CN106635962B - 一种人正常阴道上皮3d分化培养物模型的构建方法与用途 - Google Patents
一种人正常阴道上皮3d分化培养物模型的构建方法与用途 Download PDFInfo
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Abstract
本发明属于生物医药领域,公开了一种人正常阴道上皮3D分化培养物模型的构建方法与用途。提供2D生长培养基,获得分离培养自女性阴道癌旁正常组织的正常阴道上皮细胞,未导入任何外源基因,具有正常分化的生理功能。其构建人正常分化阴道上皮3D模型的方法为:2D培养基重悬单细胞,接种到气‑液培养装置中,生长培养基更换为分化培养基培养14‑21天。人阴道上皮3D气‑液培养物分化完全后,接种HSV‑2病毒液,获得HSV‑2病毒感染3D模型。这两种3D模型可用于人正常生殖道上皮的生理学研究和药物毒性安全性评价,HSV‑2病毒感染性疾病、性传播病原体感染性疾病的发病机理研究,以及抗病毒药物的研发。
Description
技术领域
本发明属于细胞生物学领域,涉及一种人正常阴道上皮3D分化培养物模型的构建方法及其用途。
背景技术
人体重要器官都是由器官特异性分化的上皮细胞为其组成成分。这些特异性分化的上皮细胞与不同器官的特异性功能直接相关。生殖道粘膜上最接近于生殖道管腔的是上皮组织层,上皮层暴露于生殖道管腔内的各种病原体中,将生殖道中的病原体与生殖系统分隔开来。因此,生殖道上皮层是机体抵抗外界病原体的第一道天然的物理屏障。而这些重要器官一旦发生病变或退化就会威胁人类的健康,因为这些重要器官是很难被替换的,不同器官的特异性细胞也不能相互取代其它器官的细胞。这些特异性分化的细胞都是很难再生的,更不要说在体外进行培养增殖了。
目前对于分离自人和哺乳动物的原代上皮细胞的体外培养仍然是很困难的。应用无血清培养基,有的只能进行短期的原代培养(存活数天,如肺和胰腺等上皮细胞),有的只能进行有限的传代培养(1-3代,如气管/角膜等上皮细胞),有的甚至根本不能体外培养(如肝、结肠、前列腺等上皮细胞),只有来自于人体皮肤的角化上皮细胞可以传代培养10代左右。而且从每个动物或活体组织样品中获得的上皮细胞产量仍然很低,包括细胞的数量、直接分离或短期培养的细胞纯度都是很低的,这些原代和传代上皮细胞的增殖速度也极为有限。为了在体外进行上皮细胞的培养,目前国外尝试通过遗传操作,如转入病毒(SV40 T或HPV16E6E7)或细胞癌基因,可以延长体外细胞存活的代数。然而遗传操作的最大缺点是会改变这些细胞的遗传背景和表型,以致让这些正常上皮细胞丢失其正常生理功能,因此这些细胞也不具备正常分化功能。
上皮组织层具有一定的极性,主要分为三层细胞:基底层、中层、表层,从而形成了两个不同的表面:顶端面和基底外侧面。顶端面主要面对外环境,而基底面则是接触底层细胞以及全身脉管系统,其形态学及分化程度在研究病原体感染激活的免疫反应中至关重要。性传播病原体如HSV-2病毒在生殖道粘膜上皮的复制以及向神经系统的传播,很大程度上依赖于极性分化的上皮细胞。由于生殖道分层极化上皮组织与其它上皮组织明显不同,对性传播病原体易感性以及药物的毒性反应有显著差异。因此,利用常规二维的细胞培养模型来研究性传播病原体感染机理始终存在着明显的局限性。另一方面,近几十年间的研究大多使用动物模型来研究病毒HSV-2疫苗,由于动物和人类之间的种属差异性,通过动物模型研发的HSV-2疫苗在人身上的效果不明显,因此这些处于动物研究水平的HSV-2疫苗很少能够运用到临床上。同时,利用动物模型并不能更进一步地研究微观的HSV-2感染致病机理。因此,建立一个接近人类生殖道上皮生理状态的研究模型意义重大。
发明内容
为了克服现有技术的缺陷,本发明的目的在于提供一种人正常阴道上皮3D分化培养物模型的构建方法,包括如下步骤:
A、人正常阴道上皮细胞原代分离培养:
(1)在病人或病人监护人知情同意的情况下,收集阴道癌患者手术切除的癌旁正常组织样品。
(2)消化液的配制:含胶原酶和分散酶均为0.2mg/mL的原代上皮细胞生长培养基;其中,原代上皮细胞生长培养基(2D培养基)的配制:DMEM与Ham’s F-12 NUTRIENT MIX按体积比3:1混合的培养基,同时添加4-6%胎牛血清、1-3nM三碘甲状腺氨酸(triiodothyronine),0.4-0.65%胰岛素铁硒传递蛋白(insulin-transferrin-selenium)试剂、4-6μg/ml铁传递蛋白(transferrin)、9-11ng/mL表皮生长因子、0.3-0.5μg/mL氢化可的松、35-45μg/mL庆大霉素、45-55nM calpeptin、35-45ng/ml重组人IL-1RA,及3μg/ml重组人R-Spondin-1。
(3)用95~100%(v/v)的乙醇洗分离的组织样品1次,再用PBS(0.01M,pH 7.4)洗2次,然后将组织放入含冰上预冷PBS的无菌培养皿中,在解剖显微镜下,用解剖镊子和剪刀去除组织中残留的脂肪。
(4)将正常阴道组织样品放入10倍于组织样品体积的含步骤(2)所配制消化液的无菌培养皿中,37℃消化1~3小时。
(5)将消化后的组织低速离心(1000rpm)5分钟,去除上清。
(6)将细胞沉淀重悬于2-5mL的0.25%(质量体积比)胰酶-EDTA中,置于冰上1小时或室温消化10分钟。
(7)然后加入10mL含10%(v/v)FBS的DMEM培养基,低速1000rmp离心5分钟,尽量将上清去除干净。
(8)加入2mL温水浴(37℃)的5mg/mL分散酶和200μL的1mg/mL DNaseI,用无菌的P1000一次性塑料枪头反复吹打样品1分钟。
(9)加入10mL含10%(v/v)FBS的DMEM,用40~70μm孔径的过滤器过滤细胞悬液,收集过滤后的细胞悬液,低速1000rmp离心5分钟,去除上清。
(10)重悬细胞沉淀于2D培养基中,接种于T25或T75的培养瓶培养,培养条件是37℃、5%CO2。
B、人正常阴道上皮细胞的传代培养:
(1)当在T25或T75的培养瓶中培养的人正常阴道上皮细胞增殖至70~90%丰度时,用1×PBS(0.01M,pH 7.4)洗涤细胞三次,再用0.05%(质量体积比)胰酶-EDTA消化单层细胞2~5分钟。
(2)加入10mL DMEM中和消化反应1~2分种。
(3)1000rmp离心5分钟,弃上清。
(4)以1:2,1:3,1:4或1:5比例重悬细胞沉淀于2D培养基,接种于培养瓶培养,培养条件是37℃、5%CO2得到人正常阴道上皮细胞。
(5)必要时可将1×106上皮细胞重悬于1-2mL的细胞冻存液(90%胎牛血清和10%DMSO,v/v)中,储存于液氮中备用。
C、人正常阴道上皮的气-液3D培养:
(1)将0.4μm的Millicell PCF insert(12mm size,Millipore)放入六孔板中,每孔最多放三个inserts。
(2)用400μl 2D培养基重悬5x105个的人阴道上皮细胞,然后接种到每个insert中。
(3)每个孔板内部即insert外围加入2ml的2D培养基。
(4)将放有insert的六孔板放入湿热培养箱中,37℃,5%CO2培养时长为48小时。
(5)将insert内部及外部中的培养基更换为3D分化培养基。
3D分化培养基配制为:DMEM与F12按体积比1:1混合的培养基,同时添加0.5-1.2μM胰岛素(Sigma-Aldrich I6634)、0.05-0.2μM铁传递蛋白(Sigma-Aldrich T0665),0.05-0.15μM氢化可的松(Sigma-Aldrich H0396),0.005-0.015μM三碘甲状腺氨酸(Sigma-Aldrich T6397),1-4μM肾上腺素(Sigma-Aldrich E4642),0.1-1ng/mL表皮生长因子,2x10–8-8x 10–8M视黄酸(Sigma-Aldrich R2625),0.1-1μM磷酸乙醇胺(Sigma-AldrichP0503),0.1-1μM氨基乙醇(Sigma-Aldrich E0135),1-5μM硫酸锌(Sigma-Aldrich Z0251),100U/mL青霉素G硫酸(Sigma-Aldrich P3032),100μg/mL链霉素硫酸盐(Sigma-AldrichS9137),0.5-1.5mM氯化钙(Sigma-Aldrich C3881)。
(6)将放有insert的培养皿放入湿热培养箱中,培养15-17小时,使insert内部的细胞与细胞之间形成紧密连接。
(7)移除insert内部及外部所有的培养基,外部的培养皿中加入3D分化培养基,开始分化培养。
(8)在湿热培养箱中培养阴道上皮细胞14-21天,培养条件为37℃,5%CO2,每2-3天更换insert外围的培养基。
优选地,上述步骤A(2)2D培养基的配制为:DMEM与Ham’s F-12NUTRIENT MIX按体积比3:1混合的培养基,同时添加5%胎牛血清、2nM三碘甲状腺氨酸(triiodothyronine),0.5%胰岛素铁硒传递蛋白(insulin-transferrin-selenium)试剂、5μg/ml铁传递蛋白(transferrin)、10ng/mL表皮生长因子、0.4μg/mL氢化可的松、40μg/mL庆大霉素、50nMcalpeptin、40ng/ml重组人IL-1RA,及3μg/ml重组人R-Spondin-1。
优选地,上述步骤C(5)3D分化培养基的配制为:DMEM与F12按体积比1:1混合的培养基,同时添加0.87μM胰岛素(Sigma-Aldrich I6634)、0.125μM铁传递蛋白(Sigma-Aldrich T0665),0.1μM氢化可的松(Sigma-Aldrich H0396),0.01μM三碘甲状腺氨酸(Sigma-Aldrich T6397),2.7μM肾上腺素(Sigma-Aldrich E4642),0.50ng/mL表皮生长因子,5x 10–8M视黄酸(Sigma-Aldrich R2625),0.5μM磷酸乙醇胺(Sigma-Aldrich P0503),0.5μM氨基乙醇(Sigma-Aldrich E0135),3.0μM硫酸锌(Sigma-Aldrich Z0251),100U/mL青霉素G硫酸(Sigma-Aldrich P3032),100μg/mL链霉素硫酸盐(Sigma-Aldrich S9137)。
在本发明的一个实施例中,将分化完成的人正常阴道上皮3D培养物放入4%多聚甲醛(wt/vol)中,4℃固定过夜,根据标准制作流程制作石蜡病理切片,H&E染色后显微镜下观察,如图1所示,正常阴道组织(A)和3D分化培养物(B)具有相似的结构特征,主要分为三层,最上层的角质层、中间的移行层和最下层的基底层,表明我们所建立的3D分化培养物不仅可以在体外正常分化,且具有与正常阴道组织相似的结构特征。
在本发明的一个实施例中,将获得的人正常阴道上皮3D培养物的石蜡病理切片进行DAB免疫组化分析,如图2所示,正常的阴道上皮组织和体外建立的3D分化培养物具有相同的特异性蛋白标志物和细胞紧密连接相关蛋白的表达特点(主要体现在蛋白分子的表达分布和表达量上),即在基底层表达CK14、p63和Laminin,在基底层和移形层中表达E-cadherin和DSG-1,且DSG-1的表达量都是高于E-cadherin,同样地不表达CK18蛋白。
在本发明的一个实施例中,将HSV-2病毒感染人正常阴道上皮3D培养物,观察不同时间点H&E染色的形态学变化,如图3所示,随着HSV-2病毒感染时间的延长,体外构建的3D分化培养物呈现出相应病理学变化,主要表现为病毒感染后,破坏程度依次加重,从最初角质层变得松散开始,到波及中间的移行层直至最后基底层受到严重破坏。
在本发明的一个实施例中,将HSV-2病毒感染人正常阴道上皮3D培养物,观察不同时间点的DAB免疫组化标记细胞紧密连接相关蛋白及阴道上皮细胞特异性分子表达量的变化,图4所示,病毒在感染3D分化培养物不同时间点后,依然保留了细胞紧密连接相关蛋白的表达,通过观察上述图中相应蛋白的表达,可反映出HSV-2病毒感染3D分化培养物不同时间点,对3D分化培养物的破坏程度依次递增。
在本发明的一个实施例中,将HSV-2病毒感染人正常阴道上皮3D培养物,观察不同时间点的免疫荧光检测HSV病毒特异性标志蛋白gB的表达量的变化,如图5所示,在不同时间点,HSV-2病毒均成功感染人正常阴道上皮3D培养物并进入细胞后复制表达,因此可以检测到病毒晚期复制蛋白gB的表达,且呈现出周期性的表达变化趋势。
因此,本发明提供了具有正常极性分化的3D培养模型,可作为研究性传播病原体包括病毒感染的一种体外研究系统,能在三维的条件下重现上皮细胞在体内环境的生长和分化情况,能更好模拟体内环境和机体正常生理状态,成为研究病毒或病原体感染、预防和治疗性疫苗、抗病毒药物以及基因治疗载体的绝佳体外模型和系统。
本发明的另一个目的是提供人正常阴道上皮细胞3D培养模型在制备抗性传播病原体药物中的应用。
本发明相对于现有技术具有如下优点和效果:
1.本发明提供的2D培养基,可以原代分离培养自人的正常阴道组织的人正常阴道上皮细胞,该细胞未导入任何外源基因,为人的正常细胞;利用本发明提供的3D分化培养基,通过气-液3D培养条件下获得的3D培养物,显示具有正常分化的生理功能。
2.本发明提供的3D模型可用于人正常生殖道上皮的生理学研究和药物毒性安全性评价。
3.本发明提供的人正常阴道上皮3D分化培养物的HSV-2病毒感染模型,可应用于HSV-2病毒感染性疾病以及性传播病原体感染性疾病的发病机理研究,以及抗病毒药物的研发。
附图说明
图1是人正常阴道上皮细胞在气-液3D培养条件下可正常分化(与镜像的正常阴道组织A对比);
图2是人正常阴道上皮细胞在气-液3D培养条件下正常分化后,特异性蛋白标志物(CK14、CK18、p63)及细胞紧密连接相关蛋白(Laminin,E-Cadherin、DSG-1)的表达(与镜像的正常阴道组织对比);
图3是HSV-2病毒感染人正常阴道上皮3D培养物不同时间点的H&E染色形态学变化图(0hr p.i.、12hr p.i.、24hr p.i.、48hr p.i.、72hr p.i.、96hr p.i.);
图4是HSV-2病毒感染人正常阴道上皮3D培养物不同时间点(0hr p.i.、12hrp.i.、24hr p.i.、48hr p.i.、72hr p.i.、96hr p.i.)的DAB免疫组化标记紧密连接相关蛋白表达变化图(A为Laminin,B为E-cadherin,C为Dsg-1);
图5是HSV-2病毒感染人正常阴道上皮3D培养物不同时间点免疫荧光检测HSV病毒特异性标志蛋白gB的表达(0hr p.i.、12hr p.i.、24hr p.i(A图)、48hr p.i.、72hr p.i.、96hr p.i.(B图))。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。
【实施例1】人正常阴道上皮细胞原代分离培养
(1)在病人或病人监护人知情同意的情况下,收集阴道癌患者手术切除的癌旁正常组织样品。
(2)消化液的配制:含胶原酶和分散酶均为0.2mg/mL的原代上皮细胞生长培养基;其中,原代上皮细胞生长培养基(2D培养基)的成分包括:DMEM与Ham’s F-12NUTRIENT MIX按体积比3:1混合的培养基,同时添加5%胎牛血清、2nM三碘甲状腺氨酸(triiodothyronine),0.5%胰岛素铁硒传递蛋白(insulin-transferrin-selenium)试剂、5μg/ml铁传递蛋白(transferrin)、10ng/mL表皮生长因子、0.4μg/mL氢化可的松、40μg/mL庆大霉素、50nM calpeptin、40ng/ml重组人IL-1RA,及3μg/ml重组人R-Spondin-1。
(3)用95~100%(v/v)的乙醇洗分离的组织样品1次,再用PBS(0.01M,pH 7.4)洗2次,然后将组织放入含冰上预冷PBS的无菌培养皿中,在解剖显微镜下,用解剖镊子和剪刀去除组织中残留的脂肪。
(4)将正常阴道组织样品放入10倍于组织样品体积的含步骤(2)所配制消化液的无菌培养皿中,37℃消化1~3小时。
(5)将消化后的组织低速离心(1000rpm)5分钟,去除上清。
(6)将细胞沉淀重悬于2-5mL的0.25%(质量体积比)胰酶-EDTA中,置于冰上1小时或室温消化10分钟。
(7)然后加入10mL含10%(v/v)FBS的DMEM培养基,低速1000rmp离心5分钟,尽量将上清去除干净。
(8)加入2mL温水浴(37℃)的5mg/mL分散酶和200μL的1mg/mL DNaseI,用无菌的P1000一次性塑料枪头反复吹打样品1分钟。
(9)加入10mL含10%(v/v)FBS的DMEM,用40~70μm孔径的过滤器过滤细胞悬液,收集过滤后的细胞悬液,低速1000rmp离心5分钟,去除上清。
(10)重悬细胞沉淀于2D培养基中,接种于T25或T75的培养瓶培养,培养条件是37℃、5%CO2。
【实施例2】人正常阴道上皮细胞的传代培养
(1)当在T25或T75的培养瓶中培养的人正常阴道上皮细胞增殖至70~90%丰度时,用1×PBS(0.01M,pH 7.4)洗涤细胞三次,再用0.05%(质量体积比)胰酶-EDTA消化单层细胞2~5分钟。
(2)加入10mL DMEM中和消化反应1~2分种。
(3)1000rmp离心5分钟,弃上清。
(4)以1:2,1:3,1:4或1:5比例重悬细胞沉淀于2D培养基,接种于培养瓶培养,培养条件是37℃、5%CO2得到人正常阴道上皮细胞。
(5)必要时可将1×106上皮细胞重悬于1-2mL的细胞冻存液(90%胎牛血清和10%DMSO,v/v)中,储存于液氮中备用。
【实施例3】人正常阴道上皮的气-液3D培养
(1)将0.4μm的Millicell PCF insert(12mm size,Millipore)放入六孔板中,每孔最多放三个inserts。
(2)用400μl 2D培养基重悬5x105个的人阴道上皮细胞,然后接种到每个insert中。
(3)每个孔板内部即insert外围加入2ml的2D培养基。
(4)将放有insert的六孔板放入湿热培养箱中,37℃,5%CO2培养时长为48小时。
(5)将insert内部及外部中的培养基更换为3D分化培养基。
3D分化培养基的配制为:DMEM与F12按体积比1:1混合的培养基,同时添加0.87μM胰岛素(Sigma-Aldrich I6634)、0.125μM铁传递蛋白(Sigma-Aldrich T0665),0.1μM氢化可的松(Sigma-Aldrich H0396),0.01μM三碘甲状腺氨酸(Sigma-Aldrich T6397),2.7μM肾上腺素(Sigma-Aldrich E4642),0.50ng/mL表皮生长因子,5x 10–8M视黄酸(Sigma-AldrichR2625),0.5μM磷酸乙醇胺(Sigma-Aldrich P0503),0.5μM氨基乙醇(Sigma-AldrichE0135),3.0μM硫酸锌(Sigma-Aldrich Z0251),100U/mL青霉素G硫酸(Sigma-AldrichP3032),100μg/mL链霉素硫酸盐(Sigma-Aldrich S9137),1.0mM氯化钙(Sigma-AldrichC3881)。
(6)将放有insert的培养皿放入湿热培养箱中,培养15-17小时,使insert内部的细胞与细胞之间形成紧密连接。
(7)移除insert内部及外部所有的培养基,外部的培养皿中加入3D分化培养基,开始分化培养。
(8)在湿热培养箱中培养阴道上皮细胞14-21天,培养条件为37℃,5%CO2,每2-3天更换insert外围的培养基。
(9)分化完成后,将insert放入4%多聚甲醛(wt/vol)中,4℃固定过夜。
(10)吸走insert内部的4%多聚甲醛,用新的手术刀将膜切下,并将膜小心铺在滴有4%多聚甲醛的光滑平皿上,将膜剪成两半,并将边缘部分剪成小块。
(11)将小块的膜放入两个4%的多聚甲醛湿润的化妆棉中,再放入包埋盒。
(12)立即将包埋盒放入含有70%乙醇的容器内,根据标准制作流程制作石蜡病理切片。
【实施例4】人正常阴道上皮细胞在气-液3D培养条件下呈现正常分化,具有正常阴道组织相似的结构
(1)切片:将人阴道上皮细胞的气-液3D分化培养物或者阴道组织,固定后连续切片,切片厚度为5μm;
(2)捞片:将切好的石蜡切片置入37℃温水中,待石蜡中的组织完全伸展开来,即用载玻片捞起固定;
(3)烤片:将固定有组织切片的载玻片置入烘箱中,烘箱温度为70℃,2h,然后置于室温冷却;
(4)脱蜡:将切片分别先后浸入二甲苯I和二甲苯II中,时间分别为5min;
(5)获水:将石蜡切片分别置于梯度乙醇的时间分别为无水乙醇5min,95%乙醇5min,80%乙醇5min,70%乙醇5min,水洗1min;
(6)苏木素染色:苏木塑染液染色10min,水洗1min,盐酸乙醇的分化时间为10s;
(7)蓝化:水洗1s;
(8)伊红染色:伊红染液染色30s;
(9)分色与脱水:先水洗1min,再置于梯度乙醇中,分别为80%乙醇20s,95%乙醇20s,无水乙醇I 20s,无水乙醇II 1min;
(10)透明:将石蜡切片置于二甲苯中10min;
(11)封片:中性树脂封片。
如图1所示,正常阴道组织(A)和3D分化培养物(B)具有相似的结构特征,主要分为三层,最上层的角质层、中间的移行层和最下层的基底层,表明我们所建立的3D分化培养物不仅可以在体外正常分化,且具有与正常阴道组织相似的结构特征。
【实施例5】人正常阴道上皮细胞在气-液3D培养条件下正常分化,且表达与正常阴道组织相同的分化标志蛋白
(1)石蜡切片脱蜡至水:将石蜡切片置于新鲜的二甲苯中,15分钟×2次,除去多余的液体后,分别置于梯度乙醇中;依次为无水乙醇浸泡3分钟×2次;95%乙醇中,浸泡3分钟;85%乙醇中,浸泡3分钟;75%乙醇中,浸泡3分钟;自来水冲洗1分钟;PBS溶液冲洗3分钟×3次;
(2)用3%的H2O2 1份+分析甲醇9份混合配制过氧化物酶阻断剂,室温下灭活10min,蒸馏水浸洗3次,每次1min;
(3)抗原微波修复:将切片浸入浓度为0.01M,PH 6.0柠檬酸缓冲液,微波中最大火力加热至沸腾,微波中最大活力温度为98℃-100℃,冷却,冷却时间约为5min-10min,不烫手即可,反复两次;
(4)血清封闭:封闭(用荧光笔画出包含组织的区域):将切片置于湿盒中,室温下,使用10%FBS封闭,时间为37℃,30min,封闭完毕后甩去多余液体;
(5)一抗孵育:在各组织的圆圈中滴加用PBS、抗原稀释液或者封闭液稀释的抗体,一抗分别为CK14,CK18,p63,Laminin,E-cadherin和DSG-1,一抗的稀释比例为1:100,滴加约50μL,应完全覆盖组织,置于湿盒中,放入4℃中冰箱孵育过夜,时间为15h以上,取出后37℃复温,取出倾去多于液体,用PBS洗,PBS浸洗时间为5min×4次;
(6)甩去多余PBS,滴加反应增强液,室温孵育或37℃孵育,20min,倾去多于抗体,PBS洗5min×4次;
(7)二抗孵育:滴加约50μL酶标二抗,稀释比例为1:100,室温或者37℃孵育,孵育时间为30min,取出倾去多于液体,用新鲜的PBS浸洗5min×4次;
(8)DAB显色:DAB显色液应按照试剂盒说明书现配现用,显微镜下控制反应时间,显色反应时间约3-5min,染色完毕后用蒸馏水浸洗,1min×3次;
(9)苏木素复染,复染时间为10min,取出,过水后,在1%盐酸乙醇中分化,分化时间为3-5s,自来水或者1%的氨水返蓝,时间为1min;
(10)脱水透明:将石蜡切片依次置于梯度酒精中,分别为70%乙醇20s;80%乙醇20s;90%乙醇20s;95%乙醇20s;无水乙醇Ⅰ1min;无水乙醇Ⅱ1min;二甲苯Ⅰ2min;二甲苯Ⅱ2min。
(11)通风晾干,中性树胶封片。
如图2所示,正常的阴道上皮组织和体外建立的3D分化培养物具有相同的特异性蛋白标志物和细胞紧密连接相关蛋白的表达特点(主要体现在蛋白分子的表达分布和表达量上),即在基底层表达CK14、p63和Laminin,在基底层和移形层中表达E-cadherin和DSG-1,且DSG-1的表达量都是高于E-cadherin,同样地不表达CK18蛋白。
【实施例6】HSV-2病毒感染人阴道上皮的气-液3D分化培养物
(1)阴道上皮3D培养物分化完全(上述气-液3D培养14-21天)后,向insert中3D培养物的顶端面加入200μl的HSV-2病毒液,HSV-2病毒接种量为4x105PFU/孔;
(2)在温箱中孵育两个小时后,移去insert内部的病毒液,将insert重新放入37℃,5%CO2的培养箱中,再形成气液分化界面;
(3)HSV-2病毒感染3D培养分化物后分别在0hr p.i.、12hr p.i.、24hr p.i.、48hrp.i.、72hr p.i.、96hr p.i.时间点收样;
(4)将不同时间点的insert置于4%多聚甲醛(wt/vol)中固定,再按照标准制作流程制作石蜡病理切片后进行H&E染色,观察分析3D分化的阴道上皮培养物接种病毒感染后的情况。
【实施列7】HSV-2病毒感染人正常阴道上皮3D培养物不同时间点H&E染色的形态学变化
(1)切片:将人阴道上皮细胞的气-液3D分化培养物固定后连续切片,切片厚度为5μm;
(2)捞片:将切好的石蜡切片置入37℃温水中,待石蜡中的组织完全伸展开来,即用载玻片捞起固定;
(3)烤片:将固定有组织切片的载玻片置入烘箱中,烘箱温度为70℃,2h,然后置于室温冷却;
(4)脱蜡:将切片分别先后浸入二甲苯I和二甲苯II中,时间分别为5min;
(5)获水:将石蜡切片分别置于梯度乙醇的时间分别为无水乙醇5min,95%乙醇5min,80%乙醇5min,70%乙醇5min,水洗1min;
(6)苏木素染色:苏木塑染液染色10min,水洗1min,盐酸乙醇的分化时间为10s;
(7)蓝化:水洗1s;
(8)伊红染色:伊红染液染色30s;
(9)分色与脱水:先水洗1min,再置于梯度乙醇中,分别为80%乙醇20s,95%乙醇20s,无水乙醇I 20s,无水乙醇II 1min;
(10)透明:将石蜡切片置于二甲苯中10min;
(11)封片:中性树脂封片。
如图3所示,随着HSV-2病毒感染时间的延长,体外构建的3D分化培养物呈现出相应病理学变化,主要表现为病毒感染后,破坏程度依次加重,从最初角质层变得松散开始,到波及中间的移行层直至最后基底层受到严重破坏。
【实施例8】HSV-2感染人阴道上皮的气-液3D分化培养物不同时间点的DAB免疫组化标记细胞紧密连接相关蛋白及阴道上皮细胞特异性分子表达量的变化
(1)石蜡切片脱蜡至水:将石蜡切片置于新鲜的二甲苯中,15分钟×2次,除去多余的液体后,分别置于梯度乙醇中;依次为无水乙醇浸泡3分钟×2次;95%乙醇中,浸泡3分钟;85%乙醇中,浸泡3分钟;75%乙醇中,浸泡3分钟;自来水冲洗1分钟;PBS溶液冲洗3分钟×3次;
(2)用3%的H2O2 1份+分析甲醇9份混合配制过氧化物酶阻断剂,室温下灭活10min,蒸馏水浸洗3次,每次1min;
(3)抗原微波修复:将切片浸入浓度为0.01M,PH 6.0柠檬酸缓冲液,微波中最大火力加热至沸腾,微波中最大活力温度为98℃-100℃,冷却,冷却时间约为5min-10min,不烫手即可,反复两次;
(4)血清封闭:封闭(用荧光笔画出包含组织的区域):将切片置于湿盒中,室温下,使用10%FBS封闭,时间为37℃,30min,封闭完毕后甩去多余液体;
(5)一抗孵育:在各组织的圆圈中滴加用PBS、抗原稀释液或者封闭液稀释的抗体,一抗分别为CK14,CK18,p63,Laminin,E-cadherin和DSG-1,一抗的稀释比例为1:100,滴加约50μL,应完全覆盖组织,置于湿盒中,放入4℃中冰箱孵育过夜,时间为15h以上,取出后37℃复温,取出倾去多于液体,用PBS洗,PBS浸洗时间为5min×4次;
(6)甩去多余PBS,滴加反应增强液,室温孵育或37℃孵育,20min,倾去多于抗体,PBS洗5min×4次;
(7)二抗孵育:滴加约50μL酶标二抗,稀释比例为1:100,室温或者37℃孵育,孵育时间为30min,取出倾去多于液体,用新鲜的PBS浸洗5min×4次;
(8)DAB显色:DAB显色液应按照试剂盒说明书现配现用,显微镜下控制反应时间,显色反应时间约3-5min,染色完毕后用蒸馏水浸洗,1min×3次;
(9)苏木素复染,复染时间为10min,取出,过水后,在1%盐酸乙醇中分化,分化时间为3-5s,自来水或者1%的氨水返蓝,时间为1min;
(10)脱水透明:将石蜡切片依次置于梯度酒精中,分别为70%乙醇20s;80%乙醇20s;90%乙醇20s;95%乙醇20s;无水乙醇Ⅰ1min;无水乙醇Ⅱ1min;二甲苯Ⅰ2min;二甲苯Ⅱ2min。
(11)通风晾干,中性树胶封片。
图4所示,病毒在感染3D分化培养物不同时间点后,依然保留了细胞紧密连接相关蛋白的表达,通过观察上述图中相应蛋白的表达,可反映出HSV-2病毒感染3D分化培养物不同时间点,对3D分化培养物的破坏程度依次递增。
【实施例9】HSV-2病毒感染人正常阴道上皮3D培养物不同时间点的免疫荧光检测HSV病毒特异性标志蛋白gB的表达量的变化
(1)切片:将经过不同时间点病毒孵育过的人阴道上皮的气-液3D分化培养物固定后连续切片,切片厚度为5μm;
(2)捞片:将切好的石蜡切片置入37℃温水中,待石蜡中的组织完全伸展开来,即用载玻片捞起固定;
(3)烤片:将固定有组织切片的载玻片置入烘箱中,70℃,2h,然后置于室温冷却;
(4)脱蜡:将切片分别先后浸入二甲苯I和二甲苯II中,时间分别为5min;
(5)获水:将切片依次浸入梯度乙醇。时间分别为无水乙醇5min、95%乙醇5min、80%乙醇5min、75%乙醇5min、水洗1min;
(6)抗原微波修复:将切片浸入浓度为0.01M,PH 6.0柠檬酸缓冲液,微波中最大活力温度为98℃-100℃,加热至沸腾,冷却,冷却时间约为5min-10min,不烫手即可,反复两次;
(7)内源性酶灭活:1份30%H2O2加9份蒸馏水配制成3%H2O2,灭活内源性酶的时间为室温10min,水洗的时间为每次3min;3次;
(8)将切片自然冷却至室温,PBS洗涤3次,每次洗5min;
(9)封闭(用荧光笔画出包含组织的区域):使用10%FBS封闭,时间为37℃1h,封闭完毕后甩去多余液体;
(10)一抗的稀释比例为1:100,滴加一抗,37℃1h,或者4℃过夜(次日复温37℃1h);
(11)PBST洗涤3次,洗涤时间分别为5min;
(12)二抗的稀释比例为1:100,滴加荧光二抗,37℃,1h;
(13)PBST洗涤3次,分别为5min,吸干;
(14)DAPI复染10min,;
(15)PBST洗涤3次,洗涤时间分别为5min;
(16)荧光抗淬灭剂封片,镜检;
如图5所示,在不同时间点,HSV-2病毒均成功感染人正常阴道上皮3D培养物并进入细胞后复制表达,因此可以检测到病毒晚期复制蛋白gB的表达,且呈现出周期性的表达变化趋势。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (3)
1.一种人正常阴道上皮3D分化培养物模型的构建方法,包括如下步骤:
A、人正常阴道上皮细胞原代分离培养:
(1)在病人或病人监护人知情同意的情况下,收集阴道癌患者手术切除的癌旁正常组织样品;
(2) 消化液的配制:含胶原酶和分散酶均为0.2 mg/mL的原代上皮细胞生长培养基;其中,原代上皮细胞生长2D培养基的成分包括:DMEM与Ham’s F-12 NUTRIENT MIX按体积比3:1混合的培养基,同时添加4-6%胎牛血清、1-3nM三碘甲状腺氨酸、0.4-0.65%胰岛素铁硒传递蛋白、4-6μg/ml铁传递蛋白、9-11ng/mL表皮生长因子、0.3-0.5μg/mL氢化可的松、35-45μg/mL庆大霉素、45-55nM calpeptin、35-45ng/ml重组人IL-1RA,及3μg/ml重组人R-Spondin-1;
(3)用95~100%的乙醇洗分离的组织样品1次,再用0.01M,pH 7.4的PBS洗2次,然后将组织放入含冰上预冷PBS的无菌培养皿中,在解剖显微镜下,用解剖镊子和剪刀去除组织中残留的脂肪;
(4)将正常阴道组织样品放入10倍于组织样品体积的含步骤(2)所配制消化液的无菌培养皿中,37℃消化1~3小时;
(5)将消化后的组织低速离心1000 rpm 5分钟,去除上清;
(6)将细胞沉淀重悬于2-5 mL的0.25%胰酶-EDTA中,置于冰上1小时或室温消化10分钟;
(7)然后加入10 mL含10% FBS的DMEM培养基,低速1000 rmp离心5分钟,尽量将上清去除干净;
(8)加入2 mL37℃温水浴的5 mg/mL分散酶和200μL 的1 mg/mL DNase I,用无菌的P1000一次性塑料枪头反复吹打样品1分钟;
(9)加入10 mL含10% FBS的DMEM,用40~70 μm孔径的过滤器过滤细胞悬液,收集过滤后的细胞悬液,低速1000 rmp离心 5分钟,去除上清;
(10)重悬细胞沉淀于2D培养基中,接种于T25或T75的培养瓶培养,培养条件是37℃、5%CO2;
B、人正常阴道上皮细胞的传代培养:
(1)当在T25或T75的培养瓶中培养的人正常阴道上皮细胞增殖至70~90%丰度时,用1×0.01M,pH 7.4 PBS洗涤细胞三次,再用0.05% 胰酶-EDTA消化单层细胞2~5分钟;
(2)加入10 mL DMEM中和消化反应1~2分种;
(3)1000 rmp离心5分钟,弃上清;
(4)以1:2,1:3,1:4或1:5比例重悬细胞沉淀于2D培养基,接种于培养瓶培养,培养条件是37℃、5% CO2得到人正常阴道上皮细胞;
(5)必要时可将1×106上皮细胞重悬于1-2 mL的含90%胎牛血清和10% DMSO的细胞冻存液中,储存于液氮中备用;
C、人正常阴道上皮的气-液3D培养:
(1)将0.4µm的Millipore公司的12 mm Millicell PCF insert, 放入六孔板中,每孔最多放三个inserts;
(2)用400µl 2D培养基重悬5x105个的人阴道上皮细胞,然后接种到每个insert中;
(3)每个孔板内部即insert外围加入2ml的2D培养基;
(4)将放有insert的六孔板放入湿热培养箱中,37℃,5%CO2培养时长为48小时;
(5)将insert内部及外部中的培养基更换为3D分化培养基;
3D分化培养基的配制:DMEM与F12按体积比1:1混合的培养基,同时添加0.5-1.2 μM胰岛素、0.05-0.2μM铁传递蛋白,0.05-0.15μM氢化可的松,0.005-0.015μM三碘甲状腺氨酸,1-4μM肾上腺素,0.1-1ng/mL表皮生长因子,2 x 10–8 -8 x 10–8 M视黄酸,0.1-1 μM磷酸乙醇胺,0.1-1 μM氨基乙醇,1-5μM硫酸锌,100 U/mL青霉素G硫酸,100μg/mL链霉素硫酸盐,0.5-1.5 mM氯化钙;
(6)将放有insert的培养皿放入湿热培养箱中,培养15-17小时,使insert内部的细胞与细胞之间形成紧密连接;
(7)移除insert内部及外部所有的培养基,外部的培养皿中加入3D分化培养基,开始分化培养;
(8)在湿热培养箱中培养阴道上皮细胞14-21天,培养条件为37℃,5%CO2,每2-3天更换insert外围的培养基。
2.根据权利要求1所述的人正常阴道上皮3D分化培养物模型的构建方法,其特征在于,步骤A(2)所述的2D培养基的配制为:DMEM与Ham’s F-12 NUTRIENT MIX按体积比3:1混合的培养基,同时添加5%胎牛血清、2nM三碘甲状腺氨酸、0.5%胰岛素铁硒传递蛋白、5μg/ml铁传递蛋白、10ng/mL表皮生长因子、0.4μg/mL氢化可的松、40μg/mL庆大霉素、50nMcalpeptin、40ng/ml重组人IL-1RA,及3μg/ml重组人R-Spondin-1。
3.根据权利要求1所述的人正常阴道上皮3D分化培养物模型的构建方法,其特征在于,步骤C(5)所述的3D分化培养基的配制为:DMEM与F12按体积比1:1混合的培养基,同时添加0.87 μM胰岛素、0.125μM铁传递蛋白,0.1μM氢化可的松,0.01μM三碘甲状腺氨酸,2.7μM肾上腺素,0.50ng/mL表皮生长因子,5 x 10–8 M视黄酸,0.5 μM磷酸乙醇胺,0.5 μM氨基乙醇,3.0μM硫酸锌,100 U/mL青霉素G硫酸,100μg/mL链霉素硫酸盐。
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