不同前列腺癌细胞高效启动子筛选和双萤光素酶慢病毒载体的构建
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摘要
研究背景
前列腺癌是成年男性最常见的恶性肿瘤之一,在美国,其发病率据男性恶性肿瘤首位,每年死于前列腺癌的男性在因肿瘤而死亡的男性中排第二位。前列腺癌因其部位深在,症状出现较晚,因而很多患者发现时均已是晚期,失去了手术治疗的机会。目前,对晚期特别是雄激素非依赖型前列腺癌尚缺乏有效的治疗方法。基因治疗(gene therapy)是指把目的基因导入靶细胞和宿主体内,通过目的基因在宿主中的表达,纠正错误的基因表达和基因表达的错误,以达到治疗目的。近10多年来,随着分子生物学技术和理论的飞速发展,使基因治疗这一新型治疗方法成为可能,并在对单基因缺陷性遗传疾病的治疗中取得了令人瞩目的成就。肿瘤的基因治疗研究也成为目前肿瘤防治研究的一个活跃领域。肿瘤基因疗法具有下列优越性：①肿瘤的选择性比放疗、化疗更强,这可通过特异性基因转导技术或特异基因的定向作用而获得；②对患瘤个体损伤小而轻；③对晚期肿瘤或转移瘤灶仍有效。然而,基因治疗在临床应用上仍有许多有待解决的难点。主要有：①缺乏对靶细胞定向导入基因的技术；②病毒载体有抗原性,会引起机体对它产生抗体,故不能长期应用；③导入基因的表达和调控问题。
纵观将外源基因导入并整合到宿主细胞的整个过程,将目的基因导入靶细胞并使之持久稳定表达是关键环节,因此介导的载体选择便显得格外有意义。目前基因治疗中所用的载体可分为两大类：非病毒载体和病毒载体。非病毒载体包括基因片段和质粒载体。而应用最广泛的是病毒载体,约85%的基因治疗临床项目采用的均是这种载体。其中慢病毒载体因其独特的生物学性状显示出在基因治疗中的巨大潜力：①慢病毒载体转导的细胞类型不受限制,既能转导分裂细胞又能转导非分裂细胞；②能够使目的基因高效的整合到宿主细胞基因组内实现长期而稳定的表达；③不会引起宿主细胞及宿主本身的免疫反应；④已经去除了原病毒的可致病区,消除了致病性。
“导入基因的表达和调控”就是使目的基因特异性的在癌细胞中高效表达从而起到治疗作用,而在正常组织细胞中不表达或几乎不表达而避免伤害正常组织。这就需要一个高效、肿瘤特异性的转录激活因子既启动子来完成这一任务。启动子是决定目的基因表达效率的关键环节。大量前期研究已经证明,同一载体同一细胞系中,如果启动子不同,则目的基因表达效率不同。而同一启动子携带目的基因相同,在不同细胞系中,表达效率也不同。因此,针对不同的肿瘤细胞,选择特异性强、高效表达的启动子作为慢病毒载体中插入基因的启动子,对于提高肿瘤基因治疗的效率,提高其靶向性和特异性具有重大意义。
在针对基因治疗的效果进行评价的细胞和动物模型中,尚缺乏易于在体外动态监测肿瘤细胞生长和转移及代谢情况的特异性标记手段,这严重影响了对基因治疗效果的评价。因此,寻找一种特异性强且不会影响肿瘤生长的标记物标记肿瘤细胞,以便从体外监测肿瘤的生长、转移等,对评价肿瘤的基因治疗效果有着重要的意义。
萤光素酶(luciferase)是自然界中能够催化底物产生生物荧光的酶的统称,在哺乳动物体内无内源性表达,检测不受细胞内其他物质影响,并且具有敏感性高,特异性好,反应迅速,操作简单,检测范围广等优点,作为报告基因在医学、生物学、环境科学等领域研究已得到广泛应用,其中最有代表性的是萤火虫萤光素酶(Firefly luciferase, Fluc)。
早在1956年,Green和MeElroy就得到了萤火虫萤光素酶(Lueiferase)的结晶。萤火虫萤光素酶是分子量为60-64kD的多肽链在Mg2+、ATP、O2存在时,催化D-萤光素(D-Luciferin)氧化脱羧,产生波峰在560nnm左右的荧光。只有在活细胞内才会产生发光现象,并且光的强度与标记细胞的数目成正比。作为经典的萤光素酶,已经在细胞内ATP水平探测、感染检测、药物筛选、细胞标记和示踪以及动物活体成像等方面都得到了广泛的应用。目前应用最多的活体光学成像动物模型是表达Fluc的动物模型。
Gaussia luciferase(Gluc)是近年来发现的一种新型萤光素酶,来源于海洋桡脚类动物(Gaussia princeps),其显著特点是可以高效分泌至细胞外,同时具有更高的检测灵敏度。Gluc与底物腔肠素的反应不依赖于ATP,结合后发出的荧光波峰在480nm左右。由于Gluc具有分子量小、易分泌、检测灵敏度高、半衰期短等优点,已成功应用于体内外的实验研究中。通过检测分泌到模型动物血液或其他体液中的Gluc活性,可以定量反映动物体内肿瘤的负荷,不仅能追踪肿瘤转移进展,而且能作为有效标记物体现肿瘤对治疗的反应。并且萤光素酶与特定底物的发光反应属于化学发光,具有特异性,不存在交叉干扰现象。
基于上述特点,为了进一步研究分泌性萤光色素酶Gluc和非分泌性萤光色素酶Fluc在细胞及动物体内的表达特性,并结合二者与慢病毒载体在外源基因导入方面的优点,本研究拟构建一种Gluc和Fluc双萤光色素酶共表达的慢病毒基因载体,并对其体内外表达特点进行研究,进而建立双萤光素酶基因标记的肿瘤细胞系和动物模型、为下一步建立一系列双萤光素酶标记的泌尿系肿瘤细胞系并对其进行体内外监测和示踪研究奠定基础。
随着分子生物学技术的迅猛发展,特别是近年来转基因技术的巨大进步,重组腺病毒载体在目的基因的导入、基因的过表达方面已获得广泛的应用。并由于其转基因效率高、转导细胞类型广泛、易于制备和纯化,且进入宿主细胞后仅瞬时表达,不整合到宿主细胞基因组等优点,近年来在基因治疗、基因免疫以及疫苗制备方面的地位越来越突出。现已成功应用于视网膜母细胞瘤、肝细胞癌、前列腺癌的基因治疗并已进入临床试验阶段。在腺病毒应用的过程中,一个十分重要的步骤是测定病毒的滴度。目前测定腺病毒滴度的方法有多种,如壳蛋白免疫法、荧光定量PCR法、OD260法、细胞病变(CPE)法和空斑法,对于带有GFP标签的腺病毒载体还有绿色荧光蛋白(GFP)标记法。其中以壳蛋白免疫法最为常用,但该法步骤繁琐,且试剂成本高,不利于普及推广。现我们以壳蛋白免疫法为标准测定方法,将上述其他几种方法与其进行比较,对测定结果进行分析,探讨各种方法的优缺点,并进一步讨论各种方法的意义及可行性。
全文分三大部分,分别论述前列腺癌不同启动子的效果差异的比较研究和双萤光素酶标记的慢病毒载体的构建及其表达特性研究,以及不同测定腺病毒滴度方法的比较。
(一)不同启动子慢病毒载体在前列腺癌细胞表达效果差异的比较研究
1.研究目的以增强型绿色荧光蛋白(EGFP)作为报告基因,比较UBIQUITIN, EF1α和CMV三种不同启动子的慢病毒载体在293A, MOLT4, PC3, DU145和RM1三种不同细胞株的表达效率,筛选出每种细胞株的高效启动子,为在今后的研究中,针对不同细胞株,选择恰当的启动子奠定基础。
2.研究方法
2.1不同启动子载体的构建和鉴定
以限制性内切酶将IRES编码区自PTY-EF1α-IRES-EGFP上切下,再连接形成PTY-EF1α-EGFP (EF1α-EGFP)慢病毒载体。PTY-CMV-EGFP (CMV-EGFP)载体是在此基础上将EF1α编码区替换为CMV编码区。
2.2不同启动子慢病毒载体的包装和滴度测定
293T细胞于含10%胎牛血清、1%的青链霉素、1%的谷氨酰胺DMEM培养基中,37℃5%CO2培养箱培养。细胞均匀铺于T25培养瓶中,24h后观察细胞分裂活跃时,将PMD2G、PPAX2以及所构建的含不同启动子的穿梭质粒共转染293T细胞。转染步骤按照lipofectamineTM 2000说明书步骤进行。60小时后收获病毒并以超速离心法纯化。病毒液-80℃保存。荧光定量法检测病毒拷贝数在Agilent3500荧光定量PCR仪上进行。病毒液进行去RNA酶处理后按照试剂盒步骤要求提取病毒核酸,质粒标准品PTY-EF1a-EGFP浓度为1.09μg/μl,换算成核酸拷贝数为1.06×1014 copies/ml 10倍倍比稀释为7个浓度梯度,同时设一个阴性对照,和待测cDNA样品同时进行扩增并检测荧光信号。依据标准品所生成的标准曲线,将所测得cDNA CT值与之相比较,可得该病毒液中病毒基因组的拷贝数,进而换算得病毒的滴度。
2.3细胞的病毒转导
293A,PC3,DU145,RM1细胞均以含10%胎牛血清的DMEM培养基培养,MOLT4细胞以含10%新生牛血清的1640培养基培养,铺于6孔板中,每种细胞4个复孔。细胞铺满6孔板板孔底面积90%分别加入5×108copy的LV-UBIQUITIN-EGFP, LV-EF1α-EGFP, LV-CMV-EGFP慢病毒病毒液或者DEPCH20(作为对照)200W。继续培养72h。
2.4倒置荧光显微镜下比较荧光强度
倒置荧光显微镜下观察细胞荧光强度。每种细胞均随机观察3个视野。比较同一细胞不同启动子的效果差异。所有照片均按照10×物镜、10×目镜放大倍数拍摄,曝光时间相同。
2.5流式细胞计数差异
将6孔板中细胞以胰酶消化后,1000rpm/min离心3min,弃上清,以500μl PBS缓冲液重悬细胞,用移液枪充分吹打混匀。FACS Calibur流式细胞计数仪计数GFP阳性细胞数。仪器自动分析结果。
2.6总RNA的提取及荧光定量检测
取流式细胞计数后的细胞悬液,按照QIAGEN RNeasy Mini Kit步骤要求提取细胞中总RNA。将提取的总RNA应用PrimeScriptTM RT reagent Kit进行逆转录,获得cDNA。分别取逆转录所得cDNA各2u1,按照SYBR(?) Premix Ex Taq荧光定量试剂盒步骤要求,配置反应体系。每个细胞每种启动子设置3个复孔,同时设立等量的18s RNA复孔作为内参,及3个复孔的阴性对照,阴性对照中cDNA样品以DEPC处理水代替,总体系大小不变。Agilent3005型荧光定量PCR检测仪进行目的片段扩增和荧光信号检测,反应条件为：第一步95℃30s,第二步95℃5s,60℃30 s,共40个循环。反应过程中自动收集荧光。
3.研究结果
3.1慢病毒载体的鉴定
PTY-EF1α-EGFP质粒载体构建成功,琼脂糖凝胶电泳显示其大于符合预期,且Mlu和SmaⅠ限制性双酶切已不能再次将其切开。PTY-UBIQUITIN-EGFP, PTY-EF1α-EGFP, PTY-CMV-EGFP含三种不同启动子的慢病毒载体除启动子部分不同外,其余部分均一致。
3.2病毒滴度测定
荧光定量反应结束后,MX3005型荧光定量PCR仪自动输出标准品cT值,并据此输出标准曲线。从标准曲线图中可以看出,各梯度稀释倍数的质粒标准品大致位于同一直线上,样品稀释梯度明显,标准曲线准确。将样品cT值与标准曲线相比较,可知各组病毒液中基因组的拷贝数。依公式：病毒基因组拷贝数/毫升(vg/ml)=(病毒核酸拷贝数/2μl)×(20(μl/2μl)×(50μl/5μl)×1ml/200μl×稀释度计算,所得结果即为各种慢病毒滴度：LV-UBIQUITIN-EGFP 5.37×109 copies/ml, LV-EF1α-EGFP 3.89×109 copies/ml, LV-CMV-EGFP 4.20×109 copies /ml。
3.3倒置荧光显微镜下293A,MOLT4,PC3,DU145,RM1在不同慢病毒转导后的荧光强度的比较
293A,MOLT4,PC3,DU 145,RM 1各细胞分别在接受LV-UBIQUITIN-EGFP, LV-EF1α-EGFP, LV-CMV-EGFP转导72小时后拍摄荧光照片的结果显示,在293A细胞中,CMV启动子介导的目的基因(EGFP)表达,明显强于UBIQUITIN启动子和EF1α启动子；在PC3细胞中,也可以观察到相同的现象。而在MOLT4细胞中,EF1α启动子的表达效率则明显高于UBIQUITIN启动子和CMV启动子。同样的,在DU145细胞中,EF1α启动子的效率也是最高的,但是该细胞株中,UBIQUITIN启动子的效率虽然低于EF1α启动子,却明显高于CMV启动子。而在RM1细胞中,三种启动子表达均不明显,但其中EF1α启动子要强于UBIQUITIN启动子和CMV启动子。
3.4不同启动子转导细胞表达效率的流式细胞计数比较
CMV启动子在293A和PC3细胞中介导的目的基因表达效率是最高的,而EF1a启动子则在MOLT4和DU-145细胞中,介导了目的基因的高效表达,效果强于其它两种启动子。在RM1细胞中,三种启动子介导目的基因表达的效率均不高,但是EF1α启动子介导的外源基因表达(平均荧光强度)要强于其它两种启动子。其结果与荧光显微镜下观察到的趋势一致。
3.5 Realtime PCR法比较编码mRNA表达量差异
在293A和PC3细胞中,LV-CMV-EGFP转导后GFP基因编码mRNA的表达量高于LV-UBIQUITIN-EGFP和LV-EF1α-EGFP转导后GFP基因编码mRNA的表达量。在MOLT4, DU145和RM1细胞中,则是LV-EF1α-EGFP转导后GFP基因编码mRNA的表达量最高。这些都与荧光显微镜观察到的结果和流式细胞计数的结果相一致。
4.讨论
慢病毒转导是效率最高的转导和表达外源基因的方法之一。但慢病毒核酸结构的差异性可以极大的影响到外源基因表达的效率。在本研究中,我们分别构建了LV-UBIQUITIN-EGFP, LV-EF1α-EGFP, LV-CMV-EGFP三种不同启动子的慢病毒载体,并转导了293A, MOLT4, PC3, DU145和RM1五种不同的细胞。比较了三种启动子在同一细胞系中效率的差异。研究结果显示：三种启动子在各细胞株中的表达效率,由强到弱,依次为：293A:CMV, UBIQUITIN, EF1α, MOLT:EF1α, UBIQUITIN, CMV, PC3:CMV, EF1α, UBIQUITIN, DU145: EF1α, UBIQUITIN, CMV, RM1细胞：EF1α, UBIQUITIN, CMV。本研究的完成为我们在将来的科研中,针对不同的细胞系,有针对性的选择高效率启动子奠定了基础。
(二)双萤光素酶标记的慢病毒载体的构建及其表达特性研究
1.研究目的
构建一种分泌型萤光素酶Gluc和非分泌型萤光素酶Fluc共表达的双报告基因慢病毒载体,并建立稳定表达的前列腺癌PC3细胞系,以此为基础建立前列腺癌皮下肿瘤模型,对其体内外表达及活体成像特点进行研究。
2.研究方法
2.1 PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc双萤光素酶共表达质粒载体的构建及鉴定
采用分子克隆技术从pAAV2neoCAG-Gluc-2A-Fluc质粒中切取CAG-Gluc-2A-Fluc片段,并插入到去掉EGFP编码基因片段的PTY-UBIQUITIN-EGFP质粒载体中。再从pTR-UF11质粒上切下GFP-HSV-Tk-neo片段,定向克隆到新构建的载体中,酶切鉴定连接方向,取正向连接的质粒载体,转化大肠杆菌DH5α,挑取氨苄青霉素抗性单菌落提取质粒,双酶切鉴定。
2.2 PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒的细胞内瞬时表达检测
293T细胞在含10%胎牛血清的DMEM培养基的24孔板中,置于37℃,5%CO2的培养箱中进行贴壁培养。转染前一天接种细胞,待293T细胞汇合度达到90%时,采用脂质体转染法将PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc转染入293T细胞,转染步骤按lipofectamineTM 2000说明书进行。将等摩尔混合的PTY-UBIQUITIN-EGFP质粒共转染设为对照。转染24h后,倒置荧光显微镜下观察转染效率。收集细胞培养上清液和细胞裂解液。细胞裂解的具体步骤：将细胞用PBS清洗3遍,每孔加入200μl细胞裂解缓冲液,冰上放置30min后,将细胞悬液移入1.5 ml离心管,12000rpm离心15s。收集上清液,并测量其体积。分别加入浓度为5ug/ml的Glue底物腔肠素Coelenterazine200μl或150μg/ml的Flue底物luciferin 200ul,混匀后,用XenogenIVIS活体成像系统进行成像。曝光时间设定为l0s。
2.3小鼠PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒流体力学注射活体成像
6只6-8周龄、体重为18-20g的雄性BALB/c小鼠随机分为2组,每组3只小鼠。在5-7 sec内,采用水动力注射法,实验组每只小鼠经尾静脉注射2.0ml含10μgPTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc表达质粒的生理盐水,对照组每只小鼠只注射2.0ml生理盐水。注射12h后,经腹腔注射麻醉剂戊巴比妥钠(60-80mg/kg),麻醉后,经尾静脉注射300μL含50μg腔肠素的PBS溶液,立刻置于活体成像系统的暗箱中收集光子,每次收集光子3min,连续3次。待小鼠体内不再产生光子后(约30min),同一只小鼠腹腔注射D-荧光素钾盐(150mg/kg),注射1min、15min、30min后,分别用活体成像系统收集光子5 sec。
2.4双萤光素酶23T3细胞内的稳定表达
利用三质粒包装系统、脂质体共转染法,将PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒包装为慢病毒载体LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc,纯化后Realtime-PCR法测定其滴度。293T细胞以1×105/孔接种于24孔板中,24h后每孔接种1×107copies的病毒液(约50μ1),37℃、5%CO2培养箱中培养72小时。双萤光素酶基因Glue和Flue在细胞水平的动态监测研究在24孔板中进行。具体方法：将慢病毒转导后的293T细胞以1×104/孔接种于24孔板中,48小时、60小时、72小时分别收集细胞培养上清及细胞裂解液。分别取20μl加入Glue底物腔肠素和Flue底物D-luciferin混匀后,用XenogenIVIS活体成像系统进行成像。曝光时间设定为10s。
2.5双萤光素酶标记的裸鼠皮下移植瘤模型建立及表达监测
按照上文所述转导293T细胞同样方法,转导PC3细胞。同时设立转导无抗性的PTY-UBIQUITIN-EGFP质粒的PC3细胞作为阴性对照。转导72小时后,更换为含10%胎牛血清的DMEM培养基,同时加入neo基因抗性筛选药物G418,浓度为300ug/ml。之后依据细胞生长情况和对照组细胞死亡情况,逐渐调整G418浓度为700ug/ml。阴性对照组处理和实验组相同。直至阴性对照组细胞95%以上以死亡时,更换为不含G418,含10%胎牛血清的DMEM培养基。倒置荧光显微镜下观察绿色荧光对照基因表达。筛选得稳定表达的PC3-Gluc-2A-Fluc细胞。4-6周龄雄性裸鼠6只,体重为18-20g,称重,随机分为2组,每组3只。饲养于SPF环境。取对数生长期的PC3-Gluc-2A-Fluc细胞胰酶消化、离心弃上清后,以PBS重悬。细胞计数板计数,调整细胞浓度为5×106/200μl。按每只裸鼠5×106/200μl的剂量,将细胞悬液接种于3只裸鼠背侧皮下。对照组每只裸鼠皮下同侧位置注射200μl生理盐水。每3天,以XenogenIVIS活体成像系统观察肿瘤生长情况一次,连续观察2周。观察时裸鼠首先尾静脉注射300μl含50μg腔肠素的PBS溶液,注射后立刻成像。曝光时间设定为3min。待裸鼠体内不再产生光子后(约30min),腹腔注射D-萤光素钾盐(150mg/kg),注射后5min以活体成像系统观察。曝光时间同样设定为3min。上述裸鼠第7天起每3天分别经尾静脉采取全血20μl,加入Gaussia luciferase Assay Kit的底物50μl,混匀后,用XenogenIVIS活体成像系统进行成像,收集光子时间为lmin。
3.研究结果
3.1 PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒获得及鉴定
重组表达质粒PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc经酶切鉴定,以KpnⅠ、SacⅠ双酶切,能分别回收到3条片段,且最小片段约3000bp大小,与理论预测值相符,为正向连接。测序结果表明Gluc-2A-Fluc融合片段的顺序、方向及序列完全正确。
3.2293T细胞中Gluc和Fluc的瞬时表达及分布特点
PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒转染293T细胞,24h后分别检测细胞培养上清液和细胞裂解液中Gluc和Fluc的活性及分布特点。Glue和Flue在细胞内外的分布特点各不相同。Glue活性主要分泌至细胞外的培养上清中,细胞内较少；而Flue细胞外活性较低,大部分的Flue存在于细胞内。
3.3 PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc动物体内瞬时表达及分布特点
活体成像结果显示,Glue和Flue在小鼠体内都能显像,但是它们具有明显不同的显像特点：注入Glue底物腔肠素后表现为全身显像,在肝脏显像不明显,而在表浅裸露部位(如口鼻和四肢)信号较强,并且显像信号迅速衰减,10min后基本消失；而注入Flue底物D-Luciferin后则主要在肝脏显像,且显像信号比较持续稳定,30min内衰减不明显。
3.4 Glue、Flue在293T细胞持续表达监测
LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc慢病毒转导接种于96孔板中的293T细胞,分别在48h、60h、72h检测细胞培养上清的Glue活性和细胞裂解液的Flue活性。结果显示双萤光素酶标记的慢病毒转导后的293T细胞,不同时间点都能检测到细胞培养上清中Glue的表达和细胞内Flue的表达。
3.5利用活体成像系统动态观察肿瘤生长
PC3-Gluc-2A-Fluc细胞接种于裸鼠背部皮下后,每3天用Xenogen IVISTM Lumina Imaging system成像系统对肿瘤的生长情况进行观察。如图2-8所示,Glue和Flue基因在底物作用下,都能够催化底物发光。且其发光均与肿瘤生长部位相同。随肿瘤生长,Flue与Glue发光均逐渐增强。Glue因底物直接注入血液的原因,发光更快达到峰值,但发光时间更短。在发光强度方面,Glue发光信号略强与Flue发光,两者处于同一数量级。
3.6小鼠全血内Glue表达持续监测
在不同的时间点经尾静脉采血,检测血液中Glue活性。结果显示,连续监测的21d内,尾静脉采20μl全血均能检测到Glue活性,且Glue活性随肿瘤的生长而逐渐增强,而生理盐水对照组不能检测到发光。
4.研究结论
4.1我们成功构建了双萤光素酶共表达慢病毒载体PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc。
4.2体外细胞实验验证表达特性及持久性
共表达载体PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc能同时高效表达分泌型的萤光素酶Gluc和非分泌型的萤光素酶Fluc,且Gluc主要分布在细胞培养上清中,Fluc主要存在于细胞裂解液中,未改变其原有表达特性。其包装为慢病毒并转导靶细胞后,Fluc和Gluc两种萤光素酶表达持久稳定。
4.3裸鼠皮下成瘤及裸质粒尾静脉注射转染法观察双萤光素酶体内表达特性及裸质粒
尾静脉裸质粒注射转染法结果证实,Fluc基因作为报告基因更适合于体内成像时的表达定位研究,而Gluc由于具有高效分泌入血的特性,在裸质粒流体力学注射转染这样的类似的手段中,用于成像定位研究时会造成定位错误的判断；可能也不适合尾静脉或心房注射方式建立肿瘤血液转移模型的定位检测。并且注射Gluc底物后其发光信号不稳定且发光时间短暂,也不利于成像分析。皮下PC3-Gluc-2a-Fluc肿瘤成瘤后监测双萤光素酶表达证实：Fluc和Gluc都能用于皮下肿瘤模型的定位监测,两种均能正确反映肿瘤的生长部位。其发光强度处于同一数量级,具有可比性。因此,在皮下肿瘤模型监测的应用中,两种萤光素酶具有同等的应用价值。
4.4裸鼠皮下成瘤模型尾静脉采集外周血监测肿瘤生长
尾静脉采集外周血动态监测肿瘤生长的结果表明：外周血中Gluc的表达量随肿瘤生长而逐渐升高,两者具有明显的相关性。可以通过检测外周血中的Gluc表达从而间接监测肿瘤的生长。
总之,本研究成功设计和构建了同时高效表达双萤光素酶的慢病毒载体PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc,并成功包装为慢病毒。并以此为基础建立了稳定表达的293T-Gluc-2A-Fluc和PC3-Gluc-2A-Fluc细胞系,以及PC3-Gluc-2A-Fluc裸鼠皮下肿瘤模型。实验证实我们建立的双萤光素酶慢病毒载体可以高效转导细胞,且转导后双萤光素酶表达稳定可靠。以这种双萤光素酶慢病毒载体建立的稳定表达细胞株,既可以利用分泌型萤光素酶Glue的分泌特性和高灵敏度,实现可以在不裂解细胞或处死动物的情况下通过检测细胞培养上清液或血液中Glue的活性来动态监测Glue的表达水平；同时又可以利用萤光素酶Flue的细胞内分布特点和发射光波长、穿透性好以及发光稳定等特点,发挥其在动物活体成像方面的优势,比单一的萤光素酶报告基因载体在细胞标记和体内示踪研究方面更具优越性。本研究为细胞及肿瘤动物模型的标记提供了一个有效的方法和工具,为进一步建立一系列萤光素酶标记的单克隆肿瘤细胞系的工作奠定了基础。
(三)腺病毒滴度不同测定方法的比较
1.目的：将目前测定腺病毒滴度常用的OD260法、荧光定量PCR法、绿色荧光蛋白标记法、壳蛋白免疫法、细胞病变(CPE)法进行比较研究,分析和讨论各种方法的优缺点。
2.方法：4种重组腺病毒(Ad-G-AT2R-EGFP、Ad-CMV-EGFP、Ad-mif-shRNA-EGFP和Ad-CBA-GFP)扩增、纯化后,同时分别用OD260值法、荧光定量PCR法、绿色荧光蛋白标记法、壳蛋白免疫法、及CPE法测定病毒滴度。并以壳蛋白免疫法测定结果作为标准,对各种方法所得结果进行比较研究。
3.结果：绿色荧光蛋白标记法、CPE法与壳蛋白免疫法同时测定4种腺病毒滴度,其结果无统计学差异(P>0.1)。荧光定量PCR法测定值明显高于壳蛋白免疫法(P<0.05),但与该法测定结果间存在良好的相关性(r=0.965),约为壳蛋白免疫法所测得结果的10倍。OD260法测得结果与壳蛋白免疫法测得结果间则无相关性。
4.结论：绿色荧光蛋白标记法、与壳蛋白免疫法是测定腺病毒感染性滴度的最为快速有效的方法。其结果客观可靠,能真实的反映病毒的实际感染力。荧光定量PCR法测定的结果反映的是完整的病毒颗粒数,能够间接的反映出病毒的感染力,且该方法操作简单,耗时短,同样有较高的实用性。
BACKGROUND
Prostate cancer(PCa)is a common disease in aged men. In America, PCa is the most common malignant illness in men, and the second leading cause of cancer death. Most PCa was not detected and diagnosed in the first time due to the deep in of prostate and the lack of obvious symptoms. Many patients were diagnosised too lated to take an redical operation. At present, there is still no effective method to treat prostate cancer, especially the androgen independent prostate cancer. Chemotherapy had led to stabilization of disease and improvement of symptoms but did not increase survival rate. The method of gene therapy is to transduct specific gene into target cells and fixes the problem in gene expression through exogenous gene. In the recent 10 years, as the development of molecular biology, gene therapy has made great pogoress in cancer therapy and succeeded in curing some monogenic diseases. Gene therapy has several advantages:①high selectivity comparing to Chemotherapy and radiotherapy, which can be obtained by using targeting gene.②less side effect③efficient even to the advanced stage cancer and metastasis. How ever, lots of things are still to be solved:①the lack of tumor-specific technology to direct the expression of therapeutic genes specifically to the tumor;①the antigenicity of the viral vector, which can deduce the immune response of body, makes it a temporary theatment;③the management of the exogenous gene expression.
In the progress of exogenous gene transduction and expression in target cells, the keypoint is to tranduce the vector into target cells and the get stable expression, so it is critical to choose the suitable vector. For now, there are mainly two kinds of vectors:non-viral vectors and viral vectors. Non-viral vectors includes gene sequence and plasmid vector. This kind of vectors is used in limited areas for the low expression efficiency. In contrast, the viral vectors have been widely used, it is estimated that 85% of the total gene therapy was carried on viral vectors. Amoung them, lentiviral vector is the most perspective one with great potential:①no limitation of the cell types:either dividing cell or not;②transducing the exogenous gene into target cell to obtain stable expression;③free of immunogenicity;④free of pathopoiesia areas.
The purpose of regulation of exogenous gene in cancer gene therapy is to target the exogenous gene efficiently and specically expressed in tumor cells without hurting the normal tissues. Transcriptional targeting of gene therapy is a novel and prospect method for cancer's treatment.It is a strategy to enhance the specificity of gene expression to target tumor cells of interest.This targeting approach is based on the use of tissue or tumor-specific promoters(TSP)in a heterologous context to direct the expression of therapeutic genes specifically to the tumor. An efficient gene therapy regimen requires transgene expression in the tumor and absence of expression in relevant normal tissues, especially in the liver.This combination of'tumor on" and "liver off" profile can result in increasing the therapeutic index and limiting the toxicity of vectors and transgenes in vivo.Many promoters have already been evaluated for transcriptional targeting in cancer gene therapy to be more sufficient specific to avoid gene expression in normal tissues,while shows noticeable expression levels in tumor cells. Promotor is the key of the efficiency of exogenous gene. It has been demonstrated in many researches that the same promotor shows obvious difference in efficiency in the different cell lines. Take the cytomegalovirus(CMV) promoter for example, it is highly effective in 293 cell lines, in C127 cell lines,however, its efficiency drops sharply. So it is crucial to choose the effective promoter according to different cancer cells. By doing this,the effective and selective expression of exogenous gene can be crchieved.
In the evalution of the gene therapeutic effect in animal models, we still a method of ex-vivo monitoring of in-vivo processes of the cancer cells,without killing the subjects until the end of our investigation. This means less animals will be needed during the total experiment and more exact diction. The luciferase is just this kind of marks we need.
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence. There is no endogenous expression in mammalian cell and its detection is not affected by the influence of other substances in cells.Luciferase has characteristics of high sensitivity and specificity,rapid response,simple operation,wide detection range, so as a reporter gene has been widely used in medicine, biology, environmental science,etc. One famous example is firefly luciferase (Fluc) from firefly Photinus pyralis.
As early as in 1956, Green and MeElroy got the Flue crystallization. Flue is a molecular weight of 60～64kD polypeptide chain. In a reaction that requires oxygen, magnesium, and ATP, Fluc oxidizes its substrate, luciferin, to produce light with a broad emission spectrum and a peak at approximately 560 nm. Only in living cells produce light-emitting phenomenon, and the light intensity is proportional to the number of labeled cells. As a most commonly used luciferase, Fluc has been widespread application in ATP rapid microbiology, infectious invasion,drug screening,cell marking and tracing as well as in vivo animal imaging, etc. Flue, optimized for expression in mammalian cells, is the most commonly used luciferase for in vivo imaging.
Discovered in recent years as a new type of luciferase, Gaussia luciferase (Glue) derived from the Gaussia princeps, its distinguishing feature is efficiently secreted from cells, also has a higher detection sensitivity.Gluc reacts with substrate, coelenterazine, to produce blue light with an emission peak at approximately 480nm,which is ATP-independent.As Glue has small molecular weight, secretion characteristic, detection sensitivity, a short half-life, etc,it has been successfully applied in the experimental study of in vitro and in vivo.By testing animal blood or other body fluids of Glue activity,Gluc can be quantitatively reflected tumor burden in animals,which not only track the progress of tumor metastasis, but also can serve as an effective marker of tumor response to treatment reflects.Luciferase with a specific substrate light-emitting reactions are chemiluminescent,which is specific and no cross-interference.
Based on these characteristics,in order to combine the advantages of secreted luciferase Glue and non-secreted luciferase Fluc,this study was to construct a novel dual luciferase co-expression vector and studied its expression characteristics in vivo and in vitro, for the next step to establish dual-luciferase gene markers of tumor cells and animal models, and lay the foundation for monitoring and tracing study later.
The article is component of two parts, the first part will discuss the optimal promoter choices for lentiviral vector-mediated transduction of different prostate cell lines; the second part details the design of the dual luciferase lebeled lentivirus vector with its character of expression in vitro and in vivo.
Part 1:optimal promoter choices for lentiviral vector-mediated transduction of different prostate cell lines
Objectives
To compare the expression of GFP driven by three different promoters carried by lentiviral vectors in 293A, MOLT-4, DU145, PC3 and RM1 cell lines. Demonstrate that different promoters showed various driven activities among different cell lines. So that promoter selection and proper host cells could be considered in order to achieve high level transgene expression in the further experiment.
Methods
1. construction of PTY-EF1α-EGFP, PTY-CMV-GFP and PTY-UBIQUITIN-GFP
PTY-EF1α-EGFP (EF1α-GFP) was constructed by excising the IRES sequence from PTY-EF1α-IRES-GFP. PTY-CMV-GFP (CMV-GFP) were constructed by replacing the EF1αpromoter in PTY-EF1α-GFP with human cytomegalovirus promoter.
2. package and titer lentivirus with different promotor
293T cells were planted in T25 flask and transfected with shuttle plasmid PMD2G, PSPAX2 and packaging plasmid PPAX2 following the instruction of lipofectamineTM2000. Harvest virus 60h after transfection. Purified the lentivirus with ultracentrifuge, stored at -80℃. Some samples was subjected to quantitative PCR analysis using the stragene 3500 system, to quantify genomic titer. Lentivirus was serially diluted and sequentially digested with DNase I. Viral RNA was extracted with Roche viral RNAmini kit. A standard amplification curve was set up at range from 105 to 108 copies and the amplification curve corresponding to each initial template copy number was obtained.
3. vitro transduction of different cells
293T and 293A cells, DU145, PC3 cells and RM1 cells were cultured according to the instruction of ATCC.293A, DU145, PC3, RM1 and MOLT-4 cells were plated at the appropriate density (70-80% confluence the following day). Medium was removed and cells were transduced with maintaining medium containing 108 particles of each recombinant lenti virus per well for 6-well plates and incubated at 37℃,5% CO2 for 3 days before assay.
4. Visualization of GFP
GFP fluorescence were directly imaged on an Olympus Model BX41 fluorescent microscope (Olympus, Tokyo, Japan). All cell images were captured at×10 magnification and All fluorescent images were collected using an identical exposure time.
5. Flow cytometry Analysis of EGFP fluorescence in transduced cells was performed by flow cytometry. Briefly, cells were harvested with Trypsin-EDTA Solution. Cells were washed and resuspended in PBS for analysis on a FACS Calibur (Becton Dickinson, MA, USA). Samples were analyzed using CellQuest oftware (Becton Dickinson, MA, USA).
6. Total RNA extraction and quantitative real-time PCR analyses
Total RNA was isolated from the transduced cultures using RNeasy Mini Kit (Qiagen,Valencia,CA). RNA concentration was quantified by the ultraviolet spectrum at 260 nm, and reverse transcribed using PrimeScript 1st Strand cDNA Synthesis Kits (TaKaRa) according to the manufacturer's instructions. Synthesized cDNA corresponding to 100 ng of total RNA, was used for real-time PCR. Specific primers for the GFP and ribosomal RNA (18sRNA; internalcontrol) genes were used in real-time RT-PCR analysis. All primers were synthesized from Invitrogen company. The primers used were as follows:EGFP-F:5'-GAGCTGAAGGGCATCGACTT-3', EGFP-R:5'-CTTGTGCCCCAGGATGTTG-3'; 18sRNA, forward primer: 5'-CAGCCACCCGAGATTGAGCA-3', reverse primer:5'-TAGTAGCGACGGGCGGTGTG-3'.The SYBR green real-time PCR assays for each target gene were performed on cDNA samples using an Stragene 3005 Detection system (Agilent Biosystems).18s RNA assays were run in parallel for each sample. Amplication was carried out in optical 96-well reaction plates (Agilent Biosystems)with each well containing 2μl of cDNA template,1μl of sense primer (10μM),1μl of antisense primer (10μM),12.5μl of SYBR Premix Ex TaqTM (TaKaRa),0.4μl of ROX and DEPC- treated water (8.1μl). The conditions for real-time PCR were one cycle of 95℃for 30 s, followed by 40 cycles of 95℃for 5 s and 60℃for 30 s.
Results
1. Identification of lentiviral vector
Recombinant plasmid PTY-EF1α-EGFP and PTY-CMV-EGFP was identified by restriction enzyme digestion with Mlu和SmaI. No fragment can be obtained again, which indicates that the vectors have been built successfully.
2. Titer of lentiviraus produced
Compare the cTvalue of samples with standard curve to get the exact copy in every ml of viral solution. The titers of the lentivirus are listed below: LV-UBIQUITIN-EGFP 5.71*108copy/ml, LV-EFla-EGFP5.53*108copy/ml, LV-CMV-EGFP 5.87*108.
3. Evaluation of transgene expression by lentiviral vectors in vitro using flourscent microscopy
293A, DU145, PC3, RM1 and MOLT-4 cells were transduced with 109 copies/ml of either LV-UBIQUITIN-EGFP, LV-EFla-EGFP or LV-CMV-EGFP and,72h later, we compared activity of the UBIQUITIN, EF1αand CMV promoters in various cell lines. GFP expression was analyzed by visualizing green fluorescence. All three lentiviral vectors were capable of producing gene transfer to cell lines, but in various cell lines, they showed different driven activities. In 293A cell lines, all three CMV, EF1αand ubiquitin promoters showed high activities. EGFP expression driven by CMV promoter peaked earlier than the two other promoters (not shown) and showed the highest activity. Also in PC3 cells, CMV promoter showed the highest activity. In the contrast, the CMV promoter showed much lower activity than the two other promoters in MOLT-4 cells. In the present study the CMV promoter showed a different driven activity in vitro among different cells tested, in fact, we found that it is the weakest promoter tested in vitro among MOLT-4 cells and DU145 cells. However, in DU145 cells, EF1αpromoter showed the highest activity, UBIQUITIN promoter comes the second. The CMV promoter shows the weakest activity among them. In RM1 cells, no promoter showed high activity, but the EF1αpromoter activity is higher than the other two.
4. Evaluation of transgene expression by lentiviral vectors in vitro using flow cytometry
To determine whether the level of EGFP expression is only dependent on the strength of promoters,293 T cells were transduced with. LV-UBIQUITIN-EGFP, LV-EF1α-EGFP or LV-CMV-EGFP vector.72h after transfection, the cells are digested and resuspended in PBS. Cell concentration per milliliter was determined using a hemocytometer. All the cells were tested using a Aliquots of 200μL of each sample was analyzed on a FACScan cytometer (Becton Dickinson, San Jose, CA) equipped with an argon ion laser set at 488 nm line and fluorescence detector wavelength of 530/30 bandpass using Summit Software with the Cicero upgrade (Cytomation, Ft. Collins, CO). The results shows that the GFP expression from LV-UBIQUITIN-EGFP, LV-EFla-EGFP and LV-CMV-EGFP vectors are varied. The data showed that 293A cells harboring CMV-EGFP, UBIQUITIN-EGFP and EF1α-EGFP vectors displayed high, medium, and low intensity of green fluorescence, respectively. The results suggests that the CMV-EGFP, UBIQUITIN-EGFP and EF1α-EGFP promoters are listed in the order of promoter activity from the highest to the lowest in the 293 A cells, totally corresponds well with the results in the past chapter. Similar results can be draw in other cells. In MOLT4 cells, the order from high to low is; while CMV EF1α, UBIQUITIN in descending order in PC3 and EF1α, UBIQUITIN, CMV in DU145. In RM1 still no promoter shows high activity, but the EF1αpromoter is still higher, then comes the UBIQUITIN promoter, CMV promotor is the last one.
5. Evaluation of transgene expression by lentiviral vectors in vitro by Realtime-PCR
The lentiviral stocks were further analyzed byquantitative PCR to determine the DNA titer and the same DNA titers of LV-UBIQUITIN-EGFP, LV-EF1α-EGFP and LV-CMV-EGFP lentiviral stocks were used to transducer 293A, MOLT4, PC3, DU145 and RM1 cells. Negative controls were set up with DEPC H2O correspondingly. The results showed that the EF1αpromoter exhibited the highest activity in MOLT4, PC3, DU145 and RM1 cell lines. The CMV promoter appeared to be the strongest in 293A cells. All the results corresponded well with the results of flow cytometry except the order in PC3. This may be caused of the different expression in mRNA and protein.
Discussion
The lentiviral transduction is one of the most effective methods to overexpress transgenes. However, the influence of the lentiviral DNA context on overexpression still has to be considered. In this study, we have shown that the GFP expression from the UBIQUITIN, EF1αand CMV promoters encoded in three lentiviral vectors, reveals sigficant differences in 293A, MOLT4, PC3, DU145 and RM1 cells. The differential expression of GFP reporter may be due to the specific characters of these promotors as the other parts of the lentivius are the same. The results indicate that the differential expression of GFP between the three promotors in different cells is due to the lentiviral genomic sequences but not the plasmid topology. In PC3, the results of Realtime-PCR did not correlate well with that of flow cytometry and flourscent microscopy. This may be caused by the differenct expression in mRNA and protein. Some mRNA seemed were not translated into protein. The CMV promoter may not always be the best choice for transgene overexpression in all cell types. By comparing to other promoters, while CMV promoter gave much more than 6-fold of GFP expression than human house keeping gene EFla promoter in 293A and PC3 cells, the GFP production by EF1αpromoter revealed the highest intensity in MOLT4, DU145 and RM1 cells.
Part 2:design of the dual luciferase lebeled lentivirus vector with its character of expression in vitro and in vivo
Objectives
To constructe a novel dual luciferase co-expression lentiviral vector, which expressed simultaneously secreted luciferase Gluc and non-secreted luciferase Fluc, transducer 293T and PC3 cells with it, establish Subcutaneous tumor model of prostate cancer and study its expression characteristics in vitro and in vivo.
Methods
1. Construction of co-expressed plasmid PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc with its identification
The sequence CAG-Gluc-2A-Fluc was deprived from plasmid pAAV2neoCAG-Gluc-2A-Fluc and incerted ito PTY-UBIQUITIN-EGFP vector deprived off GFP coding gene. Sequence GFP-HSV-Tk-neo was then deprived from plasmid pTR-UF11 and cloned into it. The production was identified and then transformed into E. coli DH5a, picked a single colony which is ampicillin resistance,extracted plasmid, and then identified by restriction enzyme digestion and sequencing.
2. PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc transient transfection and character of expression
293T cells in 10% FBS DMEM medium, placed in 37℃,5% CO2 of incubator for adherent culture. When degree of 293T cells confluence reached 90%, the plasmid was transfected into 293 T cells and the operation steps were carried out according to the instructions of lipofectamineTM2000, control plasmid PTY- UBIQUITIN-EGFP was co-transfected with the equal copies. Expression and distribution characters of Gluc and Fluc in PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc in vitro were carried out in 24-well plates. Transfection after a 24h, cell culture supernatant and cell lysate was collected. Steps of cell lysis were as follows:washed the cells three times with PBS, added 200μl cell lysis buffer to each hole, placed on ice 30min, then moved cell suspension to 1.5 ml centrifuge tubes, centrifuged for 15 sec at 12 000 rpm,after that collected supernatant and measured its volume. Detection of Gluc and Fluc activity in cell culture supernatant or cell lysate was done in accordance with instructions of Gaussia luciferase Assay kit and Luciferase Assay System. Coelenterazine (concentration of 5ug/ml,200μl) or of Fluc substrate luciferin (150μg/ml,200ul) were added, images were taken instantly with XenogenIVIS vivo imaging system. Time for collecting photon counts was 10 sec.
3. Bioluminescence imaging of mice injected with PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc through tail vein
6 six to eight-week-old, weighing 16～20 grams of male BALB/c mice were randomly divided into two groups,3 mice each group. Within 5-7 sec, through tail vein, each mouse in the experimental group was hydrodynamically injected 2.0ml normal saline containing 10μg UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc through tail vein expression plasmid, while only injecting 2.0ml normal saline per mouse in the control group.
4. Detect activity of Gluc and Flue in vitro
Using three-plasmid packaging system, and lipfectimine transfection method, the PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc plasmid was packaged into lentiviral vector LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, then purified and Realtime-PCR method was used for the determination of its titer.293T cells was panted with a density of 1×105/hole in 24-well plates, transduced with 1×107copy per well of virus solution (approximately 50μl), incubated in a 37℃,5% CO2 incubator for 72 hours. Dual Glue and Flue activity dynamic monitoring was carried out in 24 well plates. After lentiviral transduction,293T cells were plated in 24-well plates,1×104/hole,48 hours later the cells were collected every 12 hours for both culture supernatants and cell lysates. According to kit instructions, add Coelenterazine (concentration of 5ug/ml,200μl) or of Flue substrate luciferin (150μg/ml,200ul), images were taken instantly with XenogenIVIS vivo imaging system. The photon collection time is 10 sec. Continuous observation was taken on for 72h.
5. Establishment of dual luciferase labeled subcutaneous transplantation tumor model in nude mice and the dynamic monitoring of their expressions
Transduce PC3 cells in the same way which 293T cells were transduced as described above. Negative control was set up with transduction of lentivral vector LV-UBIQUITIN-EGFP, without neo gene, into PC3 cells. Replace the medium with DMEM containing 10% fetal bovine serum medium 72 hours after transduction, while adding G418, with the concentration of 300ug/ml. Adjust the G418 concentration gradually to 700ug/ml. Negative control group was treated the same with experiment group. When 95% of negative control cells died, replace the medium with DMEM containing 10% fetal bovine serum medium free of G418. GFP was observed under fluorescent microscope. The cells left was PC3-Gluc-2A-Fluc with stable expression of dual luciferase. Coelenterazine (concentration of 5ug/ml,200μl) or of Fluc substrate luciferin (150μg/ml,200ul), images were taken instantly with XenogenIVIS vivo imaging system. Exposure time is set to 3min, repeat this triplex.6 nude mice (male,4-6 week old) were fed in the SPF environment. PC3-Gluc-2A-Fluc resuspended in PBS (5×106/ml,200μl/mice) was injected subcutaneously into the flank of nude mice. The control group mices were inject with 200μl NS at the same location. Photos were taken every 3 days with XenogenIVIS system to observe the tumor growth, the observation lasted continuously for 2 weeks. Extract 20μl blood of the mice via the tail vein every 3 days since day 7, add Gaussia luciferase Assay Kit substrate 50μl, Coelenterazine (concentration of 5ug/ml,200μl) images were taken instantly with XenogenIVIS vivo imaging system, with a collection time of 1min.
Results
1. Identification of PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A--Fluc plasmid
Recombinant plasmid PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc was identified by restriction enzyme digestion with KpnⅠand SacⅠ. We obtained 3 fragments, a size of 2274bp fragment was our target fragment. sequencing results showed that the direction and sequence of GFP-HSV-Tk-neo-CAG-GIuc-2A-Fluc fragment was entirely correct.
2. Expression and distribution characters of Glue and Flue in vitro
Expression and distribution characteristics of Glue and Flue of transient transfection with PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc in 293T cells were namely:24 hours post transfection, the expression of Glue and Flue can both be detected in cell supernatants and cell lysates, most of Glue was mainly detected in the culture media most Flue was mostly within cells;the activity of Glue in the supernatant increased gradually with time while the Flue activity in cells almost kept stable.
3. Bioluminescence imaging of mice
In vivo imaging results showed that, both Glue and Flue can take imaged in vivo imaging system, but they have distinctly different imaging characteristics:after the injection of Glue substrate coelenterazine, images show that luminescence can be seen throughout the body, the signal from liver shows no difference in contrast with other organs, while in superficial exposed parts (such as the nose and mouth, and limbs) signals were strong, and the imaging signals decreased sharply within 10 minutes; Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour.
4.Stable expression of Glue and Flue in vitro
LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc transducd 293T cells in 96-well plates,, in 24h,36h,48h,60h,72h detect Glue activity in cell culture supernatant and Flue activity in cell lysate respectively. The results showed that after transduction of 293T cells with dual luciferase, Glue activity and Flue activity can either be detected in cell culture or cell lysates. intracellular expression of and Glue activities in the supernatant gradually increased with time, while Flue activity in cells remained stable. Cells and cell supernatant were added respectively with Glue substrate Coelenterazine and Flue substrate Luciferin, then instantly taken images in XenogenIVIS imaging system. The results showed:in the cell supernatant and the cells the expression of GLuc and Fluc activity can both be detected respectively. GLuc signal in supernatant is stronger than signal of Flue intracellular. With Continuous observation of 30min, Glue images disappeared within 15min, Fluc image remained stable within 30min.
5. Dynamic observation of tumor growth in vivo with dual luciferase
PC3-Gluc-2A-Fluc cells were into the flank of nude mice subcutaneously, the growth of tumors was observed every 3 days with the Xenogen IVISTM Lumina Imaging system imaging system. The results show that Glue and Flue can both catalyze the light-emitting substrate respectively. As the tumor growth, Flue and Glue light-emitting gradually increased. Because Glue substrate Coelenterazine was directly jnjected into blood, the signal of Glue lasted for just a short time, while the signal of Flue lasted much longer. In the luminous intensity, Glue signal is slightly stronger than Fluc signal; both are in the same order of magnitude.
6. Dynamic monitoring of Gluc activity in blood
20μl of blood taken through tail-vein was assayed for luciferase activity with Gaussia luciferase Assay kit using a luminometer at various time points. The results showed that at least within 21 days 20μl whole blood by tail vein was able to detect Glue activity, Glue signal growed as the tumor growed.
Conclusions
1.We have successfully constructed a dual-luciferase expression vector PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-GIuc-2A-Fluc.
2.Cells in vitro experimental verification
co-expression LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc lentival vector can mediate simultaneously high-level expression of secreted report gene Glue and the non-secreted report gene Flue, and Glue mainly distributed in the cell culture supernatant, Flue mainly exists in the cell lysate, indicating expression and distribution characteristics of Glue and Flue were not changed. After transduced with LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, cells expressed dual luciferase stably and consistantly.
3.In vivo imaging of dual luciferase
Tail vein injection of plasmid transfection method confirmed, Flue as a reporter gene is more suitable for in vivo localization of gene expression, while Glue has the character of secretion into the blood with high efficiency. Because of this, if Glue was used in ways like hydrodynamic method, errors may occur when using it for localization. Glue may also not be suitable for establishment and imaging localization of the tumor metastasis animal model of blood through tail vein or atrial injection. After injection of Glue substrate, the luminescence signal appears to be instability and temporary, which is not quite convenient to imaging analysis. Subcutaneous PC3-Gluc-2a-Fluc tumor surveillance luciferase expression confirmed that Flue and Glue subcutaneous tumor model can both be used to monitor the positioning of the tumor in vivo. Both of them can correctly reflect the position and scale of tumor growth. Their luminous intensity are in the same order of magnitude. Therefore, Glue and Flue contributes equally to the monitoring of subcutaneous tumor model.
4. Test of blood through tail vein of tumor model in nude mice to monitor tumor growth
Dynamic monitoring of Glue in blood through tail vein in reflacting tumor growth shows that:expression of Glue in peripheral blood gradually increased with tumor growth, with a significant correlation between the two. Peripheral blood can be used to monitor Gluc activity in reflacting tumor growth indirectly.
In conclusion, we successfully construceted the dual luciferase expression lentiviral vector LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, established the 293T-Gluc-2A-Fluc cell lines and PC3-Gluc-2A-Fluc cell lines and established animal models with these cell lines. The new lentiviral vector combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing. On one hand, we can use Gluc, which has secretion characteristics and high sensitivity, to dynamically monitor the expression levels of Gluc by detecting Gluc activit in cell culture supernatant or blood without clearaging cells or sacrificing animals; On the other hand, we can use Fluc, which distributes in the intracellular and emits spectra long, penetrates well, and imaged stablily, to localize biological processes in vivo animal imaging.
Part 3:A Comparison and Analysis of Methods for Titering Adenoviruses
1. Object:To compare different methods commonly used for titering adenovirus and discuss the advantages and drawbacks of each method.
2. Methods:Four types of recombined adenovirus were amplified and purified. Each is then simultaneously titered by optical absorbance, Real-time PCR, green fluorescent protein (GFP) labeled method, immunoassay, and cytopathic effect (CPE). Results are then compared.
3. Results:No significant difference was found between the titer amounts derived from the GFP labeled method, immunoassay, and cytopathic effect method (P>0.1) There is a position correlation between the titer amounts (r=0.965), even though Real-Time PCR resulted in ten times the vg/ml amount compared to the ifu/ml amount obtained from the immunoassay.
4. Conclusion:These results indicate that the GFP labeled method and the immunoassay can quickly determine the titer amount. Real-time PCR cannot titer the real infectious titer of the adenovirus, but the results indicate that its approach has good correlation with that of immunoassay, so it can also reflect the infectious titer of adenovirus, though not that accurate. As the Realtime PCR method takes the least time, it still worth using.
前列腺癌是成年男性最常见的恶性肿瘤之一,在美国,其发病率据男性恶性肿瘤首位,每年死于前列腺癌的男性在因肿瘤而死亡的男性中排第二位。前列腺癌因其部位深在,症状出现较晚,因而很多患者发现时均已是晚期,失去了手术治疗的机会。目前,对晚期特别是雄激素非依赖型前列腺癌尚缺乏有效的治疗方法。基因治疗(gene therapy)是指把目的基因导入靶细胞和宿主体内,通过目的基因在宿主中的表达,纠正错误的基因表达和基因表达的错误,以达到治疗目的。近10多年来,随着分子生物学技术和理论的飞速发展,使基因治疗这一新型治疗方法成为可能,并在对单基因缺陷性遗传疾病的治疗中取得了令人瞩目的成就。肿瘤的基因治疗研究也成为目前肿瘤防治研究的一个活跃领域。肿瘤基因疗法具有下列优越性：①肿瘤的选择性比放疗、化疗更强,这可通过特异性基因转导技术或特异基因的定向作用而获得；②对患瘤个体损伤小而轻；③对晚期肿瘤或转移瘤灶仍有效。然而,基因治疗在临床应用上仍有许多有待解决的难点。主要有：①缺乏对靶细胞定向导入基因的技术；②病毒载体有抗原性,会引起机体对它产生抗体,故不能长期应用；③导入基因的表达和调控问题。
纵观将外源基因导入并整合到宿主细胞的整个过程,将目的基因导入靶细胞并使之持久稳定表达是关键环节,因此介导的载体选择便显得格外有意义。目前基因治疗中所用的载体可分为两大类：非病毒载体和病毒载体。非病毒载体包括基因片段和质粒载体。而应用最广泛的是病毒载体,约85%的基因治疗临床项目采用的均是这种载体。其中慢病毒载体因其独特的生物学性状显示出在基因治疗中的巨大潜力：①慢病毒载体转导的细胞类型不受限制,既能转导分裂细胞又能转导非分裂细胞；②能够使目的基因高效的整合到宿主细胞基因组内实现长期而稳定的表达；③不会引起宿主细胞及宿主本身的免疫反应；④已经去除了原病毒的可致病区,消除了致病性。
“导入基因的表达和调控”就是使目的基因特异性的在癌细胞中高效表达从而起到治疗作用,而在正常组织细胞中不表达或几乎不表达而避免伤害正常组织。这就需要一个高效、肿瘤特异性的转录激活因子既启动子来完成这一任务。启动子是决定目的基因表达效率的关键环节。大量前期研究已经证明,同一载体同一细胞系中,如果启动子不同,则目的基因表达效率不同。而同一启动子携带目的基因相同,在不同细胞系中,表达效率也不同。因此,针对不同的肿瘤细胞,选择特异性强、高效表达的启动子作为慢病毒载体中插入基因的启动子,对于提高肿瘤基因治疗的效率,提高其靶向性和特异性具有重大意义。
在针对基因治疗的效果进行评价的细胞和动物模型中,尚缺乏易于在体外动态监测肿瘤细胞生长和转移及代谢情况的特异性标记手段,这严重影响了对基因治疗效果的评价。因此,寻找一种特异性强且不会影响肿瘤生长的标记物标记肿瘤细胞,以便从体外监测肿瘤的生长、转移等,对评价肿瘤的基因治疗效果有着重要的意义。
萤光素酶(luciferase)是自然界中能够催化底物产生生物荧光的酶的统称,在哺乳动物体内无内源性表达,检测不受细胞内其他物质影响,并且具有敏感性高,特异性好,反应迅速,操作简单,检测范围广等优点,作为报告基因在医学、生物学、环境科学等领域研究已得到广泛应用,其中最有代表性的是萤火虫萤光素酶(Firefly luciferase, Fluc)。
早在1956年,Green和MeElroy就得到了萤火虫萤光素酶(Lueiferase)的结晶。萤火虫萤光素酶是分子量为60-64kD的多肽链在Mg2+、ATP、O2存在时,催化D-萤光素(D-Luciferin)氧化脱羧,产生波峰在560nnm左右的荧光。只有在活细胞内才会产生发光现象,并且光的强度与标记细胞的数目成正比。作为经典的萤光素酶,已经在细胞内ATP水平探测、感染检测、药物筛选、细胞标记和示踪以及动物活体成像等方面都得到了广泛的应用。目前应用最多的活体光学成像动物模型是表达Fluc的动物模型。
Gaussia luciferase(Gluc)是近年来发现的一种新型萤光素酶,来源于海洋桡脚类动物(Gaussia princeps),其显著特点是可以高效分泌至细胞外,同时具有更高的检测灵敏度。Gluc与底物腔肠素的反应不依赖于ATP,结合后发出的荧光波峰在480nm左右。由于Gluc具有分子量小、易分泌、检测灵敏度高、半衰期短等优点,已成功应用于体内外的实验研究中。通过检测分泌到模型动物血液或其他体液中的Gluc活性,可以定量反映动物体内肿瘤的负荷,不仅能追踪肿瘤转移进展,而且能作为有效标记物体现肿瘤对治疗的反应。并且萤光素酶与特定底物的发光反应属于化学发光,具有特异性,不存在交叉干扰现象。
基于上述特点,为了进一步研究分泌性萤光色素酶Gluc和非分泌性萤光色素酶Fluc在细胞及动物体内的表达特性,并结合二者与慢病毒载体在外源基因导入方面的优点,本研究拟构建一种Gluc和Fluc双萤光色素酶共表达的慢病毒基因载体,并对其体内外表达特点进行研究,进而建立双萤光素酶基因标记的肿瘤细胞系和动物模型、为下一步建立一系列双萤光素酶标记的泌尿系肿瘤细胞系并对其进行体内外监测和示踪研究奠定基础。
随着分子生物学技术的迅猛发展,特别是近年来转基因技术的巨大进步,重组腺病毒载体在目的基因的导入、基因的过表达方面已获得广泛的应用。并由于其转基因效率高、转导细胞类型广泛、易于制备和纯化,且进入宿主细胞后仅瞬时表达,不整合到宿主细胞基因组等优点,近年来在基因治疗、基因免疫以及疫苗制备方面的地位越来越突出。现已成功应用于视网膜母细胞瘤、肝细胞癌、前列腺癌的基因治疗并已进入临床试验阶段。在腺病毒应用的过程中,一个十分重要的步骤是测定病毒的滴度。目前测定腺病毒滴度的方法有多种,如壳蛋白免疫法、荧光定量PCR法、OD260法、细胞病变(CPE)法和空斑法,对于带有GFP标签的腺病毒载体还有绿色荧光蛋白(GFP)标记法。其中以壳蛋白免疫法最为常用,但该法步骤繁琐,且试剂成本高,不利于普及推广。现我们以壳蛋白免疫法为标准测定方法,将上述其他几种方法与其进行比较,对测定结果进行分析,探讨各种方法的优缺点,并进一步讨论各种方法的意义及可行性。
全文分三大部分,分别论述前列腺癌不同启动子的效果差异的比较研究和双萤光素酶标记的慢病毒载体的构建及其表达特性研究,以及不同测定腺病毒滴度方法的比较。
(一)不同启动子慢病毒载体在前列腺癌细胞表达效果差异的比较研究
1.研究目的以增强型绿色荧光蛋白(EGFP)作为报告基因,比较UBIQUITIN, EF1α和CMV三种不同启动子的慢病毒载体在293A, MOLT4, PC3, DU145和RM1三种不同细胞株的表达效率,筛选出每种细胞株的高效启动子,为在今后的研究中,针对不同细胞株,选择恰当的启动子奠定基础。
2.研究方法
2.1不同启动子载体的构建和鉴定
以限制性内切酶将IRES编码区自PTY-EF1α-IRES-EGFP上切下,再连接形成PTY-EF1α-EGFP (EF1α-EGFP)慢病毒载体。PTY-CMV-EGFP (CMV-EGFP)载体是在此基础上将EF1α编码区替换为CMV编码区。
2.2不同启动子慢病毒载体的包装和滴度测定
293T细胞于含10%胎牛血清、1%的青链霉素、1%的谷氨酰胺DMEM培养基中,37℃5%CO2培养箱培养。细胞均匀铺于T25培养瓶中,24h后观察细胞分裂活跃时,将PMD2G、PPAX2以及所构建的含不同启动子的穿梭质粒共转染293T细胞。转染步骤按照lipofectamineTM 2000说明书步骤进行。60小时后收获病毒并以超速离心法纯化。病毒液-80℃保存。荧光定量法检测病毒拷贝数在Agilent3500荧光定量PCR仪上进行。病毒液进行去RNA酶处理后按照试剂盒步骤要求提取病毒核酸,质粒标准品PTY-EF1a-EGFP浓度为1.09μg/μl,换算成核酸拷贝数为1.06×1014 copies/ml 10倍倍比稀释为7个浓度梯度,同时设一个阴性对照,和待测cDNA样品同时进行扩增并检测荧光信号。依据标准品所生成的标准曲线,将所测得cDNA CT值与之相比较,可得该病毒液中病毒基因组的拷贝数,进而换算得病毒的滴度。
2.3细胞的病毒转导
293A,PC3,DU145,RM1细胞均以含10%胎牛血清的DMEM培养基培养,MOLT4细胞以含10%新生牛血清的1640培养基培养,铺于6孔板中,每种细胞4个复孔。细胞铺满6孔板板孔底面积90%分别加入5×108copy的LV-UBIQUITIN-EGFP, LV-EF1α-EGFP, LV-CMV-EGFP慢病毒病毒液或者DEPCH20(作为对照)200W。继续培养72h。
2.4倒置荧光显微镜下比较荧光强度
倒置荧光显微镜下观察细胞荧光强度。每种细胞均随机观察3个视野。比较同一细胞不同启动子的效果差异。所有照片均按照10×物镜、10×目镜放大倍数拍摄,曝光时间相同。
2.5流式细胞计数差异
将6孔板中细胞以胰酶消化后,1000rpm/min离心3min,弃上清,以500μl PBS缓冲液重悬细胞,用移液枪充分吹打混匀。FACS Calibur流式细胞计数仪计数GFP阳性细胞数。仪器自动分析结果。
2.6总RNA的提取及荧光定量检测
取流式细胞计数后的细胞悬液,按照QIAGEN RNeasy Mini Kit步骤要求提取细胞中总RNA。将提取的总RNA应用PrimeScriptTM RT reagent Kit进行逆转录,获得cDNA。分别取逆转录所得cDNA各2u1,按照SYBR(?) Premix Ex Taq荧光定量试剂盒步骤要求,配置反应体系。每个细胞每种启动子设置3个复孔,同时设立等量的18s RNA复孔作为内参,及3个复孔的阴性对照,阴性对照中cDNA样品以DEPC处理水代替,总体系大小不变。Agilent3005型荧光定量PCR检测仪进行目的片段扩增和荧光信号检测,反应条件为：第一步95℃30s,第二步95℃5s,60℃30 s,共40个循环。反应过程中自动收集荧光。
3.研究结果
3.1慢病毒载体的鉴定
PTY-EF1α-EGFP质粒载体构建成功,琼脂糖凝胶电泳显示其大于符合预期,且Mlu和SmaⅠ限制性双酶切已不能再次将其切开。PTY-UBIQUITIN-EGFP, PTY-EF1α-EGFP, PTY-CMV-EGFP含三种不同启动子的慢病毒载体除启动子部分不同外,其余部分均一致。
3.2病毒滴度测定
荧光定量反应结束后,MX3005型荧光定量PCR仪自动输出标准品cT值,并据此输出标准曲线。从标准曲线图中可以看出,各梯度稀释倍数的质粒标准品大致位于同一直线上,样品稀释梯度明显,标准曲线准确。将样品cT值与标准曲线相比较,可知各组病毒液中基因组的拷贝数。依公式：病毒基因组拷贝数/毫升(vg/ml)=(病毒核酸拷贝数/2μl)×(20(μl/2μl)×(50μl/5μl)×1ml/200μl×稀释度计算,所得结果即为各种慢病毒滴度：LV-UBIQUITIN-EGFP 5.37×109 copies/ml, LV-EF1α-EGFP 3.89×109 copies/ml, LV-CMV-EGFP 4.20×109 copies /ml。
3.3倒置荧光显微镜下293A,MOLT4,PC3,DU145,RM1在不同慢病毒转导后的荧光强度的比较
293A,MOLT4,PC3,DU 145,RM 1各细胞分别在接受LV-UBIQUITIN-EGFP, LV-EF1α-EGFP, LV-CMV-EGFP转导72小时后拍摄荧光照片的结果显示,在293A细胞中,CMV启动子介导的目的基因(EGFP)表达,明显强于UBIQUITIN启动子和EF1α启动子；在PC3细胞中,也可以观察到相同的现象。而在MOLT4细胞中,EF1α启动子的表达效率则明显高于UBIQUITIN启动子和CMV启动子。同样的,在DU145细胞中,EF1α启动子的效率也是最高的,但是该细胞株中,UBIQUITIN启动子的效率虽然低于EF1α启动子,却明显高于CMV启动子。而在RM1细胞中,三种启动子表达均不明显,但其中EF1α启动子要强于UBIQUITIN启动子和CMV启动子。
3.4不同启动子转导细胞表达效率的流式细胞计数比较
CMV启动子在293A和PC3细胞中介导的目的基因表达效率是最高的,而EF1a启动子则在MOLT4和DU-145细胞中,介导了目的基因的高效表达,效果强于其它两种启动子。在RM1细胞中,三种启动子介导目的基因表达的效率均不高,但是EF1α启动子介导的外源基因表达(平均荧光强度)要强于其它两种启动子。其结果与荧光显微镜下观察到的趋势一致。
3.5 Realtime PCR法比较编码mRNA表达量差异
在293A和PC3细胞中,LV-CMV-EGFP转导后GFP基因编码mRNA的表达量高于LV-UBIQUITIN-EGFP和LV-EF1α-EGFP转导后GFP基因编码mRNA的表达量。在MOLT4, DU145和RM1细胞中,则是LV-EF1α-EGFP转导后GFP基因编码mRNA的表达量最高。这些都与荧光显微镜观察到的结果和流式细胞计数的结果相一致。
4.讨论
慢病毒转导是效率最高的转导和表达外源基因的方法之一。但慢病毒核酸结构的差异性可以极大的影响到外源基因表达的效率。在本研究中,我们分别构建了LV-UBIQUITIN-EGFP, LV-EF1α-EGFP, LV-CMV-EGFP三种不同启动子的慢病毒载体,并转导了293A, MOLT4, PC3, DU145和RM1五种不同的细胞。比较了三种启动子在同一细胞系中效率的差异。研究结果显示：三种启动子在各细胞株中的表达效率,由强到弱,依次为：293A:CMV, UBIQUITIN, EF1α, MOLT:EF1α, UBIQUITIN, CMV, PC3:CMV, EF1α, UBIQUITIN, DU145: EF1α, UBIQUITIN, CMV, RM1细胞：EF1α, UBIQUITIN, CMV。本研究的完成为我们在将来的科研中,针对不同的细胞系,有针对性的选择高效率启动子奠定了基础。
(二)双萤光素酶标记的慢病毒载体的构建及其表达特性研究
1.研究目的
构建一种分泌型萤光素酶Gluc和非分泌型萤光素酶Fluc共表达的双报告基因慢病毒载体,并建立稳定表达的前列腺癌PC3细胞系,以此为基础建立前列腺癌皮下肿瘤模型,对其体内外表达及活体成像特点进行研究。
2.研究方法
2.1 PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc双萤光素酶共表达质粒载体的构建及鉴定
采用分子克隆技术从pAAV2neoCAG-Gluc-2A-Fluc质粒中切取CAG-Gluc-2A-Fluc片段,并插入到去掉EGFP编码基因片段的PTY-UBIQUITIN-EGFP质粒载体中。再从pTR-UF11质粒上切下GFP-HSV-Tk-neo片段,定向克隆到新构建的载体中,酶切鉴定连接方向,取正向连接的质粒载体,转化大肠杆菌DH5α,挑取氨苄青霉素抗性单菌落提取质粒,双酶切鉴定。
2.2 PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒的细胞内瞬时表达检测
293T细胞在含10%胎牛血清的DMEM培养基的24孔板中,置于37℃,5%CO2的培养箱中进行贴壁培养。转染前一天接种细胞,待293T细胞汇合度达到90%时,采用脂质体转染法将PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc转染入293T细胞,转染步骤按lipofectamineTM 2000说明书进行。将等摩尔混合的PTY-UBIQUITIN-EGFP质粒共转染设为对照。转染24h后,倒置荧光显微镜下观察转染效率。收集细胞培养上清液和细胞裂解液。细胞裂解的具体步骤：将细胞用PBS清洗3遍,每孔加入200μl细胞裂解缓冲液,冰上放置30min后,将细胞悬液移入1.5 ml离心管,12000rpm离心15s。收集上清液,并测量其体积。分别加入浓度为5ug/ml的Glue底物腔肠素Coelenterazine200μl或150μg/ml的Flue底物luciferin 200ul,混匀后,用XenogenIVIS活体成像系统进行成像。曝光时间设定为l0s。
2.3小鼠PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒流体力学注射活体成像
6只6-8周龄、体重为18-20g的雄性BALB/c小鼠随机分为2组,每组3只小鼠。在5-7 sec内,采用水动力注射法,实验组每只小鼠经尾静脉注射2.0ml含10μgPTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc表达质粒的生理盐水,对照组每只小鼠只注射2.0ml生理盐水。注射12h后,经腹腔注射麻醉剂戊巴比妥钠(60-80mg/kg),麻醉后,经尾静脉注射300μL含50μg腔肠素的PBS溶液,立刻置于活体成像系统的暗箱中收集光子,每次收集光子3min,连续3次。待小鼠体内不再产生光子后(约30min),同一只小鼠腹腔注射D-荧光素钾盐(150mg/kg),注射1min、15min、30min后,分别用活体成像系统收集光子5 sec。
2.4双萤光素酶23T3细胞内的稳定表达
利用三质粒包装系统、脂质体共转染法,将PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒包装为慢病毒载体LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc,纯化后Realtime-PCR法测定其滴度。293T细胞以1×105/孔接种于24孔板中,24h后每孔接种1×107copies的病毒液(约50μ1),37℃、5%CO2培养箱中培养72小时。双萤光素酶基因Glue和Flue在细胞水平的动态监测研究在24孔板中进行。具体方法：将慢病毒转导后的293T细胞以1×104/孔接种于24孔板中,48小时、60小时、72小时分别收集细胞培养上清及细胞裂解液。分别取20μl加入Glue底物腔肠素和Flue底物D-luciferin混匀后,用XenogenIVIS活体成像系统进行成像。曝光时间设定为10s。
2.5双萤光素酶标记的裸鼠皮下移植瘤模型建立及表达监测
按照上文所述转导293T细胞同样方法,转导PC3细胞。同时设立转导无抗性的PTY-UBIQUITIN-EGFP质粒的PC3细胞作为阴性对照。转导72小时后,更换为含10%胎牛血清的DMEM培养基,同时加入neo基因抗性筛选药物G418,浓度为300ug/ml。之后依据细胞生长情况和对照组细胞死亡情况,逐渐调整G418浓度为700ug/ml。阴性对照组处理和实验组相同。直至阴性对照组细胞95%以上以死亡时,更换为不含G418,含10%胎牛血清的DMEM培养基。倒置荧光显微镜下观察绿色荧光对照基因表达。筛选得稳定表达的PC3-Gluc-2A-Fluc细胞。4-6周龄雄性裸鼠6只,体重为18-20g,称重,随机分为2组,每组3只。饲养于SPF环境。取对数生长期的PC3-Gluc-2A-Fluc细胞胰酶消化、离心弃上清后,以PBS重悬。细胞计数板计数,调整细胞浓度为5×106/200μl。按每只裸鼠5×106/200μl的剂量,将细胞悬液接种于3只裸鼠背侧皮下。对照组每只裸鼠皮下同侧位置注射200μl生理盐水。每3天,以XenogenIVIS活体成像系统观察肿瘤生长情况一次,连续观察2周。观察时裸鼠首先尾静脉注射300μl含50μg腔肠素的PBS溶液,注射后立刻成像。曝光时间设定为3min。待裸鼠体内不再产生光子后(约30min),腹腔注射D-萤光素钾盐(150mg/kg),注射后5min以活体成像系统观察。曝光时间同样设定为3min。上述裸鼠第7天起每3天分别经尾静脉采取全血20μl,加入Gaussia luciferase Assay Kit的底物50μl,混匀后,用XenogenIVIS活体成像系统进行成像,收集光子时间为lmin。
3.研究结果
3.1 PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒获得及鉴定
重组表达质粒PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc经酶切鉴定,以KpnⅠ、SacⅠ双酶切,能分别回收到3条片段,且最小片段约3000bp大小,与理论预测值相符,为正向连接。测序结果表明Gluc-2A-Fluc融合片段的顺序、方向及序列完全正确。
3.2293T细胞中Gluc和Fluc的瞬时表达及分布特点
PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc质粒转染293T细胞,24h后分别检测细胞培养上清液和细胞裂解液中Gluc和Fluc的活性及分布特点。Glue和Flue在细胞内外的分布特点各不相同。Glue活性主要分泌至细胞外的培养上清中,细胞内较少；而Flue细胞外活性较低,大部分的Flue存在于细胞内。
3.3 PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc动物体内瞬时表达及分布特点
活体成像结果显示,Glue和Flue在小鼠体内都能显像,但是它们具有明显不同的显像特点：注入Glue底物腔肠素后表现为全身显像,在肝脏显像不明显,而在表浅裸露部位(如口鼻和四肢)信号较强,并且显像信号迅速衰减,10min后基本消失；而注入Flue底物D-Luciferin后则主要在肝脏显像,且显像信号比较持续稳定,30min内衰减不明显。
3.4 Glue、Flue在293T细胞持续表达监测
LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc慢病毒转导接种于96孔板中的293T细胞,分别在48h、60h、72h检测细胞培养上清的Glue活性和细胞裂解液的Flue活性。结果显示双萤光素酶标记的慢病毒转导后的293T细胞,不同时间点都能检测到细胞培养上清中Glue的表达和细胞内Flue的表达。
3.5利用活体成像系统动态观察肿瘤生长
PC3-Gluc-2A-Fluc细胞接种于裸鼠背部皮下后,每3天用Xenogen IVISTM Lumina Imaging system成像系统对肿瘤的生长情况进行观察。如图2-8所示,Glue和Flue基因在底物作用下,都能够催化底物发光。且其发光均与肿瘤生长部位相同。随肿瘤生长,Flue与Glue发光均逐渐增强。Glue因底物直接注入血液的原因,发光更快达到峰值,但发光时间更短。在发光强度方面,Glue发光信号略强与Flue发光,两者处于同一数量级。
3.6小鼠全血内Glue表达持续监测
在不同的时间点经尾静脉采血,检测血液中Glue活性。结果显示,连续监测的21d内,尾静脉采20μl全血均能检测到Glue活性,且Glue活性随肿瘤的生长而逐渐增强,而生理盐水对照组不能检测到发光。
4.研究结论
4.1我们成功构建了双萤光素酶共表达慢病毒载体PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc。
4.2体外细胞实验验证表达特性及持久性
共表达载体PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc能同时高效表达分泌型的萤光素酶Gluc和非分泌型的萤光素酶Fluc,且Gluc主要分布在细胞培养上清中,Fluc主要存在于细胞裂解液中,未改变其原有表达特性。其包装为慢病毒并转导靶细胞后,Fluc和Gluc两种萤光素酶表达持久稳定。
4.3裸鼠皮下成瘤及裸质粒尾静脉注射转染法观察双萤光素酶体内表达特性及裸质粒
尾静脉裸质粒注射转染法结果证实,Fluc基因作为报告基因更适合于体内成像时的表达定位研究,而Gluc由于具有高效分泌入血的特性,在裸质粒流体力学注射转染这样的类似的手段中,用于成像定位研究时会造成定位错误的判断；可能也不适合尾静脉或心房注射方式建立肿瘤血液转移模型的定位检测。并且注射Gluc底物后其发光信号不稳定且发光时间短暂,也不利于成像分析。皮下PC3-Gluc-2a-Fluc肿瘤成瘤后监测双萤光素酶表达证实：Fluc和Gluc都能用于皮下肿瘤模型的定位监测,两种均能正确反映肿瘤的生长部位。其发光强度处于同一数量级,具有可比性。因此,在皮下肿瘤模型监测的应用中,两种萤光素酶具有同等的应用价值。
4.4裸鼠皮下成瘤模型尾静脉采集外周血监测肿瘤生长
尾静脉采集外周血动态监测肿瘤生长的结果表明：外周血中Gluc的表达量随肿瘤生长而逐渐升高,两者具有明显的相关性。可以通过检测外周血中的Gluc表达从而间接监测肿瘤的生长。
总之,本研究成功设计和构建了同时高效表达双萤光素酶的慢病毒载体PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc,并成功包装为慢病毒。并以此为基础建立了稳定表达的293T-Gluc-2A-Fluc和PC3-Gluc-2A-Fluc细胞系,以及PC3-Gluc-2A-Fluc裸鼠皮下肿瘤模型。实验证实我们建立的双萤光素酶慢病毒载体可以高效转导细胞,且转导后双萤光素酶表达稳定可靠。以这种双萤光素酶慢病毒载体建立的稳定表达细胞株,既可以利用分泌型萤光素酶Glue的分泌特性和高灵敏度,实现可以在不裂解细胞或处死动物的情况下通过检测细胞培养上清液或血液中Glue的活性来动态监测Glue的表达水平；同时又可以利用萤光素酶Flue的细胞内分布特点和发射光波长、穿透性好以及发光稳定等特点,发挥其在动物活体成像方面的优势,比单一的萤光素酶报告基因载体在细胞标记和体内示踪研究方面更具优越性。本研究为细胞及肿瘤动物模型的标记提供了一个有效的方法和工具,为进一步建立一系列萤光素酶标记的单克隆肿瘤细胞系的工作奠定了基础。
(三)腺病毒滴度不同测定方法的比较
1.目的：将目前测定腺病毒滴度常用的OD260法、荧光定量PCR法、绿色荧光蛋白标记法、壳蛋白免疫法、细胞病变(CPE)法进行比较研究,分析和讨论各种方法的优缺点。
2.方法：4种重组腺病毒(Ad-G-AT2R-EGFP、Ad-CMV-EGFP、Ad-mif-shRNA-EGFP和Ad-CBA-GFP)扩增、纯化后,同时分别用OD260值法、荧光定量PCR法、绿色荧光蛋白标记法、壳蛋白免疫法、及CPE法测定病毒滴度。并以壳蛋白免疫法测定结果作为标准,对各种方法所得结果进行比较研究。
3.结果：绿色荧光蛋白标记法、CPE法与壳蛋白免疫法同时测定4种腺病毒滴度,其结果无统计学差异(P>0.1)。荧光定量PCR法测定值明显高于壳蛋白免疫法(P<0.05),但与该法测定结果间存在良好的相关性(r=0.965),约为壳蛋白免疫法所测得结果的10倍。OD260法测得结果与壳蛋白免疫法测得结果间则无相关性。
4.结论：绿色荧光蛋白标记法、与壳蛋白免疫法是测定腺病毒感染性滴度的最为快速有效的方法。其结果客观可靠,能真实的反映病毒的实际感染力。荧光定量PCR法测定的结果反映的是完整的病毒颗粒数,能够间接的反映出病毒的感染力,且该方法操作简单,耗时短,同样有较高的实用性。
BACKGROUND
Prostate cancer(PCa)is a common disease in aged men. In America, PCa is the most common malignant illness in men, and the second leading cause of cancer death. Most PCa was not detected and diagnosed in the first time due to the deep in of prostate and the lack of obvious symptoms. Many patients were diagnosised too lated to take an redical operation. At present, there is still no effective method to treat prostate cancer, especially the androgen independent prostate cancer. Chemotherapy had led to stabilization of disease and improvement of symptoms but did not increase survival rate. The method of gene therapy is to transduct specific gene into target cells and fixes the problem in gene expression through exogenous gene. In the recent 10 years, as the development of molecular biology, gene therapy has made great pogoress in cancer therapy and succeeded in curing some monogenic diseases. Gene therapy has several advantages:①high selectivity comparing to Chemotherapy and radiotherapy, which can be obtained by using targeting gene.②less side effect③efficient even to the advanced stage cancer and metastasis. How ever, lots of things are still to be solved:①the lack of tumor-specific technology to direct the expression of therapeutic genes specifically to the tumor;①the antigenicity of the viral vector, which can deduce the immune response of body, makes it a temporary theatment;③the management of the exogenous gene expression.
In the progress of exogenous gene transduction and expression in target cells, the keypoint is to tranduce the vector into target cells and the get stable expression, so it is critical to choose the suitable vector. For now, there are mainly two kinds of vectors:non-viral vectors and viral vectors. Non-viral vectors includes gene sequence and plasmid vector. This kind of vectors is used in limited areas for the low expression efficiency. In contrast, the viral vectors have been widely used, it is estimated that 85% of the total gene therapy was carried on viral vectors. Amoung them, lentiviral vector is the most perspective one with great potential:①no limitation of the cell types:either dividing cell or not;②transducing the exogenous gene into target cell to obtain stable expression;③free of immunogenicity;④free of pathopoiesia areas.
The purpose of regulation of exogenous gene in cancer gene therapy is to target the exogenous gene efficiently and specically expressed in tumor cells without hurting the normal tissues. Transcriptional targeting of gene therapy is a novel and prospect method for cancer's treatment.It is a strategy to enhance the specificity of gene expression to target tumor cells of interest.This targeting approach is based on the use of tissue or tumor-specific promoters(TSP)in a heterologous context to direct the expression of therapeutic genes specifically to the tumor. An efficient gene therapy regimen requires transgene expression in the tumor and absence of expression in relevant normal tissues, especially in the liver.This combination of'tumor on" and "liver off" profile can result in increasing the therapeutic index and limiting the toxicity of vectors and transgenes in vivo.Many promoters have already been evaluated for transcriptional targeting in cancer gene therapy to be more sufficient specific to avoid gene expression in normal tissues,while shows noticeable expression levels in tumor cells. Promotor is the key of the efficiency of exogenous gene. It has been demonstrated in many researches that the same promotor shows obvious difference in efficiency in the different cell lines. Take the cytomegalovirus(CMV) promoter for example, it is highly effective in 293 cell lines, in C127 cell lines,however, its efficiency drops sharply. So it is crucial to choose the effective promoter according to different cancer cells. By doing this,the effective and selective expression of exogenous gene can be crchieved.
In the evalution of the gene therapeutic effect in animal models, we still a method of ex-vivo monitoring of in-vivo processes of the cancer cells,without killing the subjects until the end of our investigation. This means less animals will be needed during the total experiment and more exact diction. The luciferase is just this kind of marks we need.
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence. There is no endogenous expression in mammalian cell and its detection is not affected by the influence of other substances in cells.Luciferase has characteristics of high sensitivity and specificity,rapid response,simple operation,wide detection range, so as a reporter gene has been widely used in medicine, biology, environmental science,etc. One famous example is firefly luciferase (Fluc) from firefly Photinus pyralis.
As early as in 1956, Green and MeElroy got the Flue crystallization. Flue is a molecular weight of 60～64kD polypeptide chain. In a reaction that requires oxygen, magnesium, and ATP, Fluc oxidizes its substrate, luciferin, to produce light with a broad emission spectrum and a peak at approximately 560 nm. Only in living cells produce light-emitting phenomenon, and the light intensity is proportional to the number of labeled cells. As a most commonly used luciferase, Fluc has been widespread application in ATP rapid microbiology, infectious invasion,drug screening,cell marking and tracing as well as in vivo animal imaging, etc. Flue, optimized for expression in mammalian cells, is the most commonly used luciferase for in vivo imaging.
Discovered in recent years as a new type of luciferase, Gaussia luciferase (Glue) derived from the Gaussia princeps, its distinguishing feature is efficiently secreted from cells, also has a higher detection sensitivity.Gluc reacts with substrate, coelenterazine, to produce blue light with an emission peak at approximately 480nm,which is ATP-independent.As Glue has small molecular weight, secretion characteristic, detection sensitivity, a short half-life, etc,it has been successfully applied in the experimental study of in vitro and in vivo.By testing animal blood or other body fluids of Glue activity,Gluc can be quantitatively reflected tumor burden in animals,which not only track the progress of tumor metastasis, but also can serve as an effective marker of tumor response to treatment reflects.Luciferase with a specific substrate light-emitting reactions are chemiluminescent,which is specific and no cross-interference.
Based on these characteristics,in order to combine the advantages of secreted luciferase Glue and non-secreted luciferase Fluc,this study was to construct a novel dual luciferase co-expression vector and studied its expression characteristics in vivo and in vitro, for the next step to establish dual-luciferase gene markers of tumor cells and animal models, and lay the foundation for monitoring and tracing study later.
The article is component of two parts, the first part will discuss the optimal promoter choices for lentiviral vector-mediated transduction of different prostate cell lines; the second part details the design of the dual luciferase lebeled lentivirus vector with its character of expression in vitro and in vivo.
Part 1:optimal promoter choices for lentiviral vector-mediated transduction of different prostate cell lines
Objectives
To compare the expression of GFP driven by three different promoters carried by lentiviral vectors in 293A, MOLT-4, DU145, PC3 and RM1 cell lines. Demonstrate that different promoters showed various driven activities among different cell lines. So that promoter selection and proper host cells could be considered in order to achieve high level transgene expression in the further experiment.
Methods
1. construction of PTY-EF1α-EGFP, PTY-CMV-GFP and PTY-UBIQUITIN-GFP
PTY-EF1α-EGFP (EF1α-GFP) was constructed by excising the IRES sequence from PTY-EF1α-IRES-GFP. PTY-CMV-GFP (CMV-GFP) were constructed by replacing the EF1αpromoter in PTY-EF1α-GFP with human cytomegalovirus promoter.
2. package and titer lentivirus with different promotor
293T cells were planted in T25 flask and transfected with shuttle plasmid PMD2G, PSPAX2 and packaging plasmid PPAX2 following the instruction of lipofectamineTM2000. Harvest virus 60h after transfection. Purified the lentivirus with ultracentrifuge, stored at -80℃. Some samples was subjected to quantitative PCR analysis using the stragene 3500 system, to quantify genomic titer. Lentivirus was serially diluted and sequentially digested with DNase I. Viral RNA was extracted with Roche viral RNAmini kit. A standard amplification curve was set up at range from 105 to 108 copies and the amplification curve corresponding to each initial template copy number was obtained.
3. vitro transduction of different cells
293T and 293A cells, DU145, PC3 cells and RM1 cells were cultured according to the instruction of ATCC.293A, DU145, PC3, RM1 and MOLT-4 cells were plated at the appropriate density (70-80% confluence the following day). Medium was removed and cells were transduced with maintaining medium containing 108 particles of each recombinant lenti virus per well for 6-well plates and incubated at 37℃,5% CO2 for 3 days before assay.
4. Visualization of GFP
GFP fluorescence were directly imaged on an Olympus Model BX41 fluorescent microscope (Olympus, Tokyo, Japan). All cell images were captured at×10 magnification and All fluorescent images were collected using an identical exposure time.
5. Flow cytometry Analysis of EGFP fluorescence in transduced cells was performed by flow cytometry. Briefly, cells were harvested with Trypsin-EDTA Solution. Cells were washed and resuspended in PBS for analysis on a FACS Calibur (Becton Dickinson, MA, USA). Samples were analyzed using CellQuest oftware (Becton Dickinson, MA, USA).
6. Total RNA extraction and quantitative real-time PCR analyses
Total RNA was isolated from the transduced cultures using RNeasy Mini Kit (Qiagen,Valencia,CA). RNA concentration was quantified by the ultraviolet spectrum at 260 nm, and reverse transcribed using PrimeScript 1st Strand cDNA Synthesis Kits (TaKaRa) according to the manufacturer's instructions. Synthesized cDNA corresponding to 100 ng of total RNA, was used for real-time PCR. Specific primers for the GFP and ribosomal RNA (18sRNA; internalcontrol) genes were used in real-time RT-PCR analysis. All primers were synthesized from Invitrogen company. The primers used were as follows:EGFP-F:5'-GAGCTGAAGGGCATCGACTT-3', EGFP-R:5'-CTTGTGCCCCAGGATGTTG-3'; 18sRNA, forward primer: 5'-CAGCCACCCGAGATTGAGCA-3', reverse primer:5'-TAGTAGCGACGGGCGGTGTG-3'.The SYBR green real-time PCR assays for each target gene were performed on cDNA samples using an Stragene 3005 Detection system (Agilent Biosystems).18s RNA assays were run in parallel for each sample. Amplication was carried out in optical 96-well reaction plates (Agilent Biosystems)with each well containing 2μl of cDNA template,1μl of sense primer (10μM),1μl of antisense primer (10μM),12.5μl of SYBR Premix Ex TaqTM (TaKaRa),0.4μl of ROX and DEPC- treated water (8.1μl). The conditions for real-time PCR were one cycle of 95℃for 30 s, followed by 40 cycles of 95℃for 5 s and 60℃for 30 s.
Results
1. Identification of lentiviral vector
Recombinant plasmid PTY-EF1α-EGFP and PTY-CMV-EGFP was identified by restriction enzyme digestion with Mlu和SmaI. No fragment can be obtained again, which indicates that the vectors have been built successfully.
2. Titer of lentiviraus produced
Compare the cTvalue of samples with standard curve to get the exact copy in every ml of viral solution. The titers of the lentivirus are listed below: LV-UBIQUITIN-EGFP 5.71*108copy/ml, LV-EFla-EGFP5.53*108copy/ml, LV-CMV-EGFP 5.87*108.
3. Evaluation of transgene expression by lentiviral vectors in vitro using flourscent microscopy
293A, DU145, PC3, RM1 and MOLT-4 cells were transduced with 109 copies/ml of either LV-UBIQUITIN-EGFP, LV-EFla-EGFP or LV-CMV-EGFP and,72h later, we compared activity of the UBIQUITIN, EF1αand CMV promoters in various cell lines. GFP expression was analyzed by visualizing green fluorescence. All three lentiviral vectors were capable of producing gene transfer to cell lines, but in various cell lines, they showed different driven activities. In 293A cell lines, all three CMV, EF1αand ubiquitin promoters showed high activities. EGFP expression driven by CMV promoter peaked earlier than the two other promoters (not shown) and showed the highest activity. Also in PC3 cells, CMV promoter showed the highest activity. In the contrast, the CMV promoter showed much lower activity than the two other promoters in MOLT-4 cells. In the present study the CMV promoter showed a different driven activity in vitro among different cells tested, in fact, we found that it is the weakest promoter tested in vitro among MOLT-4 cells and DU145 cells. However, in DU145 cells, EF1αpromoter showed the highest activity, UBIQUITIN promoter comes the second. The CMV promoter shows the weakest activity among them. In RM1 cells, no promoter showed high activity, but the EF1αpromoter activity is higher than the other two.
4. Evaluation of transgene expression by lentiviral vectors in vitro using flow cytometry
To determine whether the level of EGFP expression is only dependent on the strength of promoters,293 T cells were transduced with. LV-UBIQUITIN-EGFP, LV-EF1α-EGFP or LV-CMV-EGFP vector.72h after transfection, the cells are digested and resuspended in PBS. Cell concentration per milliliter was determined using a hemocytometer. All the cells were tested using a Aliquots of 200μL of each sample was analyzed on a FACScan cytometer (Becton Dickinson, San Jose, CA) equipped with an argon ion laser set at 488 nm line and fluorescence detector wavelength of 530/30 bandpass using Summit Software with the Cicero upgrade (Cytomation, Ft. Collins, CO). The results shows that the GFP expression from LV-UBIQUITIN-EGFP, LV-EFla-EGFP and LV-CMV-EGFP vectors are varied. The data showed that 293A cells harboring CMV-EGFP, UBIQUITIN-EGFP and EF1α-EGFP vectors displayed high, medium, and low intensity of green fluorescence, respectively. The results suggests that the CMV-EGFP, UBIQUITIN-EGFP and EF1α-EGFP promoters are listed in the order of promoter activity from the highest to the lowest in the 293 A cells, totally corresponds well with the results in the past chapter. Similar results can be draw in other cells. In MOLT4 cells, the order from high to low is; while CMV EF1α, UBIQUITIN in descending order in PC3 and EF1α, UBIQUITIN, CMV in DU145. In RM1 still no promoter shows high activity, but the EF1αpromoter is still higher, then comes the UBIQUITIN promoter, CMV promotor is the last one.
5. Evaluation of transgene expression by lentiviral vectors in vitro by Realtime-PCR
The lentiviral stocks were further analyzed byquantitative PCR to determine the DNA titer and the same DNA titers of LV-UBIQUITIN-EGFP, LV-EF1α-EGFP and LV-CMV-EGFP lentiviral stocks were used to transducer 293A, MOLT4, PC3, DU145 and RM1 cells. Negative controls were set up with DEPC H2O correspondingly. The results showed that the EF1αpromoter exhibited the highest activity in MOLT4, PC3, DU145 and RM1 cell lines. The CMV promoter appeared to be the strongest in 293A cells. All the results corresponded well with the results of flow cytometry except the order in PC3. This may be caused of the different expression in mRNA and protein.
Discussion
The lentiviral transduction is one of the most effective methods to overexpress transgenes. However, the influence of the lentiviral DNA context on overexpression still has to be considered. In this study, we have shown that the GFP expression from the UBIQUITIN, EF1αand CMV promoters encoded in three lentiviral vectors, reveals sigficant differences in 293A, MOLT4, PC3, DU145 and RM1 cells. The differential expression of GFP reporter may be due to the specific characters of these promotors as the other parts of the lentivius are the same. The results indicate that the differential expression of GFP between the three promotors in different cells is due to the lentiviral genomic sequences but not the plasmid topology. In PC3, the results of Realtime-PCR did not correlate well with that of flow cytometry and flourscent microscopy. This may be caused by the differenct expression in mRNA and protein. Some mRNA seemed were not translated into protein. The CMV promoter may not always be the best choice for transgene overexpression in all cell types. By comparing to other promoters, while CMV promoter gave much more than 6-fold of GFP expression than human house keeping gene EFla promoter in 293A and PC3 cells, the GFP production by EF1αpromoter revealed the highest intensity in MOLT4, DU145 and RM1 cells.
Part 2:design of the dual luciferase lebeled lentivirus vector with its character of expression in vitro and in vivo
Objectives
To constructe a novel dual luciferase co-expression lentiviral vector, which expressed simultaneously secreted luciferase Gluc and non-secreted luciferase Fluc, transducer 293T and PC3 cells with it, establish Subcutaneous tumor model of prostate cancer and study its expression characteristics in vitro and in vivo.
Methods
1. Construction of co-expressed plasmid PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc with its identification
The sequence CAG-Gluc-2A-Fluc was deprived from plasmid pAAV2neoCAG-Gluc-2A-Fluc and incerted ito PTY-UBIQUITIN-EGFP vector deprived off GFP coding gene. Sequence GFP-HSV-Tk-neo was then deprived from plasmid pTR-UF11 and cloned into it. The production was identified and then transformed into E. coli DH5a, picked a single colony which is ampicillin resistance,extracted plasmid, and then identified by restriction enzyme digestion and sequencing.
2. PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc transient transfection and character of expression
293T cells in 10% FBS DMEM medium, placed in 37℃,5% CO2 of incubator for adherent culture. When degree of 293T cells confluence reached 90%, the plasmid was transfected into 293 T cells and the operation steps were carried out according to the instructions of lipofectamineTM2000, control plasmid PTY- UBIQUITIN-EGFP was co-transfected with the equal copies. Expression and distribution characters of Gluc and Fluc in PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc in vitro were carried out in 24-well plates. Transfection after a 24h, cell culture supernatant and cell lysate was collected. Steps of cell lysis were as follows:washed the cells three times with PBS, added 200μl cell lysis buffer to each hole, placed on ice 30min, then moved cell suspension to 1.5 ml centrifuge tubes, centrifuged for 15 sec at 12 000 rpm,after that collected supernatant and measured its volume. Detection of Gluc and Fluc activity in cell culture supernatant or cell lysate was done in accordance with instructions of Gaussia luciferase Assay kit and Luciferase Assay System. Coelenterazine (concentration of 5ug/ml,200μl) or of Fluc substrate luciferin (150μg/ml,200ul) were added, images were taken instantly with XenogenIVIS vivo imaging system. Time for collecting photon counts was 10 sec.
3. Bioluminescence imaging of mice injected with PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc through tail vein
6 six to eight-week-old, weighing 16～20 grams of male BALB/c mice were randomly divided into two groups,3 mice each group. Within 5-7 sec, through tail vein, each mouse in the experimental group was hydrodynamically injected 2.0ml normal saline containing 10μg UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc through tail vein expression plasmid, while only injecting 2.0ml normal saline per mouse in the control group.
4. Detect activity of Gluc and Flue in vitro
Using three-plasmid packaging system, and lipfectimine transfection method, the PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc plasmid was packaged into lentiviral vector LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, then purified and Realtime-PCR method was used for the determination of its titer.293T cells was panted with a density of 1×105/hole in 24-well plates, transduced with 1×107copy per well of virus solution (approximately 50μl), incubated in a 37℃,5% CO2 incubator for 72 hours. Dual Glue and Flue activity dynamic monitoring was carried out in 24 well plates. After lentiviral transduction,293T cells were plated in 24-well plates,1×104/hole,48 hours later the cells were collected every 12 hours for both culture supernatants and cell lysates. According to kit instructions, add Coelenterazine (concentration of 5ug/ml,200μl) or of Flue substrate luciferin (150μg/ml,200ul), images were taken instantly with XenogenIVIS vivo imaging system. The photon collection time is 10 sec. Continuous observation was taken on for 72h.
5. Establishment of dual luciferase labeled subcutaneous transplantation tumor model in nude mice and the dynamic monitoring of their expressions
Transduce PC3 cells in the same way which 293T cells were transduced as described above. Negative control was set up with transduction of lentivral vector LV-UBIQUITIN-EGFP, without neo gene, into PC3 cells. Replace the medium with DMEM containing 10% fetal bovine serum medium 72 hours after transduction, while adding G418, with the concentration of 300ug/ml. Adjust the G418 concentration gradually to 700ug/ml. Negative control group was treated the same with experiment group. When 95% of negative control cells died, replace the medium with DMEM containing 10% fetal bovine serum medium free of G418. GFP was observed under fluorescent microscope. The cells left was PC3-Gluc-2A-Fluc with stable expression of dual luciferase. Coelenterazine (concentration of 5ug/ml,200μl) or of Fluc substrate luciferin (150μg/ml,200ul), images were taken instantly with XenogenIVIS vivo imaging system. Exposure time is set to 3min, repeat this triplex.6 nude mice (male,4-6 week old) were fed in the SPF environment. PC3-Gluc-2A-Fluc resuspended in PBS (5×106/ml,200μl/mice) was injected subcutaneously into the flank of nude mice. The control group mices were inject with 200μl NS at the same location. Photos were taken every 3 days with XenogenIVIS system to observe the tumor growth, the observation lasted continuously for 2 weeks. Extract 20μl blood of the mice via the tail vein every 3 days since day 7, add Gaussia luciferase Assay Kit substrate 50μl, Coelenterazine (concentration of 5ug/ml,200μl) images were taken instantly with XenogenIVIS vivo imaging system, with a collection time of 1min.
Results
1. Identification of PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A--Fluc plasmid
Recombinant plasmid PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc was identified by restriction enzyme digestion with KpnⅠand SacⅠ. We obtained 3 fragments, a size of 2274bp fragment was our target fragment. sequencing results showed that the direction and sequence of GFP-HSV-Tk-neo-CAG-GIuc-2A-Fluc fragment was entirely correct.
2. Expression and distribution characters of Glue and Flue in vitro
Expression and distribution characteristics of Glue and Flue of transient transfection with PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc in 293T cells were namely:24 hours post transfection, the expression of Glue and Flue can both be detected in cell supernatants and cell lysates, most of Glue was mainly detected in the culture media most Flue was mostly within cells;the activity of Glue in the supernatant increased gradually with time while the Flue activity in cells almost kept stable.
3. Bioluminescence imaging of mice
In vivo imaging results showed that, both Glue and Flue can take imaged in vivo imaging system, but they have distinctly different imaging characteristics:after the injection of Glue substrate coelenterazine, images show that luminescence can be seen throughout the body, the signal from liver shows no difference in contrast with other organs, while in superficial exposed parts (such as the nose and mouth, and limbs) signals were strong, and the imaging signals decreased sharply within 10 minutes; Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour.
4.Stable expression of Glue and Flue in vitro
LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc transducd 293T cells in 96-well plates,, in 24h,36h,48h,60h,72h detect Glue activity in cell culture supernatant and Flue activity in cell lysate respectively. The results showed that after transduction of 293T cells with dual luciferase, Glue activity and Flue activity can either be detected in cell culture or cell lysates. intracellular expression of and Glue activities in the supernatant gradually increased with time, while Flue activity in cells remained stable. Cells and cell supernatant were added respectively with Glue substrate Coelenterazine and Flue substrate Luciferin, then instantly taken images in XenogenIVIS imaging system. The results showed:in the cell supernatant and the cells the expression of GLuc and Fluc activity can both be detected respectively. GLuc signal in supernatant is stronger than signal of Flue intracellular. With Continuous observation of 30min, Glue images disappeared within 15min, Fluc image remained stable within 30min.
5. Dynamic observation of tumor growth in vivo with dual luciferase
PC3-Gluc-2A-Fluc cells were into the flank of nude mice subcutaneously, the growth of tumors was observed every 3 days with the Xenogen IVISTM Lumina Imaging system imaging system. The results show that Glue and Flue can both catalyze the light-emitting substrate respectively. As the tumor growth, Flue and Glue light-emitting gradually increased. Because Glue substrate Coelenterazine was directly jnjected into blood, the signal of Glue lasted for just a short time, while the signal of Flue lasted much longer. In the luminous intensity, Glue signal is slightly stronger than Fluc signal; both are in the same order of magnitude.
6. Dynamic monitoring of Gluc activity in blood
20μl of blood taken through tail-vein was assayed for luciferase activity with Gaussia luciferase Assay kit using a luminometer at various time points. The results showed that at least within 21 days 20μl whole blood by tail vein was able to detect Glue activity, Glue signal growed as the tumor growed.
Conclusions
1.We have successfully constructed a dual-luciferase expression vector PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-GIuc-2A-Fluc.
2.Cells in vitro experimental verification
co-expression LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc lentival vector can mediate simultaneously high-level expression of secreted report gene Glue and the non-secreted report gene Flue, and Glue mainly distributed in the cell culture supernatant, Flue mainly exists in the cell lysate, indicating expression and distribution characteristics of Glue and Flue were not changed. After transduced with LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, cells expressed dual luciferase stably and consistantly.
3.In vivo imaging of dual luciferase
Tail vein injection of plasmid transfection method confirmed, Flue as a reporter gene is more suitable for in vivo localization of gene expression, while Glue has the character of secretion into the blood with high efficiency. Because of this, if Glue was used in ways like hydrodynamic method, errors may occur when using it for localization. Glue may also not be suitable for establishment and imaging localization of the tumor metastasis animal model of blood through tail vein or atrial injection. After injection of Glue substrate, the luminescence signal appears to be instability and temporary, which is not quite convenient to imaging analysis. Subcutaneous PC3-Gluc-2a-Fluc tumor surveillance luciferase expression confirmed that Flue and Glue subcutaneous tumor model can both be used to monitor the positioning of the tumor in vivo. Both of them can correctly reflect the position and scale of tumor growth. Their luminous intensity are in the same order of magnitude. Therefore, Glue and Flue contributes equally to the monitoring of subcutaneous tumor model.
4. Test of blood through tail vein of tumor model in nude mice to monitor tumor growth
Dynamic monitoring of Glue in blood through tail vein in reflacting tumor growth shows that:expression of Glue in peripheral blood gradually increased with tumor growth, with a significant correlation between the two. Peripheral blood can be used to monitor Gluc activity in reflacting tumor growth indirectly.
In conclusion, we successfully construceted the dual luciferase expression lentiviral vector LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, established the 293T-Gluc-2A-Fluc cell lines and PC3-Gluc-2A-Fluc cell lines and established animal models with these cell lines. The new lentiviral vector combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing. On one hand, we can use Gluc, which has secretion characteristics and high sensitivity, to dynamically monitor the expression levels of Gluc by detecting Gluc activit in cell culture supernatant or blood without clearaging cells or sacrificing animals; On the other hand, we can use Fluc, which distributes in the intracellular and emits spectra long, penetrates well, and imaged stablily, to localize biological processes in vivo animal imaging.
Part 3:A Comparison and Analysis of Methods for Titering Adenoviruses
1. Object:To compare different methods commonly used for titering adenovirus and discuss the advantages and drawbacks of each method.
2. Methods:Four types of recombined adenovirus were amplified and purified. Each is then simultaneously titered by optical absorbance, Real-time PCR, green fluorescent protein (GFP) labeled method, immunoassay, and cytopathic effect (CPE). Results are then compared.
3. Results:No significant difference was found between the titer amounts derived from the GFP labeled method, immunoassay, and cytopathic effect method (P>0.1) There is a position correlation between the titer amounts (r=0.965), even though Real-Time PCR resulted in ten times the vg/ml amount compared to the ifu/ml amount obtained from the immunoassay.
4. Conclusion:These results indicate that the GFP labeled method and the immunoassay can quickly determine the titer amount. Real-time PCR cannot titer the real infectious titer of the adenovirus, but the results indicate that its approach has good correlation with that of immunoassay, so it can also reflect the infectious titer of adenovirus, though not that accurate. As the Realtime PCR method takes the least time, it still worth using.
引文
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