盧莎，譚文杰，張玲，鄧瑤，陶格斯，蔡敏，李戀，沈曉玲

(1內(nèi)蒙古醫(yī)科大學(xué)，呼和浩特010110；2中國(guó)疾病預(yù)防控制中心病毒病預(yù)防控制所)

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脂質(zhì)體轉(zhuǎn)染法與電轉(zhuǎn)染法在丙型肝炎病毒RNA轉(zhuǎn)染人肝癌細(xì)胞中的應(yīng)用效果比較

盧莎1,2，譚文杰2，張玲2，鄧瑤2，陶格斯1,2，蔡敏1，李戀1，沈曉玲1

(1內(nèi)蒙古醫(yī)科大學(xué)，呼和浩特010110；2中國(guó)疾病預(yù)防控制中心病毒病預(yù)防控制所)

目的比較脂質(zhì)體轉(zhuǎn)染法與電轉(zhuǎn)染法在丙型肝炎病毒(HCV)RNA轉(zhuǎn)染人肝癌細(xì)胞中的應(yīng)用效果。方法培養(yǎng)人肝癌細(xì)胞Huh7.5-CD81并分為A、B組及對(duì)照組。A組采用電轉(zhuǎn)染質(zhì)粒pHebei(E1E2)/JFH1來(lái)源的HCV RNA；B組采用脂質(zhì)體轉(zhuǎn)染質(zhì)粒pHebei(E1E2)/JFH1來(lái)源的HCV RNA；對(duì)照組采用脂質(zhì)體轉(zhuǎn)染復(fù)制缺陷質(zhì)粒pJFH1/GND轉(zhuǎn)錄獲得的HCV RNA。光學(xué)顯微鏡下觀察各組轉(zhuǎn)染后細(xì)胞形態(tài)，采用免疫熒光法(IFA)檢測(cè)三組轉(zhuǎn)染效率，real-time PCR法檢測(cè)三組細(xì)胞培養(yǎng)液上清中的HCV RNA，采用TCID50法檢測(cè)A、B組細(xì)胞培養(yǎng)液上清中的丙型肝炎病毒(HCVcc)感染滴度。結(jié)果A組轉(zhuǎn)染使用細(xì)胞數(shù)為1×106，轉(zhuǎn)染后第2天細(xì)胞損傷過(guò)半，邊緣毛糙；B組轉(zhuǎn)染使用細(xì)胞數(shù)為2×105，轉(zhuǎn)染后第2天細(xì)胞基本無(wú)損傷。A組轉(zhuǎn)染效率為9.98%±0.83%，B組轉(zhuǎn)染效率為2.58%±0.39%，兩組轉(zhuǎn)染效率相比，P<0.01；對(duì)照組未檢出熒光陽(yáng)性細(xì)胞。A、B組轉(zhuǎn)染后細(xì)胞培養(yǎng)液上清中HCV RNA拷貝數(shù)均呈現(xiàn)先下降后升高的趨勢(shì)，最終穩(wěn)定于106copies/mL。A組轉(zhuǎn)染后21 d、B組轉(zhuǎn)染后31 d方能檢測(cè)到HCV感染滴度，A、B組收獲HCV最高感染滴度均為104ffu/mL。結(jié)論脂質(zhì)體轉(zhuǎn)染和電轉(zhuǎn)染兩種方法均可建立嵌合HCV的人肝癌細(xì)胞培養(yǎng)體系，脂質(zhì)體轉(zhuǎn)染法對(duì)細(xì)胞的損傷較小，電轉(zhuǎn)染法轉(zhuǎn)染效率較高、收獲HCVcc的時(shí)間較短，但兩種方法收獲的HCVcc感染滴度無(wú)明顯差異。

丙型肝炎病毒；電穿孔；脂質(zhì)體；RNA轉(zhuǎn)染

丙型肝炎病毒(HCV)可引起人類丙型肝炎。最新的靶向HCV蛋白抑制劑療效較好，但價(jià)格昂貴，不良反應(yīng)較多，因此針對(duì)HCV的疫苗及抗病毒藥物仍在探索中[1～4]。HCV體外細(xì)胞培養(yǎng)系統(tǒng)是重要的研究工具[5～7]，其構(gòu)建策略包括建立全基因或亞基因組HCV感染性克隆、應(yīng)用電轉(zhuǎn)染或脂質(zhì)體轉(zhuǎn)染技術(shù)將HCV RNA或DNA轉(zhuǎn)移到容納細(xì)胞內(nèi)。電轉(zhuǎn)染或脂質(zhì)體轉(zhuǎn)染同屬經(jīng)典方法，各有優(yōu)勢(shì)[8～13]。電轉(zhuǎn)染效率高(20%～30%)但對(duì)細(xì)胞損傷大，脂質(zhì)體轉(zhuǎn)染效率在10%左右但對(duì)細(xì)胞損傷小，利于傳代培養(yǎng)。我們前期實(shí)驗(yàn)構(gòu)建了含有中國(guó)分離株包膜蛋白基因的嵌合HCV全長(zhǎng)質(zhì)粒[14]，體外轉(zhuǎn)錄獲得病毒RNA，同時(shí)應(yīng)用脂質(zhì)體轉(zhuǎn)染法和電轉(zhuǎn)染法，觀察并比較兩種方法建立嵌合HCV人肝癌細(xì)胞培養(yǎng)體系的效果。

1　材料與方法

1.1質(zhì)粒、細(xì)胞及其他實(shí)驗(yàn)材料質(zhì)粒pHebei(E1E2)/JFH1、復(fù)制缺陷質(zhì)粒pJFH1/GND由本實(shí)驗(yàn)室保存(源自Wakita T教授)。人肝癌細(xì)胞Huh7.5-CD81為本實(shí)驗(yàn)室保存。所有培養(yǎng)液均購(gòu)自GIBCO公司。MEGAscript T7 Kit、轉(zhuǎn)染試劑DMRIE-C購(gòu)自Invitrogen公司。鼠抗HCV Core單抗購(gòu)自ABR公司。FITC標(biāo)記的羊抗鼠多抗購(gòu)自中杉金橋生物有限公司。紅外熒光標(biāo)記的羊抗鼠多抗購(gòu)自Rockland公司。

1.2細(xì)胞分組與質(zhì)粒轉(zhuǎn)染方法將嵌合中國(guó)分離株包膜蛋白基因的HCV質(zhì)粒pHebei(E1E2)/JFH1經(jīng)體外轉(zhuǎn)錄，獲得嵌合病毒全長(zhǎng)RNA。將Huh7.5-CD81細(xì)胞分為A、B組及對(duì)照組。A組采用電轉(zhuǎn)染法：25T細(xì)胞培養(yǎng)皿接種細(xì)胞，用不含抗生素、含10%胎牛血清的DMEM培養(yǎng)；次日，待細(xì)胞生長(zhǎng)至70%～80%融合成片時(shí)，提取5 μg pHebei(E1E2)/JFH1的RNA；轉(zhuǎn)染前用0.25%胰酶消化細(xì)胞，預(yù)冷的cytomix電轉(zhuǎn)緩沖液清洗細(xì)胞2次，500 r/min離心5 min后收集細(xì)胞；加入200 μL緩沖液重懸細(xì)胞，移至0.4 mm的預(yù)冷電轉(zhuǎn)杯中，電轉(zhuǎn)條件為135 V、970 μF；電轉(zhuǎn)結(jié)束，將電轉(zhuǎn)杯中的細(xì)胞迅速轉(zhuǎn)入準(zhǔn)備好的25T細(xì)胞培養(yǎng)瓶中，37 ℃、5% CO2培養(yǎng)過(guò)夜，次日去除漂浮死細(xì)胞，更換新鮮培養(yǎng)基。B組采用脂質(zhì)體轉(zhuǎn)染法[15]：在6孔板中接種Huh7.5-CD81細(xì)胞(1×105/孔)，用不含抗生素、含10%胎牛血清的DMEM培養(yǎng)；次日，待細(xì)胞生長(zhǎng)到70%～80%融合成片時(shí)，用脂質(zhì)體DMRIE-C(Invitroge公司)作轉(zhuǎn)染試劑，依照廠商提供的方法轉(zhuǎn)染5 μg pHebei(E1E2)/JFH1的RNA。對(duì)照組經(jīng)脂質(zhì)體轉(zhuǎn)染5 μg pJFH1/GND的RNA。

1.3轉(zhuǎn)染后細(xì)胞形態(tài)觀察于光學(xué)顯微鏡下觀察細(xì)胞形態(tài)、邊緣、透明度、折光性等特點(diǎn)。

1.4轉(zhuǎn)染效率測(cè)算A、B組及對(duì)照組分別于轉(zhuǎn)染后3 d或4 d及每次傳代后48 h采用免疫熒光法(IFA)檢測(cè)病毒Core蛋白，在熒光顯微鏡下觀察并計(jì)數(shù)熒光陽(yáng)性細(xì)胞，計(jì)算轉(zhuǎn)染效率。

1.5培養(yǎng)液上清中HCV RNA檢測(cè)轉(zhuǎn)染后細(xì)胞每次傳代時(shí)收集培養(yǎng)液上清，用TRIzol試劑提取細(xì)胞培養(yǎng)液上清中的RNA，采用real-time PCR法檢測(cè)HCV mRNA。

1.6A、B組細(xì)胞培養(yǎng)液上清中HCV感染滴度檢測(cè)將A、B組細(xì)胞以1×103/孔接種于96孔板，第2天對(duì)細(xì)胞培養(yǎng)液上清(待測(cè)病毒液)進(jìn)行10倍比稀釋，最高稀釋度定為106，每個(gè)稀釋度設(shè)6個(gè)復(fù)孔；每孔加入培養(yǎng)液上清100 μL，37 ℃孵育5 h；用PBS清洗細(xì)胞3次，然后加入100 μL DMEM完全培養(yǎng)基繼續(xù)培養(yǎng)；細(xì)胞長(zhǎng)滿后行IFA檢測(cè)，觀察感染病灶(foci)，以半數(shù)復(fù)孔出現(xiàn)foci的最高稀釋度計(jì)算HCV感染滴度。

2　結(jié)果

2.1HCV RNA轉(zhuǎn)染后細(xì)胞狀態(tài)比較A組轉(zhuǎn)染使用細(xì)胞數(shù)為1×106，轉(zhuǎn)染后第2天細(xì)胞損傷過(guò)半，細(xì)胞50%融合成片，邊緣毛糙；B組轉(zhuǎn)染使用細(xì)胞數(shù)為2×105，轉(zhuǎn)染后第2天細(xì)胞基本無(wú)損傷，80%融合成片。詳見(jiàn)圖1。

注：1為A組電轉(zhuǎn)染后細(xì)胞，2為B組脂質(zhì)體轉(zhuǎn)染后細(xì)胞，3為對(duì)照組細(xì)胞。

圖1HCV RNA轉(zhuǎn)染后細(xì)胞形態(tài)(100×)

2.2各組轉(zhuǎn)染效率比較A組轉(zhuǎn)染后4 d轉(zhuǎn)染效率為9.98%±0.83%，25 d后熒光陽(yáng)性細(xì)胞百分比為80%～90%；B組轉(zhuǎn)染后3 d轉(zhuǎn)染效率為2.58%±0.39%，36 d后熒光陽(yáng)性細(xì)胞百分比為80%～90%；兩組轉(zhuǎn)染效率相比，P<0.01。對(duì)照組未檢出熒光陽(yáng)性細(xì)胞。

2.3各組細(xì)胞培養(yǎng)液上清中HCV RNA水平比較A、B組轉(zhuǎn)染后4 d細(xì)胞培養(yǎng)液上清中HCV RNA拷貝數(shù)開(kāi)始降低，于轉(zhuǎn)染后14～19 d降到最低，而后逐漸升高，最終均維持在106copies/mL，A、B組差異無(wú)統(tǒng)計(jì)學(xué)意義；對(duì)照組轉(zhuǎn)染6 d后HCV RNA拷貝數(shù)降到103以下，10 d后未檢出。見(jiàn)圖2。

圖2　各組轉(zhuǎn)染后不同時(shí)間細(xì)胞培養(yǎng)液上清中HCV RNA水平比較

2.4A、B組細(xì)胞培養(yǎng)液上清中HCV感染滴度比較受檢測(cè)方法靈敏度影響，A組轉(zhuǎn)染后21 d、B組轉(zhuǎn)染后31 d方能檢測(cè)到HCV感染滴度。隨時(shí)間延長(zhǎng)，病毒滴度逐漸增高并趨于穩(wěn)定。A、B組轉(zhuǎn)染后收獲最高HCV感染滴度均為104ffu/mL，兩組相比，P>0.05。見(jiàn)圖3。

圖3　A、B組轉(zhuǎn)染后不同時(shí)間細(xì)胞培養(yǎng)液上清中HCV感染滴度比較

3　討論

脂質(zhì)體轉(zhuǎn)染法的原理是通過(guò)轉(zhuǎn)染試劑提供脂質(zhì)雙分子層包裹外源基因形成囊狀小泡，繼而與細(xì)胞膜融合，完成外源基因?qū)?。該法操作?jiǎn)單、細(xì)胞毒性小，但轉(zhuǎn)染效率受細(xì)胞類型及外源基因性質(zhì)影響較大。電轉(zhuǎn)染法利用儀器瞬時(shí)產(chǎn)生高強(qiáng)度電場(chǎng)從而改變細(xì)胞膜完整性和通透性，吸收周圍介質(zhì)中的外源性分子進(jìn)入細(xì)胞內(nèi)，具有重復(fù)性好、安全性高、適用性廣的優(yōu)勢(shì)，但對(duì)細(xì)胞損傷較大[16]。與脂質(zhì)體轉(zhuǎn)染法相比，電轉(zhuǎn)染法因使用瞬間高電壓刺激可能使轉(zhuǎn)染后細(xì)胞大量死亡。已有研究[17]發(fā)現(xiàn)，細(xì)胞存活率達(dá)50%時(shí)的電場(chǎng)參數(shù)可獲得最佳轉(zhuǎn)染效率。

本研究分別采用脂質(zhì)體轉(zhuǎn)染和電轉(zhuǎn)染兩種方法將嵌合HCV全基因RNA導(dǎo)入Huh7.5-CD81，建立了體外細(xì)胞培養(yǎng)模型。本研究結(jié)果顯示，A組轉(zhuǎn)染效率為9.98%±0.83%，B組轉(zhuǎn)染效率為2.58%±0.39%，A組轉(zhuǎn)染效率高于B組；對(duì)照組中HCV不能復(fù)制因此沒(méi)有HCV Core蛋白產(chǎn)生，故未檢出熒光陽(yáng)性細(xì)胞。我們對(duì)三組細(xì)胞培養(yǎng)液上清中的HCV RNA進(jìn)行檢測(cè)，發(fā)現(xiàn)A、B組HCV RNA拷貝數(shù)因本底R(shí)NA的影響呈現(xiàn)先下降后升高的趨勢(shì)，最終穩(wěn)定在106copies/mL；對(duì)照組轉(zhuǎn)染6 d后HCV RNA拷貝數(shù)降到本底以下，10 d后未檢出。A、B兩組最終收獲的HCVcc感染滴度相似，均為104ffu/mL，可見(jiàn)，轉(zhuǎn)染效率并非感染滴度的決定因素。

電轉(zhuǎn)染法轉(zhuǎn)染效率高，可使收獲HCVcc的時(shí)間縮短，但其使用細(xì)胞量大，需損失50%甚至更多的細(xì)胞方能保證轉(zhuǎn)染效率，且需多次摸索電壓、電流、脈沖時(shí)間等轉(zhuǎn)染條件，本實(shí)驗(yàn)中A組的電轉(zhuǎn)染參數(shù)最終確定為135 V、970 μF、10 ms。對(duì)貼壁細(xì)胞進(jìn)行電轉(zhuǎn)染操作程序復(fù)雜，需多次轉(zhuǎn)移細(xì)胞，污染風(fēng)險(xiǎn)增大，轉(zhuǎn)染后細(xì)胞損傷明顯，不利于后期連續(xù)傳代培養(yǎng)。而脂質(zhì)體轉(zhuǎn)染法細(xì)胞用量小，轉(zhuǎn)染后細(xì)胞損傷小，操作簡(jiǎn)單，雖然轉(zhuǎn)染效率較低，但對(duì)于同一HCV毒株，最終收獲的HCVcc感染滴度與電轉(zhuǎn)染法相比并無(wú)明顯差異。

總之，脂質(zhì)體轉(zhuǎn)染和電轉(zhuǎn)染兩種方法均可建立嵌合HCV的人肝癌細(xì)胞培養(yǎng)體系，脂質(zhì)體轉(zhuǎn)染法對(duì)細(xì)胞損傷較小，電轉(zhuǎn)染法轉(zhuǎn)染效率較高、收獲HCVcc的時(shí)間縮短，但兩種方法最終得到的HCVcc感染滴度無(wú)明顯差異。

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Application effects of electroporation and lipofection in HCV RNA transfecting human hepatoma cells

LU Sha1,TAN Wenjie,ZHANG Ling,DENG Yao,TAO Gesi,CAI Min,LI Lian,SHEN Xiaoling

(1 Inner Mongolia Medical University,Hohhot 010059,China)

ObjectiveTo observe the application effects of electroporation and lipofection in hepatitis C virus (HCV) RNA transfecting human hepatoma cells.MethodsHuman hepatoma cells (Huh7.5-CD81) were cultured and divided into groups A,B and control group.Cells in the group A were transfected with HCV RNA of pHebei (E1E2)/JFH1 by electroporation,cells in the group B were transfected with HCV RNA of pHebei (E1E2)/JFH1 by lipofection,and cells in the control group were transfected with HCV RNA of replicating defective pJFH1/GND.Cell morphology was observed using an optical microscope in each group after transfection.Immunofluorescence was employed to detect the transfectional efficiency.RNA copies in the supernatant were tested by real-time PCR.TCID50 was applied to detect HCV infectious titer.ResultsThe number of cells used in the group A were 1×106,and more than half were damaged with rough edges one day after transfection; the number of cells in the group B were 2×105and almost no damage was found one day after transfection.Transfectional efficiency of group A was 9.98%±0.83%,and that was 2.58%±0.39% in the group B,P<0.01.Fluorescence positive cells in the control group were not detected.Meantime,HCV RNA copy number in the supernatant of groups A and B first decreased,then increased and eventually reached at 106copies/mL.HCV infection titer was detected successfully at 21days after transfection in the group A and at 31days in the group B.The highest infectious titer of groups A and B was both 104ffu/mL.ConclusionsElectroporation and lipofection both can be used to establish HCV cell culture system.Lipofection displays less damage to cells.Although electroporation demonstrates higher transfectional efficiency and short time of obtaining HCVcc,but there is no difference in infectious titer of obtained HCVcc.

hepatitis C virus; electroporation; liposomes; RNA transfection

國(guó)家自然科學(xué)基金資助項(xiàng)目(81373229)；傳染病科技重大專項(xiàng)(2013ZX10004601，2013ZX10004609)；內(nèi)蒙古醫(yī)科大學(xué)科技百萬(wàn)項(xiàng)目(NY2011BW003)。

盧莎(1980-)，女，碩士，講師，主要研究方向?yàn)獒t(yī)學(xué)微生物學(xué)。E-mail：huder@sina.com

簡(jiǎn)介：沈曉玲(1964-),女，教授，主要研究方向?yàn)槊庖邔W(xué)與分子病毒學(xué)。E-mail: shenxl1@aliyun.com

10.3969/j.issn.1002-266X.2016.19.005

R373.9

A

1002-266X(2016)19-0015-04

2015-12-18)