綿羊肺腺瘤病毒囊膜蛋白引起綿羊絨毛膜滋養(yǎng)層細(xì)胞的惡性轉(zhuǎn)化

趙 娟，徐斯日古楞，李慧萍，劉淑英，2*

(1.內(nèi)蒙古農(nóng)業(yè)大學(xué) 獸醫(yī)學(xué)院，呼和浩特 010018；2.農(nóng)業(yè)部動(dòng)物臨床診療技術(shù)重點(diǎn)實(shí)驗(yàn)室，呼和浩特 010018)

綿羊肺腺瘤病毒(Jaagsiekte sheep retrovirus, JSRV)是引起綿羊肺腺瘤病(ovine pulmonary adenocarcinoma，OPA)的病原，該病是綿羊肺分泌上皮細(xì)胞發(fā)生惡性轉(zhuǎn)化的一種傳染性肺癌[1-2]。綿羊肺腺瘤病毒含有標(biāo)準(zhǔn)的逆轉(zhuǎn)錄病毒基因gag、pro、pol和env[3]。研究報(bào)道在羔羊的接種試驗(yàn)中，該病毒能夠在10 d內(nèi)迅速誘導(dǎo)腫瘤，類(lèi)似于攜帶致癌基因的急性轉(zhuǎn)化逆轉(zhuǎn)錄病毒[4]。JSRV-env基因主要編碼病毒的囊膜蛋白(Env)，具有表面蛋白(surface protein, SU)和跨膜蛋白(transmembrane protein, TM)兩個(gè)功能區(qū)[5]。SU負(fù)責(zé)與細(xì)胞表面的特異性受體結(jié)合，并且TM負(fù)責(zé)感染時(shí)病毒與細(xì)胞膜的融合。研究表明JSRV-env可作為致癌基因，單獨(dú)的JSRV Env蛋白可以轉(zhuǎn)化小鼠NIH3T3細(xì)胞[6]、大鼠208F細(xì)胞[7]、雞成纖維細(xì)胞[8]和犬上皮細(xì)胞[9]，并且可以在小鼠[10-11]和綿羊[12]中誘導(dǎo)肺癌，因此，JSRV的囊膜蛋白(Env)具有引起細(xì)胞轉(zhuǎn)化而致癌的罕見(jiàn)特征，但Env的致癌作用機(jī)制仍未解析清楚。

在綿羊基因組中存在大約27拷貝的與綿羊肺腺瘤病毒(JSRV)基因結(jié)構(gòu)非常相似的內(nèi)源性逆轉(zhuǎn)錄病毒(enJSRV)[13]，為了與之區(qū)別，JSRV也稱(chēng)外源性綿羊肺腺瘤病毒(exogenous Jaagsiekte sheep retrovirus，exJSRV)。研究發(fā)現(xiàn)，exJSRV和enJSRV之間的序列除了三個(gè)區(qū)域的變異(VR1、VR2和VR3)以外具有高度同源性，其中VR3則映射到env基因部分，即TM蛋白的細(xì)胞質(zhì)尾區(qū)[14]，而TM區(qū)的YXXM基序在所有轉(zhuǎn)化的JSRV中是必需的，但enJSRV的Env蛋白中不存在YXXM基序功能區(qū)，也無(wú)轉(zhuǎn)化功能[15]。而且缺失和/或突變?cè)囼?yàn)已經(jīng)證明TM蛋白的細(xì)胞質(zhì)尾區(qū)是轉(zhuǎn)化必不可少的[16-17]。哺乳動(dòng)物的胎盤(pán)具有物質(zhì)交換、分泌激素和防御等重要功能，在胎盤(pán)中行使這些功能的主要細(xì)胞類(lèi)型是滋養(yǎng)層細(xì)胞[18]。本實(shí)驗(yàn)室從正常胎盤(pán)組織中分離培養(yǎng)了一株永生化的滋養(yǎng)層細(xì)胞系用于細(xì)胞轉(zhuǎn)化試驗(yàn)。因此本研究利用體外培養(yǎng)的永生化綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞，轉(zhuǎn)染重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env，用平板克隆以及軟瓊脂集落形成試驗(yàn)，觀察細(xì)胞的惡性轉(zhuǎn)化以及細(xì)胞增殖情況，為進(jìn)一步探討exJSRV Env的致癌功能提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 試驗(yàn)材料

真核表達(dá)載體pEGFP-C1由本實(shí)驗(yàn)室保存；重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env以及pEGFP-C1/enJSRV-env由本實(shí)驗(yàn)室構(gòu)建；永生化綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞由本實(shí)驗(yàn)室建立。

1.2 主要試驗(yàn)試劑

真核細(xì)胞轉(zhuǎn)染試劑LipofectamineTMLTX & PLUS購(gòu)自Invitrogen公司；限制性?xún)?nèi)切酶KpnⅠ、BamHⅠ、HindⅢ和ApaⅠ購(gòu)自TaKaRa公司；DMEM細(xì)胞培養(yǎng)液、Opti-MEM培養(yǎng)液，0.25%胰蛋白酶購(gòu)自GIBCO公司；胎牛血清(FBS)購(gòu)自澳洲；瓊脂糖購(gòu)自Invitrogen公司。

1.3 真核表達(dá)重組質(zhì)粒的鑒定

成功構(gòu)建重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env以及pEGFP-C1/enJSRV-env。重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env和pEGFP-C1/enJSRV-env分別用限制性?xún)?nèi)切酶HindⅢ、ApaⅠ和KpnⅠ、BamHⅠ水浴酶切，酶切結(jié)束后利用瓊脂糖凝膠電泳鑒定。

1.4 永生化綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞培養(yǎng)及轉(zhuǎn)染效率的測(cè)定

將永生化綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞復(fù)蘇，放入37 ℃ 5% CO2培養(yǎng)箱中培養(yǎng)，待長(zhǎng)到80%時(shí)傳代。轉(zhuǎn)染的前一天，用胰蛋白酶消化并將細(xì)胞接種在六孔板內(nèi)，加入2 mL無(wú)雙抗(青鏈霉素)的含20% FBS完全培養(yǎng)基。細(xì)胞密度應(yīng)在轉(zhuǎn)染當(dāng)天匯合度應(yīng)達(dá)到80%左右。按照 LipofectamineTMLTX & PLUS轉(zhuǎn)染試劑說(shuō)明分別轉(zhuǎn)染質(zhì)粒 pEGFP-C1/exJSRV-env、pEGFP-C1/enJSRV-env以及空載體pEGFP-C1，不作處理的正常細(xì)胞作為空白對(duì)照組。48 h后進(jìn)行免疫熒光照相，以及后續(xù)軟瓊脂集落形成試驗(yàn)和平板克隆試驗(yàn)。

1.5 軟瓊脂集落形成試驗(yàn)

轉(zhuǎn)染48 h后的細(xì)胞用0.25%胰蛋白酶消化并輕輕吹打，作活細(xì)胞計(jì)數(shù)，然后根據(jù)試驗(yàn)要求做梯度倍數(shù)稀釋。用1.2%瓊脂糖溶液均勻鋪制細(xì)胞6孔板底層，置CO2溫箱中備用。將0.7%瓊脂糖溶液與約3 000個(gè)轉(zhuǎn)染后的細(xì)胞充分混勻，注入鋪有瓊脂糖底層的平皿中，置入37 ℃、5% CO2溫箱中，培養(yǎng)10～14 d。顯微鏡下觀察集落形成。

1.6 平板克隆試驗(yàn)

對(duì)各組轉(zhuǎn)染的細(xì)胞進(jìn)行細(xì)胞計(jì)數(shù)，以每皿50個(gè)和100個(gè)細(xì)胞放入六孔板中，加入3 mL含20% FBS的完全培養(yǎng)基，每組做3組平行試驗(yàn)，置入37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)10～14 d。每3 d換一次培養(yǎng)液。待肉眼可見(jiàn)的克隆形成時(shí)，用4%多聚甲醛固定并用結(jié)晶紫染色，顯微鏡下計(jì)數(shù)大于50個(gè)細(xì)胞的克隆數(shù)，計(jì)算克隆形成率(克隆形成率=克隆數(shù)/接種數(shù)×100%)，并用SPSS軟件作統(tǒng)計(jì)學(xué)分析。

2 結(jié) 果

2.1 pEGFP-C1/exJSRV-env和pEGFP-C1/enJSRV-env質(zhì)粒酶切鑒定

將實(shí)驗(yàn)室保存的重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env和pEGFP-C1/enJSRV-env經(jīng)單、雙酶切鑒定(圖1)，結(jié)果顯示質(zhì)粒正確，可以繼續(xù)使用。

2.2 綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞的最佳轉(zhuǎn)染效率

綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞瞬時(shí)轉(zhuǎn)染重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env和pEGFP-C1/enJSRV-env48 h后，熒光顯微鏡下觀察，結(jié)果顯示,在綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞的細(xì)胞質(zhì)內(nèi)彌漫大量的綠色熒光蛋白(圖2A、B)，表明重組真核表達(dá)質(zhì)粒成功轉(zhuǎn)染綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞，可用于后續(xù)研究。

2.3 軟瓊脂集落形成試驗(yàn)

轉(zhuǎn)染重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env的綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞接觸抑制性消失，且能夠在軟瓊脂上形成集落(圖3A)，說(shuō)明綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞發(fā)生了惡性轉(zhuǎn)化；而轉(zhuǎn)染內(nèi)源性病毒的重組真核表達(dá)質(zhì)粒pEGFP-C1/enJSRV-env、空載體pEGFP-C1以及空白對(duì)照組細(xì)胞均不能在瓊脂上形成集落(圖3B、C、D)，說(shuō)明綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞并未發(fā)生惡性轉(zhuǎn)化。

M. DNA相對(duì)分子質(zhì)量標(biāo)準(zhǔn)(DL5000); 1、2. HindⅢ單酶切pEGFP-C1/exJSRV-env重組質(zhì)粒; 3、4. Hind Ⅲ和ApaⅠ雙酶切pEGFP-C1/exJSRV-env重組質(zhì)粒; 5. HindⅢ單酶切pEGFP-C1空質(zhì)粒; 6. KpnⅠ單酶切pEGFP-C1空質(zhì)粒; 7、8. KpnⅠ單酶切pEGFP-C1/enJSRV-env重組質(zhì)粒; 9、10. KpnⅠ和BamHⅠ雙酶切pEGFP-C1/enJSRV-env重組質(zhì)粒M. DNA marker (DL5000); 1, 2. Digested product of pEGFP-C1/exJSRV-env with HindⅢ; 3, 4. Digested product of pEGFP-C1/exJSRV-env with HindⅢ and ApaⅠ; 5. Digested product of pEGFP-C1 with HindⅢ; 6. Digested product of pEGFP-C1 with KpnⅠ; 7, 8. Digested product of pEGFP-C1/enJSRV-env with KpnⅠ; 9, 10. Digested product of pEGFP-C1/enJSRV-env with Kpn Ⅰ and BamHⅠ圖1 重組質(zhì)粒單、雙酶切鑒定Fig.1 Identification of recombinant plasmid by single and double restriction endonuclease digestion

2.4 平板克隆試驗(yàn)

統(tǒng)計(jì)轉(zhuǎn)染 pEGFP-C1/exJSRV-env的細(xì)胞、轉(zhuǎn)染pEGFP-C1/enJSRV-env、pEGFP-C1以及未轉(zhuǎn)染的細(xì)胞形成的克隆數(shù)(圖4)，克隆形成率結(jié)果經(jīng)SPSS軟件分析，轉(zhuǎn)染pEGFP-C1/exJSRV-env的綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞的克隆形成率極顯著高于其他三組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)(圖5)，轉(zhuǎn)染pEGFP-C1/enJSRV-env以及pEGFP-C1的細(xì)胞與未轉(zhuǎn)染的細(xì)胞之間無(wú)顯著差異(P>0.05)(圖5)。

3 討 論

目前，綿羊肺腺瘤逆轉(zhuǎn)錄病毒引起綿羊肺腺瘤病的致瘤轉(zhuǎn)化機(jī)制尚不清楚。M. Borobia等[19]將攜帶雙鏈DNA的JSRV質(zhì)粒轉(zhuǎn)染到NIH 3T3細(xì)胞中導(dǎo)致轉(zhuǎn)化，證明JSRV攜帶致癌基因。將pCMV2JS21真核表達(dá)質(zhì)粒轉(zhuǎn)染NIH 3T3細(xì)胞，形成轉(zhuǎn)化灶，而轉(zhuǎn)染對(duì)照組pCDNA3.1(-)質(zhì)粒未有轉(zhuǎn)化灶形成[19]。本實(shí)驗(yàn)室張宇飛等[20]證實(shí)將重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env轉(zhuǎn)染到293T細(xì)胞中也引起惡性轉(zhuǎn)化。以上研究結(jié)果均證明JSRV囊膜蛋白(Env)是腫瘤形成的主要因素。

A. 轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/exJSRV-env 48 h后的綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞；B. 轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/enJSRV-env 48 h后的綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞A. The ovine trophoblast cells transfected with recombinant plasmid pEGFP-C1/exJSRV-env after 48 h; B. The ovine trophoblast cells transfected with recombinant plasmid pEGFP-C1/enJSRV-env after 48 h圖2 轉(zhuǎn)染重組質(zhì)粒后的綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞Fig.2 The ovine trophoblast cells transfected with recombinant plasmid

A.轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/exJSRV-env 的細(xì)胞；B.轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/enJSRV-env 的細(xì)胞；C.轉(zhuǎn)染空載體的細(xì)胞；D.空白對(duì)照A. Cells tranfected with recombinant plasmid pEGFP-C1/exJSRV-env; B.Cells transfected with recombinant plasmid pEGFP-C1/enJSRV-env; C. Cells transfected with plasmid pEGFP-C1; D. Blank control圖3 集落形成試驗(yàn)(×100)Fig.3 Colony growth in soft agar(×100)

A. 每孔50個(gè)細(xì)胞；B. 每孔100個(gè)細(xì)胞；1.未轉(zhuǎn)染的綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞; 2.轉(zhuǎn)染空質(zhì)粒pEGFP-C1的細(xì)胞; 3.轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/enJSRV-env的細(xì)胞; 4.轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/exJSRV-env的細(xì)胞A. 50 cells per well; B. 100 cells per well; 1. The sheep trophoblast cells without treatment; 2. Cells transfected with pEGFP-C1; 3. Cells transfected with pEGFP-C1/enJSRV-env; 4. Cells transfected with pEGFP-C1/exJSRV-env圖4 轉(zhuǎn)染后細(xì)胞平板克隆試驗(yàn)Fig.4 Cells plate cloning experiments after transfection

各組間比較，***.P<0.01Asterisks indicate significand differences (***.P<0.01) when compared among the different groups圖5 克隆形成率柱狀圖Fig.5 Cloning efficiency histograms

3.1 Env引起綿羊絨毛膜滋養(yǎng)層細(xì)胞惡性轉(zhuǎn)化

為了更好地闡述JSRV Env蛋白的轉(zhuǎn)化機(jī)制，利用重組質(zhì)粒pEGFP-C1/exJSRV-env轉(zhuǎn)染綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞，通過(guò)軟瓊脂集落形成試驗(yàn)對(duì)該細(xì)胞發(fā)生的形態(tài)變化進(jìn)行分析。正常的絨毛膜滋養(yǎng)層細(xì)胞不能在半固體軟瓊脂上懸浮生長(zhǎng)形成集落，而惡性轉(zhuǎn)化的細(xì)胞獲得了錨定非依賴(lài)性生長(zhǎng)能力可以生長(zhǎng)形成集落。而本研究顯示轉(zhuǎn)染重組真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env的細(xì)胞形成集落，而轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/enJSRV-env、空質(zhì)粒pEGFP-C1以及空白對(duì)照組細(xì)胞卻不能形成集落，這提示exJSRV囊膜蛋白(Env)促使綿羊絨毛膜滋養(yǎng)層細(xì)胞發(fā)生惡性轉(zhuǎn)化。M. Borobia等[19]研究顯示將缺失gag、pro、pol編碼序列的表達(dá)質(zhì)粒pCMVJS21(pCMVJS21ΔGP，唯一完整的基因是env)轉(zhuǎn)染NIH3T3細(xì)胞產(chǎn)生轉(zhuǎn)化灶，并且將exJSRV的細(xì)胞質(zhì)尾區(qū)與enJSRV交換的嵌合體卻不能轉(zhuǎn)化NIH3T3細(xì)胞，說(shuō)明引起細(xì)胞發(fā)生轉(zhuǎn)化的必需是完整的囊膜蛋白，而本試驗(yàn)所采用的真核表達(dá)質(zhì)粒pEGFP-C1/exJSRV-env就是完整表達(dá)了SU 和TM的囊膜蛋白所發(fā)揮的作用。

3.2 Env對(duì)綿羊絨毛膜滋養(yǎng)層細(xì)胞增殖的影響

細(xì)胞增殖是檢測(cè)分裂中的細(xì)胞數(shù)量或者細(xì)胞群體發(fā)生的變化，克隆形成率反應(yīng)細(xì)胞的增值能力。據(jù)報(bào)道地方性鼻病毒(ENTV)[21]、禽血管瘤病毒(AHV)[22]的囊膜蛋白(Env)也具有類(lèi)似的致癌功能，AHV對(duì)NIH3T3細(xì)胞的增殖作用也通過(guò)AHVenv基因介導(dǎo),通過(guò)將AHVenv基因克隆到基于MuLV的逆轉(zhuǎn)錄病毒載體中證明了這一點(diǎn)，并且用該重組質(zhì)粒轉(zhuǎn)染NIH3T3細(xì)胞誘導(dǎo)細(xì)胞增殖和表型改變[22]。S. L. Liu等[9]將JSRV Env轉(zhuǎn)染犬腎細(xì)胞(MDCK)，發(fā)現(xiàn)轉(zhuǎn)染后的細(xì)胞增殖要顯著高于對(duì)照組。enJSRV-env對(duì)絨毛膜滋養(yǎng)層細(xì)胞的細(xì)胞融合也有一定的促進(jìn)作用[23]。本實(shí)驗(yàn)室杜方原等[24]將重組質(zhì)粒pcDNA4/myc-His/exJSRV-env轉(zhuǎn)染到NIH3T3細(xì)胞也發(fā)生細(xì)胞增殖。而本研究中轉(zhuǎn)染重組質(zhì)粒pEGFP-C1/exJSRV-env的細(xì)胞平板克隆試驗(yàn)的克隆形成率也顯著高于對(duì)照組，說(shuō)明了細(xì)胞在exJSRV囊膜蛋白(Env)的作用下，細(xì)胞發(fā)生增殖，與上述研究結(jié)果一致。目前研究表明在exJSRV囊膜蛋白致癌過(guò)程中可能有P53蛋白、表面蛋白A(surfactant protein A, SP-A)、增殖細(xì)胞核抗原(proliferating cell nuclear antigen, PCNA)、JSRV基質(zhì)蛋白(JSRV matrix protein, MA)[25]、鋅指蛋白111(Zinc Finger Protein, Zfp111)[26]的參與，以及囊膜蛋白致癌過(guò)程也與細(xì)胞自噬[27]和信號(hào)通路[28]相關(guān)，本試驗(yàn)結(jié)果為后續(xù)的研究提供了理論依據(jù)。

4 結(jié) 論

exJSRV囊膜蛋白能夠引起綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞發(fā)生惡性轉(zhuǎn)化以及細(xì)胞增殖。

參考文獻(xiàn)(References)：

[1] ZHANG Y F, SHI J, LIU S Y. Recent advances in the study of active endogenous retrovirus envelope glycoproteins in the mammalian placenta[J].VirolSin, 2015, 30(4):239-248.

[2] HULL S, LIM J, HAMIL A, et al. Analysis ofJaagsiektesheepretrovirus (JSRV) envelope protein domains in transformation[J].VirusGenes, 2012, 45(3):508-517.

[3] HOFACRE A, FAN H. Jaagsiekte sheep retrovirus biology and oncogenesis[J].Viruses, 2010, 2(12):2618-2648.

[4] LIU S L, MILLER A D. Oncogenic transformation by theJaagsiektesheepretrovirus envelope protein[J].Oncogene, 2007, 26(6):789-801.

[5] DEMARTINI J C, BISHOPP J V, ALLEN T E, et al.Jaagsiektesheepretrovirus proviral clone JSRVJS7,derived from the JS7 lung tumor cell line,induce ovine pulmonary carcinoma and is integrated into the surfactant protein A gene[J].JVirol, 2001, 75(9):4239-4246.

[6] YOUSSEF G,WALLACE W A H,DAGLEISH M P,et al.Ovine pulmonary adenocarcinoma:a large animal model for human lung cancer[J].ILARJ,2015,56(1):99-115.

[7] RAI S K, DUH F M,VIGDOROVICH V, et al. Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for Jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation[J].ProcNatlAcadSciUSA, 2001, 98(8):4443-4448.

[8] ALLEN T E, SHERRILL K J, CRISPELL S M, et al. The Jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain[J].JGenVirol, 2002, 83(11):2733-2742.

[9] LIU S L, MILLER A D. Transformation of Madin-Darby canine kidney epithelial cells by sheep retrovirus envelope proteins[J].JVirol, 2005, 79(2):927-933.

[10] WOOTTON S K, HALBERT C L, MILLER A D. Sheep retrovirus structural protein induces lung tumours[J].Nature, 2005, 434(7035):904-907.

[11] LINNERTH-PETRIK N M,SANTRY L A,YU D L, et al. Adeno-associated virus vector mediated expression of an oncogenic retroviral envelope protein induces lung adenocarcinomas in immunocompetent mice[J].PLoSOne, 2012, 7(12):e51400.

[12] CAPORALE M, COUSENS C, CENTORAME P, et al. Expression of the Jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep[J].JVirol, 2006, 80(16):8030-8037.

[13] LEROUX C, GIRARDI N, COTTIN S, et al. Jaagsiekte sheep retrovirus (JSRV): from virus to lung cancer in sheep[J].VetRes, 2007, 38(2):211-228.

[14] SISTIAQA-POVEDA M, LARRUSKAIN A, MATEO-ABAD M, et al. Lack of association between polymorphic copies of endogenous Jaagsiekte sheep retrovirus (enJSRVs) and ovine pulmonary adenocarcinoma[J].VetMicrobiol, 2016, 185:49-55.

[15] 張亞坤,劉淑英.綿羊肺腺瘤病毒囊膜蛋白致癌作用的研究進(jìn)展[J].中國(guó)獸醫(yī)科學(xué), 2012, 42(12):1315-1320.

ZHANG Y K, LIU S Y. Advance in tumorigenesis of Jaagsiekte sheep retrovirus’ envelope protein[J].ChineseVeterinaryScience, 2012, 42(12):1315-1320. (in Chinese)

[16] PALMARINI M, MAEDA N,MURGIA C, et al. A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells[J].JVirol, 2001, 75(22):11002-11009.

[17] HOFACRE A,FAN H.Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts[J].JVirol, 2004, 78(19):10479-10489.

[18] 張宇飛.綿羊胎盤(pán)絨毛膜滋養(yǎng)層細(xì)胞中enJSRV囊膜蛋白的細(xì)胞生物學(xué)作用[D].呼和浩特:內(nèi)蒙古農(nóng)業(yè)大學(xué), 2016.

ZHANG Y F.Cell biological function of enJSRV envelope proteins in sheep trophoblast cells[D].Hohhot:Inner Mongolia Agricultural University, 2016.(in Chinese)

[19] BOROBIA M,ORTIN A,FERRER L M, et al. Cells infected withJaagsiektesheepretrovirus are detected in the bone marrow of asymptomatic sheep[J].CanJVetRes, 2014, 78(3):237-240.

[20] 張宇飛, 劉 月, 王專(zhuān)家, 等.綿羊肺腺瘤病毒pEGFP-C1/exJSRV-env構(gòu)建及引起NIH3T3細(xì)胞惡性轉(zhuǎn)化的研究[J].病毒學(xué)報(bào), 2014, 30(3):268-277.

ZHANG Y F, LIU Y, WANG Z J, et al. Research on construction of sheep lung adenomas virus pEGFP-C1/exJSRV-envand induction of malignant transformation in NIH3T3[J].ChineseJournalofVirology, 2014, 30(3):268-277. (in Chinese)

[21] MAEDA N, FAN H. Signal transduction pathways utilized by enzootic nasal tumor virus (ENTV-1) envelope protein in transformation of rat epithelial cells resemble those used byJaagsiektesheepretrovirus[J].VirusGenes, 2008, 36(1):147-155.

[22] ZHANG Y F, SHI J, LIU S Y. Establishment and characterization of a Telomerase-Immortalized sheep trophoblast cell line[J].BiomedResInt, 2016, 2016:5808575.

[23] 張宇飛，石 晶，劉淑英. enJSRV囊膜蛋白及其受體的瞬時(shí)表達(dá)與綿羊絨毛膜滋養(yǎng)層細(xì)胞融合的誘導(dǎo)研究[J]. 畜牧獸醫(yī)學(xué)報(bào), 2015, 46(11): 1924-1933.

ZHANG Y F, SHI J, LIU S Y. Study on the Transient expression of enJSRV envelope protein and its receptor and the induction of trophoblast cell fusion in sheep[J].ActaVeterinariaetZootechnicaSinica, 2015, 46(11): 1924-1933. (in Chinese)

[24] 杜方原,陳大勇,張宇飛,等.綿羊肺腺瘤病毒囊膜蛋白的表達(dá)可促進(jìn)NIH3T3細(xì)胞增殖[J].細(xì)胞與分子免疫學(xué)雜志, 2016, 32(9):1188-1192.

DU F Y,CHEN D Y,ZHANG Y F, et al. Envelope protein ofJaagsiektesheepretrovious expressed in NIH3T3 cells promotes cell proliferation[J].ChineseJournalofCellularandMolecularImmunology, 2016, 32(9):1188-1192.(in Chinese)

[25] ILHAN F, VURAL S A, YILDIRIM S, et al. Expression of p53 protein, Jaagsiekte sheep retrovirus matrix protein, and surfactant protein in the lungs of sheep with pulmonary adenomatosis[J].JVetDiagnInvest, 2016, 28(3):249-256.

[26] HSU T, PHUNG A, CHOE K, et al. Role for a zinc finger protein (Zfp111) in transformation of 208F rat fibroblasts byJaagsiektesheepretrovirus envelope protein[J].JVirol, 2015, 89(20):10453-10466.

[27] SUN X L, DU F Y, LIU S Y. Modulation of autophagy in exJSRV-env-transfected cells through the Akt/mTOR and MAPK signaling pathway[J].BiochemBiophysResCommun, 2017, 485(3):672-678.

[28] 孫曉林,劉淑英.外源性綿羊肺腺瘤病毒囊膜蛋白激活A(yù)kt/mTOR信號(hào)通路及調(diào)控Beclin1的研究[J].中國(guó)預(yù)防獸醫(yī)學(xué)報(bào), 2017, 39(4):251-256.

SUN X L,LIU S Y.Modulation of Beclin 1 in exogenous Jaagsiekte sheep retrovirus envelope gene transfected cells through the Akt/mTOR signaling pathway[J].ChineseJournalofPreventiveVeterinaryMedicine, 2017, 39(4):251-256.(in Chinese)