杜義江，肖長(zhǎng)義，鄭 軍，胡 敏

(三峽大學(xué) 1.醫(yī)學(xué)院 組織學(xué)與胚胎學(xué)教研室， 湖北 宜昌 443002；三峽大學(xué) 第一臨床醫(yī)學(xué)院 2.中心實(shí)驗(yàn)室； 3.普外科， 湖北 宜昌 443003)

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研究論文

HepG2細(xì)胞內(nèi)HPV DNA物理狀態(tài)及表達(dá)L1蛋白

杜義江1,2,3，肖長(zhǎng)義1,2*，鄭 軍3，胡 敏2

(三峽大學(xué) 1.醫(yī)學(xué)院 組織學(xué)與胚胎學(xué)教研室， 湖北 宜昌 443002；三峽大學(xué) 第一臨床醫(yī)學(xué)院 2.中心實(shí)驗(yàn)室； 3.普外科， 湖北 宜昌 443003)

目的了解人肝癌細(xì)胞系HepG2細(xì)胞內(nèi)人乳頭瘤病毒(HPV)基因組的物理狀態(tài)，胞質(zhì)內(nèi)包涵體物質(zhì)的性質(zhì)以及晚期衣殼蛋白1(L1)表達(dá)。方法用PCR對(duì)細(xì)胞內(nèi)HPV18型E2和E6基因進(jìn)行擴(kuò)增，判斷HPV18基因組的物理狀態(tài)；用ELISA、光鏡和電鏡的免疫組化、Western blot等方法，以多價(jià)HPV L1小鼠單克隆抗體做探針，檢測(cè)HepG2細(xì)胞內(nèi)L1蛋白表達(dá)；用反轉(zhuǎn)錄PCR檢測(cè)細(xì)胞內(nèi)L1 mRNA表達(dá)。結(jié)果HepG2細(xì)胞內(nèi)HPV DNA基因組呈整合狀態(tài)；細(xì)胞裂解液中有HPV L1蛋白存在；細(xì)胞呈HPV L1陽(yáng)性反應(yīng)；胞質(zhì)內(nèi)包涵體樣物質(zhì)，由均勻的顆粒樣物質(zhì)組成，可以為膠體金標(biāo)記的HPV L1抗體所標(biāo)識(shí)。HepG2細(xì)胞裂解液中有HPV L1蛋白，在56 ku區(qū)出現(xiàn)與HeLa細(xì)胞一樣的L1特異陽(yáng)性條帶。反轉(zhuǎn)錄PCR檢測(cè)顯示HepG2細(xì)胞內(nèi)有L1 mRNA存在。結(jié)論HepG2是HPV18陽(yáng)性細(xì)胞，細(xì)胞內(nèi)HPV DNA基因組呈整合狀態(tài)。細(xì)胞內(nèi)包涵體樣物質(zhì)為HPV18 L1蛋白，HepG2細(xì)胞可以表達(dá)L1。

HepG2細(xì)胞；人乳頭瘤病毒；病毒基因組；物理狀態(tài)；L1蛋白

近來(lái)有研究證實(shí)肝癌細(xì)胞系HepG2是HPV18陽(yáng)性細(xì)胞[1]，電鏡可觀察到HepG2細(xì)胞胞質(zhì)內(nèi)有包涵體樣物質(zhì)，與宮頸癌來(lái)源的HeLa細(xì)胞胞質(zhì)內(nèi)包涵體樣物質(zhì)具有同樣的形態(tài)學(xué)特征，HeLa細(xì)胞內(nèi)包涵體物質(zhì)證實(shí)為HPV18晚期衣殼蛋白1(late capsid protein 1,L1)[2]。HepG2細(xì)胞胞質(zhì)內(nèi)包涵體物質(zhì)性質(zhì)及HPV DNA基因組在HepG2細(xì)胞內(nèi)物理狀態(tài)——游離還是整合？尚未見(jiàn)報(bào)道。

1 材料與方法

1.1 細(xì)胞與試劑

HepG2、HeLa和HaCat細(xì)胞[中國(guó)典型培養(yǎng)物保藏中心(武漢大學(xué))]，HaLa細(xì)胞為HPV18陽(yáng)性細(xì)胞，作陽(yáng)性對(duì)照； HaCat細(xì)胞是人永生表皮細(xì)胞，為HPV陰性細(xì)胞，作陰性對(duì)照。DNA提取試劑盒(北京天根生物科技有限公司)。Trnzol(TaKaRa公司)。反轉(zhuǎn)錄試劑盒及PCR Master Mix(ThermoFisher公司)。內(nèi)參照globin引物(上游：5′-ACACAACTG TGTTCACTAGC-3′；下游：5′-CAACTTCATCCACGTT CACC-3′)。HPV18型引物E2(上游：5′-CAACACG GCGACCCTACA-3′；下游：5′-GGATTCAACGGTTTC TGG-3′)，E6(上游：5′-ATGAAAA TGACAGTAAAG AC-3′；下游：5′-CATTGTCATGTATCCCACC-3′)。HPV通用引物GP5+/6+(上游：5′-TTTGTTACTGT GGTAGATACY(T/C)AC-3′；下游：5′-GAAAAATAA ACTGTAAATCATATTC-3′)，MY09/11引物(上游：5′-CGTCCMARRGGAWACTGATC-3′；下游：5′-GCMC AGGGWCATAAYAATGG-3′)，均由生工生物工程(上海)股份有限公司合成。一抗：小鼠抗HPV1、6、11、16、18、31 L1單克隆抗體(Millipore公司)。二抗：HRP標(biāo)記的羊抗鼠IgG(武漢谷歌生物科技有限公司)。

1.2 培養(yǎng)細(xì)胞總DNA、蛋白和RNA的提取

HepG2、HeLa和HaCat細(xì)胞用DNA提取試劑盒提取總DNA，-20 ℃保存；用RIPA裂解細(xì)胞，收集上清蛋白-20 ℃保存。用TRNzol提取細(xì)胞總mRNA， -80 ℃保存。

1.3 HepG2細(xì)胞內(nèi)HPV基因組物理狀態(tài)的檢測(cè)

3種細(xì)胞總DNA用PCR Master Mix 擴(kuò)增。引物為HPV 18型E2、E6和球蛋白(globin)。產(chǎn)物用瓊脂糖凝膠電泳，凝膠成像儀照相。

1.4 ELISA檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)

1.5 光鏡免疫組化檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)

3種細(xì)胞培養(yǎng)爬片，乙醇固定，去內(nèi)源性過(guò)氧化物酶。一抗(1∶1 000稀釋)、二抗(1∶1 000稀釋)孵育，生物素-抗生物素-辣根過(guò)氧化物酶復(fù)合物孵育，DAB顯色，PBS終止。蘇木素復(fù)染，鹽酸乙醇分化，自來(lái)水沖洗，脫水、透明、樹(shù)膠封片、鏡檢。

1.6 電鏡免疫組化檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)

收集對(duì)數(shù)期增殖HepG2細(xì)胞，1.5% 戊二醛固定，常規(guī)透射電鏡標(biāo)本制作。去內(nèi)源性過(guò)氧化物酶，一抗(1∶500稀釋)、二抗(1∶200稀釋的Φ10 nm膠體金標(biāo)記羊抗小鼠IgG)孵育，鈾、鉛復(fù)染，透射電鏡觀察。

1.7 Western blot檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)

3種細(xì)胞蛋白樣品用SDS-PAGE凝膠電泳、蛋白轉(zhuǎn)膜，封閉，一抗(1∶1 000稀釋)、二抗(1∶5 000稀釋)孵育，Super ECL Plus超敏發(fā)光液照相系統(tǒng)顯影。

1.8 反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測(cè)細(xì)胞內(nèi)L1 mRNA表達(dá)

3種細(xì)胞總mRNA反轉(zhuǎn)錄合成cDNA，用PCR Master Mix行PCR擴(kuò)增反應(yīng)，將cDNA擴(kuò)增成DNA。引物為globin和GP5+/6+和MY09/11。產(chǎn)物用瓊脂糖凝膠電泳，凝膠成像儀照相。

2 結(jié)果

2.1 HepG2細(xì)胞HPV18病毒基因組物理狀態(tài)PCR擴(kuò)增結(jié)果

3種細(xì)胞在蛋白標(biāo)記(marker)100 bp稍后均有條帶出現(xiàn)，為126 bp的球蛋白。在蛋白標(biāo)記300與400 bp之間HepG2、HeLa細(xì)胞均有條帶出現(xiàn)：為322 bp的E6。HaCat細(xì)胞未見(jiàn)該條帶。3種細(xì)胞均未見(jiàn)1 028 bp的E2條帶(圖1)。

At 126 bp district,the band are globin control; at 322 bp district, the bands are E6圖1 DNA PCR產(chǎn)物電泳圖Fig 1 Electropherogram of DNA PCR products

2.2 ELISA檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)的結(jié)果

HepG2和HeLa與HaCat比值均>2.1，均為陽(yáng)性反應(yīng)，P<0.01(表1)。

表1 培養(yǎng)細(xì)胞ELISA檢測(cè)結(jié)果

*P<0.01 compared with control.

2.3 光鏡免疫組化檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)的結(jié)果

HepG2、HeLa細(xì)胞胞質(zhì)和胞核均有染色，呈棕褐色陽(yáng)性反應(yīng)。HaCat細(xì)胞未見(jiàn)陽(yáng)性染色反應(yīng)(圖2)。

2.4 電鏡免疫組化檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)的結(jié)果

電鏡低倍見(jiàn)部分HepG2細(xì)胞胞質(zhì)內(nèi)有團(tuán)塊狀大小形態(tài)各異的包涵體類似物(圖3A)。高倍見(jiàn)這類團(tuán)塊周圍無(wú)包膜，與周邊胞質(zhì)成分界線清晰，由電子密度略高，直徑約為20～30 nm的圓形顆粒樣物質(zhì)密集組成；也有散在于胞質(zhì)或胞核中的(圖3B)。這些顆粒為膠體金顆粒標(biāo)記后，形成金顆粒密集區(qū)和散在金顆粒(圖4)。

There are high positive reaction in HepG2 cells and HeLa cells; both cytoplasm and nucleus are dyed to yellow; and it manifests that HPV L1 is present in HepG2 cells; there was negative reaction in HaCat cells

圖2 HepG2、HeLa和HaCat細(xì)胞HPV L1免疫組織化學(xué)檢測(cè)

Fig 2 HepG2, HeLa and HaCat cells HPV L1 immunohistochemistry assay(×200)

A.low magnification electron micrograph(×6 000)；B.high magnification electron micrograph(×40 000); the black arrowheads show many lumpish cytorrhyctes materials, which aggregated by the particle with similar size and higher electron density; these particles also can dispersed in the outside area of the cytoplasmic inclusion bodies(the short black arrowhead show in B Figure)

圖3 HepG2細(xì)胞電鏡圖

Fig 3 Electron micrographs of HepG2 cells

The black arrowheads show the gold particles in 4 figure; the HepG2 cells are reacted with HPV L1 monoclone antibody conjucted gold particles; there are many gold particles present in the district of cytorrhyctes materials; which means cytorrhyctes materials is consist of elements of HPV L1圖4 HepG2細(xì)胞免疫組化電鏡圖Fig 4 Immunohistochemistry electron micrographs of HepG2 cells(×40 000)

2.5 Western blot檢測(cè)HepG2細(xì)胞HPV L1蛋白表達(dá)的結(jié)果

在56 ku處，HepG2和HeLa細(xì)胞均有清晰條帶出現(xiàn)。HaCat細(xì)胞未見(jiàn)條帶出現(xiàn)。說(shuō)明HepG2和HeLa細(xì)胞均有HPV L1蛋白表達(dá)(圖5)。

The two bands at 56 ku district are stained by the reaction of HPV L1 monoclone antibody labeled by HRP; the three bands at 42 ku district are stained by the reaction of β-actin antibody labeled by HRP圖5 Western blot檢測(cè)HepG2細(xì)胞表達(dá)HPV L1蛋白情況Fig 5 Detection of HPV L1 by Western blot assay

2.6 細(xì)胞內(nèi)L1 mRNA表達(dá)的反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測(cè)結(jié)果

3種細(xì)胞在蛋白標(biāo)記100 bp稍后均有條帶出現(xiàn)，為126 bp的球蛋白。在蛋白標(biāo)記100與200 bp之間、400與500 bp之間HepG2、HeLa細(xì)胞均有條帶出現(xiàn)：分別是150 bp GP5+/6+擴(kuò)增產(chǎn)物和450 bp MY09/11擴(kuò)增產(chǎn)物；HaCat細(xì)胞未見(jiàn)以上條帶(圖6)。

The two bands at 56 ku district are stained by the reaction of HPV L1 monoclone antibody labeled by HRP; the three bands at 42 ku district are stained by the reaction of β-actin antibody labeled by HRP; at 126 bp district,the band are globin control; at 150 bp district, the bands are GP5+/6+; at 422 bp district, the bands are MY09/11圖6 RNA RT-PCR產(chǎn)物電泳圖Fig 6 Electropherogram of RNA RT-PCR products

3 討論

肝細(xì)胞癌(human papillomavirus，HCC)是最常見(jiàn)的原發(fā)性肝腫瘤，占全世界癌癥死亡的第3位，乙型肝炎病毒(hepatitis b virus，HBV)和丙型肝炎病毒(hepatitis c virus， HCV)持續(xù)感染是其發(fā)生的主要誘因[3]。近來(lái)發(fā)現(xiàn)肝癌患者中約有9%存在HPV16/18，肝癌細(xì)胞系HepG2是HPV18陽(yáng)性細(xì)胞，由肝細(xì)胞感染HPV后永生化衍生而來(lái)，提示HPV可能是肝癌病原體之一[2]。

HPV基因組游離狀態(tài)呈環(huán)狀，可表達(dá)E2、E6等，當(dāng)其整合進(jìn)宿主基因組時(shí)， 環(huán)狀基因從E2處斷

裂，導(dǎo)致E2基因缺失表達(dá)，PCR分析提示HPV基因組已經(jīng)整合進(jìn)HepG2細(xì)胞基因組。這將解除E2對(duì)E6、E7表達(dá)的抑制作用，使E6、E7 mRNA高水平表達(dá)，促進(jìn)惡性轉(zhuǎn)化[4]。

當(dāng)HPV DNA整合入宿主基因后，HPV L1殼蛋白則表達(dá)缺失，機(jī)體將無(wú)法識(shí)別和清除病變細(xì)胞。HPV L1的表達(dá)有利于激發(fā)人體細(xì)胞免疫，有效清除被感染的細(xì)胞，一般有HPV L1表達(dá)的腫瘤組織通常預(yù)后較好[5]。HPV感染所致腫瘤是否有L1表達(dá)，除了與腫瘤組織的分化狀態(tài)有關(guān)，也與HPV病毒基因組整合至宿主基因組的位點(diǎn)有關(guān)[6]。當(dāng)HPV基因組插入位點(diǎn)是宿主基因組的可轉(zhuǎn)錄序列時(shí)，HPV L1基因就可能被轉(zhuǎn)錄，并經(jīng)恰當(dāng)?shù)募羟校筁1蛋白得到表達(dá)。實(shí)驗(yàn)證實(shí)了HepG2細(xì)胞可表達(dá)HPV L1蛋白，包涵體樣物質(zhì)即由HPV L1蛋白組成。HPV L1作為具有病毒抗原特性的蛋白在對(duì)HPV檢測(cè)、預(yù)防甚至治療中都占有舉足輕重的作用，這將為臨床肝癌的診治提供新的思考。

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[2] 李慧弘,肖長(zhǎng)義,袁太寧，等. 宮頸癌HeLa細(xì)胞內(nèi)HepG2 蛋白表達(dá)的觀察[J]. 中國(guó)免疫學(xué)雜志, 2013, 29:260- 264.

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The physical states of HPV DNA and expression of human papillomavirus late capsid protein 1 in HepG2 cells

DU Yi-jiang1,2,3, XIAO Chang-yi1,2*, ZHENG Jun3, HU Min2

(1.Dept. of Histology and Embryology, Medicine College of China Three Gorges University, Yichang 443002; 2.Central Laboratory, the First College of Clinical Medicine Science, China Three Gorges University, Yichang 443003; 3.General Surgery,the First College of Clinical Medicine Science, China Three Gorges University, Yichang 443003, China)

Objective To find out the physical state of the human papillomavirus (HPV) genome in hepatoma cell line HepG2 cells and the regulation of HPV late capsid protein 1 (L1) expression and to explore the nature of the cytoryctes in HepG2 cells. MethodsE2 andE6 in HPV18 were detected by PCR to evaluate the physical state of HPV18 genome. HepG2 L1 expression was detected by ELISA, light microscropy and electron microscrope immunohistochemistry assays, Western blot assay using HPV L1 mice monoclonal antibody. L1 mRNA in HepG2 cells was detected by reverse transcriptional PCR (RT-PCR). Results PCR assay displayed that HPV DNA was integrated with HepG2 genome. ELISA assay showed that HPV L1 was present in lysate of HepG2 cells. Light microscropy demonstrated strong positive reaction in HepG2 cells. In microscopy, in the cytoplasm of partial HepG2 cells, there were lumpish cytorrhyctes materials which consists of very small and uniform particles and these parti-

cles were marked by HPV L1 antibody labeled by colloidal gold. Western blot analysis showed a band at 56 ku district and it was L1 specific strap which demonstrated HPV18 L1 was present in HepG2 cells. RT-PCR assay demonstrated the presence of L1 mRNA in HepG2 cells. Conclusions HepG2 cells are HPV18-positive HPV DNA genome is integrated with HepG2 cells. HepG2 cells can express L1. The cytorrhyctes in HepG2 cells are composed of HPV18 L1 indicating that L1 can be expressed in HepG2.

HepG2 cells; human papillomavirus; virus genome; physical state; late capsid protein 1

2014- 09- 25

2014- 11- 19

湖北省自然科學(xué)基金(2011CDC002)

1001-6325(2015)01-0060-05

R730.21; R735.7; R361.3

A

*通信作者(corresponding author)：xiaochy@ctgu.edu.cn