）、甘肅省科技重點研發(fā)計劃（21YF1FA222）、中央高?；緲I(yè)務(wù)費專項資金（31920210138）、教育部動物醫(yī)學生物工程創(chuàng)新團隊（IRT-17R88）

阿依木古麗·阿不都熱依木，E-mail：87032164@qq.com。通信作者喬自林，E-mail：670267497@qq.com

2021-02-19；

2021-07-27

（責任編輯 林鑒非）