CN108396007A - 体外构建奶牛血乳屏障三维模型的方法 - Google Patents
体外构建奶牛血乳屏障三维模型的方法 Download PDFInfo
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Abstract
体外构建奶牛血乳屏障三维模型的方法,包括奶牛乳腺上皮细胞和成纤维细胞培养,奶牛静脉内皮细胞培养,免疫荧光鉴定乳腺上皮细胞和内皮细胞,Transwell共培养,联合培养基的配制中以催乳素、胰岛素、表皮生长因子、牛生长激素、血管内皮生长因子、成纤维细胞生长因子作为与乳腺上皮细胞、成纤维细胞和内皮细胞生长相关的激素和细胞因子;最后在transwell小室接种上皮细胞、成纤维细胞和内皮细胞,从而成功构建了奶牛血乳屏障模型。本发明能够实时、有效的研究营养物质、药物和有毒物质进入乳腺组织和乳中分子机制,不仅填补了国内外空白,还为研究脂类、维生素、药物和有毒物质等穿过血乳屏障的分子机制提供研究平台。
Description
技术领域
本发明涉及一种体外构建奶牛血乳屏障三维模型的方法,属于血乳屏障研究技术领域。
背景技术
传统二维细胞培养只能在二维平面上对细胞进行分子以及基因水平的研究,从而了解特定分子和基因改变对细胞生物学行为的影响。动物模型尽管能更为准确地反映形态学上的特点,但难以进行大规模的分子水平上研究。本发明构建结合了二维细胞培养和动物模型的优点的三维血乳屏障模型,着重解决了血管内皮细胞、成纤维细胞和乳腺上皮细胞三种细胞接种密度、培养时间,三维联合培养基种类及添加促进血管生成的细胞因子种类、添加浓度和促乳腺上皮细胞分化培养中添加的激素和细胞因子浓度。
血乳屏障研究进展
循环系统是动物体内的运输系统,它将营养物质输送到各组织器官并将各组织器官的代谢产物通过同样的途径输入血液,同时它也运输免疫细胞保护机体不受病原微生物的入侵。营养物质、激素和免疫细胞从血液进入乳腺受血乳屏障的调控。血乳屏障由内皮细胞层(En)、内皮下基质膜(BM),由糖胺聚糖、蛋白多糖、蛋白质、胶原纤维和纤维束(C)、成纤维细胞(F)、免疫细胞如巨噬细胞(M)构成的细胞外基质(ECM)、上皮下基质膜(BM)、乳腺上皮细胞(Ep)构成,如图1分子通过血乳屏障模式图。
乳腺合成和分泌到乳汁的成分包括蛋白质、碳水化合物、膜包被的脂滴、水和离子。这些物质由不同的分泌路径产生,包括膜途径、高尔基体途径、乳脂途径、胞转途径、旁细胞途径。这些分泌途径受乳腺功能状态、激素和生长因子调控。乳中包含乳蛋白、乳糖和低浓度的钠离子和氯离子,而间质液中含有血浆蛋白和高浓度的钠离子和氯离子。血乳屏障分隔这两种液体,并且调控乳成分。对乳腺血乳屏障的研究对治疗乳腺癌的药物筛选有重要意义。除了大分子在血液和乳中穿梭,免疫细胞(中性粒细胞)穿过血乳屏障保护乳腺免受感染。
血乳屏障在调控营养物质和细胞在血液和乳中的运动方面发挥重要作用。体外构建血乳屏障模型可以进行大规模的分子水平研究。Guidry(1998)首次通过体外构建血乳屏障模型来研究中性粒细胞穿过血管壁的分子机制,如图2。在1.2μm的膜上包被胶原-成纤维细胞混合液后,接种乳腺上皮细胞,待培养8h后,将插入式培养皿翻转过来接种内皮细胞(图3)。Guidry构建的血乳屏障由单层细胞构成,至今国内外没有3D培养的血乳屏障模型。3D细胞培养是将细胞、生长因子与再造基质蛋白/骨架共培养,以模拟体内细胞生长的微环境。3D细胞培养可以使细胞更为自然的极性条件下生长,同时提高了细胞的分化水平,体外生长的细胞在形态和功能上更为接近体内的细胞。
发明内容
本发明目的是提供一种体外构建奶牛血乳屏障三维模型的方法,该方法构建结合了二维细胞培养和动物模型的优点的三维血乳屏障模型,为深入研究乳腺重要营养物质、药物和有毒物质转运的分子机制提供了研究平台。
一种体外构建奶牛血乳屏障三维模型的方法,其特征在于包括以下步骤:
1奶牛乳腺上皮细胞和成纤维细胞培养
无菌切取约2g乳腺组织,置于无菌并含的D-Hanks’液的50ml三角瓶中,温盒带回实验室;超净工作台中,将乳腺组织置于50ml无菌烧杯中,以含10×双抗的D-Hanks’液冲洗5次,洗净组织附着的乳汁、血液等杂质;将洗净组织置于10ml无菌烧杯中,以灭菌手术剪刀剪成1mm3的小组织块;将所有小组织块转移至另一无菌三角瓶中,加入20ml消化液,封盖后置于37℃摇床中,200r/min振荡消化4h,至无肉眼可见明显组织块;;将三角瓶中的消化液通过灭菌的200目金属网过滤到50ml烧杯中,弃掉未消化组织块,收集小烧杯中消化液;将滤出液等分至2个无菌10ml玻璃离心管中,封口后室温,1 500r/min,离心8min,收集细胞沉淀;分别以8ml含1×双抗的D-Hanks液洗涤细胞沉淀4次,室温,1 500r/min,离心5min;以总体积5ml,DF-12完全培养液重悬细胞沉淀,并分别铺于2个25cm2细胞培养瓶中,以DF-12完全培养液将2离心管中液体补至5.5ml,封口后置于5%CO2、37℃恒温培养箱中培养,并观察细胞生长情况;每隔2d更换一次培养液,至细胞生长密度达80%~90%时,0.05%胰蛋白酶0.02%EDTA溶液1mL消化10s,成纤维细胞对胰酶较敏感,此时消化下来的全是成纤维细;加入5mL完全培养基终止消化后,将培养液吸出接种到培养瓶中进行成纤维细胞培养;在原瓶中加入0.05%胰蛋白酶0.02%EDTA溶液1mL消化3min,加入10mL完全培养基终止消化,吸出5mL培养液接种到新培养瓶中进行乳腺上皮细胞传代培养;
2奶牛静脉内皮细胞培养
在无菌条件下取奶牛静脉,长度>20cm,挤干净血管内血液,用生理盐水冲洗动脉表面至无血色,动脉脉中灌入0.1%I型胶原酶溶液,使之充盈,37℃水浴中孵育20min,其间轻轻挤压并旋转,以使酶溶液充分接触血管内壁,然后将细胞酶溶液收集于预先装有温培养液三角瓶中;用2倍于消化液的温PBS冲洗血管,并收集于小三角瓶中,将细胞悬液装入10ml离心管,1 000r/min离心10min,弃上清,吹打悬浮,再次离心10min,重悬细胞并接种于铺有明胶的25cm2培养瓶中,置于37℃5%CO2饱和湿度培养箱中培养,24h后更换培养液,以后每隔2d换液1次;
当原代细胞融合80%以上后,加入0.05%胰蛋白酶0.02%EDTA溶液1mL消化,倒置显微镜下观察,当细胞皱缩变圆、彼此分离时弃消化液,加入细胞培养液终止消化,收集细胞悬液1 000r/min离心10min,弃上清,制成单细胞悬液;此时可用0.5%台盼蓝染色,计算活细胞百分率,并以血细胞计数板计数;调整细胞浓度,接种于培养瓶中继续培养;待细胞长满,彼此融合成片时依上述方法继续传代培养;
3免疫荧光鉴定乳腺上皮细胞和内皮细胞
分别将长满上皮细胞和内皮细胞的盖玻片置于2.5%多聚甲醛固定10min;室温下1×PBS漂洗3次,每次5min;10%羊血清37℃封闭30min后,去掉正常血清直接加羊抗牛K18抗体和CD31抗体孵育,4℃过夜,抗体用0.3%TritonX-100/PBS稀释;室温下1×PBS漂洗3次,每次5min;FITC-兔抗羊IgG 37℃孵育30min;室温下1×PBS漂洗3次,每次5min;PI复染5min后,室温下1×PBS漂洗3次,每次5min;DABCO封片液封片,激光扫描共聚焦显微镜观察、照像;阴性对照片不加一抗孵育,直接进行下步操作;
激光扫描共聚焦显微镜(laser scanning confocal microscopy,LSCM)使用双通道(PMT)检测,激发谱线分别为488nm(激发FITC标记的绿色荧光,染K18、波形蛋白和CD31)和543nm(激发PI标记的红色荧光,染核);扫描模式为xyz,以0.4μm的Z轴步距逐层扫描,点平均2次,面平均4次,两个通道同时扫描成像,overlay处理后采图保存;激光功率和PMT增益恒定;重复3次,每张切片选取5个视野观察;所有图像分辨率为1024×1024,存储格式为Tif;
4 Transwell共培养
(1)1mol/L氢氧化钠和10×磷酸盐缓冲液中和胶原,然后1:1的比例和Matrigel混合制成Matrigel-胶原凝胶,胶原保持1mg/mL;共培养体系中第三代上皮细胞和成纤维细胞以3:1的比例悬浮在3mLMatrigel-胶原混合液中(模拟体内的比例300 000:100 000),接种transwell下室,再将培养第三代的静脉内皮细胞3×104的接种于transwell上室Matrigel-胶原凝胶表面,让上下室的培养液相互通融,从而建立起内皮细胞、成纤维细胞和乳腺上皮细胞的共培养体系,在联合培养基上培养12天;
(2)联合培养基的配制
①培养基中添加激素和细胞因子的选择
以A(催乳素)、B(胰岛素)、E(表皮生长因子)、F(牛生长激素)、I(血管内皮生长因子)、J(成纤维细胞生长因子)作为与乳腺上皮细胞、成纤维细胞和内皮细胞生长相关的激素和细胞因子,后续实验将添加这6种激素和细胞因子;
②培养基中添加激素和细胞因子最佳浓度的选择
设计了正交试验L25(56),以确定上述选择添加的激素和细胞因子的最佳添加浓度;并通过极差分析我们找到了最佳添加浓度组合A4B4C1D4E3F3,即胰岛素0.3ug/ml、催乳素3ug/ml、牛生长激素8ng/ml、血管皮生长因子2.5ug/ml、成纤维细胞生长因子1.5ug/ml、表皮生长因子5ug/ml;
③联合培养基成分
含10%FBS的DMEM/F12,并在其中添加浓度为8ug/ml的牛生长激素,0.3ug/ml胰岛素,3ug/ml催乳素,血管内皮生长因子2.5ug/ml、成纤维细胞生长因子1.5ug/ml、表皮生长因子(EGF)5ug/ml;
5体外构建奶牛血乳屏障模型
在transwell小室接种上皮细胞、成纤维细胞和内皮细胞,从而成功构建了奶牛血乳屏障模型。
所述的一种体外构建奶牛血乳屏障三维模型的方法,其特征在于步骤2中的重悬细胞的培养基为含20%FBS的高糖型DMEM,并在其中添加浓度为100ug/ml的ECGS,20ug/ml的Plasmocin,100u/ml的青霉素,100ug/ml的链霉素,3ug/ml的两性霉素。
体外构建血乳屏障模型,能够实时、有效的研究营养物质、药物和有毒物质进入乳腺组织和乳中分子机制。国内外还没有报道血乳屏障三维模型研究。这项研究不仅填补了国内外研究空白,还为研究脂类、维生素、药物和有毒物质等穿过血乳屏障的分子机制提供研究平台,为生产营养、健康、安全的牛奶提供基础。
如今生产和消费“安全牛奶”已成为全社会的共识,所谓“安全牛奶”,即指无污染、对人类健康有安全保障的牛奶,主要是指除乳脂、蛋白质、色、香、味等达到国家或国际标准外,还要求牛奶无药物(农药、抗生素、激素等)残留、无病原微生物、无铅、砷等重金属残留、霉菌毒素、细菌总数不得超过国家或国际标准。黄曲霉毒素(Aflatoxins)是真菌产生的毒性代谢产物,广泛存在于各种粮食、食品和饲料中,对人类和动物表现出很强的毒性。乳品中主要含有黄曲霉毒素Ml。哺乳类动物摄入被黄曲霉毒素Ml污染的饲料或食品后,在体内的肝微粒体单氧化酶系催化下,通过细胞色素P-448调节作用,黄曲霉毒素Ml末端吠喃环C-10被经化而生成黄曲霉毒素M1。利用构建的血乳屏障三维模型能够研究黄曲霉毒素Ml穿过血乳屏障进入乳中的分子机制。
附图说明
图1是分子通过血乳屏障模式图。
图2是Guidry(1998)等构建的血乳屏障模式图。
图3奶牛乳腺上皮细胞和成纤维细胞的培养(×200),其中:
A:成纤维细胞和乳腺上皮细胞(×200);B:成纤维细胞和乳腺上皮细胞(×200);
C:纯化的成纤维细胞(×200);D:纯化的乳腺上皮细胞(×200)。
图4免疫荧光鉴定乳腺上皮细胞和内皮细胞,其中
A:CD31鉴定内皮细胞(75μM);B:角蛋白18鉴定乳腺上皮细胞(75μM)。
图5三维细胞共培养模式图。
图6紧密连接蛋白ZO-1的染色。
图7本发明的技术路线图。
具体实施方式
如附图7所示,一种体外构建奶牛血乳屏障三维模型的方法,其特征在于包括以下步骤:
1奶牛乳腺上皮细胞和成纤维细胞培养
无菌切取约2g乳腺组织,置于无菌并含的D-Hanks’液的50ml三角瓶中,温盒带回实验室;超净工作台中,将乳腺组织置于50ml无菌烧杯中,以含10×双抗的D-Hanks’液冲洗5次,洗净组织附着的乳汁、血液等杂质;将洗净组织置于10ml无菌烧杯中,以灭菌手术剪刀剪成1mm3的小组织块;将所有小组织块转移至另一无菌三角瓶中,加入20ml消化液,封盖后置于37℃摇床中,200r/min振荡消化4h,至无肉眼可见明显组织块;;将三角瓶中的消化液通过灭菌的200目金属网过滤到50ml烧杯中,弃掉未消化组织块,收集小烧杯中消化液;将滤出液等分至2个无菌10ml玻璃离心管中,封口后室温,1 500r/min,离心8min,收集细胞沉淀;分别以8ml含1×双抗的D-Hanks液洗涤细胞沉淀4次,室温,1 500r/min,离心5min;以总体积5ml,DF-12完全培养液重悬细胞沉淀,并分别铺于2个25cm2细胞培养瓶中,以DF-12完全培养液将2离心管中液体补至5.5ml,封口后置于5%CO2、37℃恒温培养箱中培养,并观察细胞生长情况;每隔2d更换一次培养液,至细胞生长密度达80%~90%时,0.05%胰蛋白酶0.02%EDTA溶液1mL消化10s,成纤维细胞对胰酶较敏感,此时消化下来的全是成纤维细;加入5mL完全培养基终止消化后,将培养液吸出接种到培养瓶中进行成纤维细胞培养;在原瓶中加入0.05%胰蛋白酶0.02%EDTA溶液1mL消化3min,加入10mL完全培养基终止消化,吸出5mL培养液接种到新培养瓶中进行乳腺上皮细胞传代培养;
2奶牛静脉内皮细胞培养
在无菌条件下取奶牛静脉,长度>20cm,挤干净血管内血液,用生理盐水冲洗动脉表面至无血色,动脉脉中灌入0.1%I型胶原酶溶液,使之充盈,37℃水浴中孵育20min,其间轻轻挤压并旋转,以使酶溶液充分接触血管内壁,然后将细胞酶溶液收集于预先装有温培养液三角瓶中;用2倍于消化液的温PBS冲洗血管,并收集于小三角瓶中,将细胞悬液装入10ml离心管,1 000r/min离心10min,弃上清,吹打悬浮,再次离心10min,重悬细胞并接种于铺有明胶的25cm2培养瓶中,置于37℃5%CO2饱和湿度培养箱中培养,24h后更换培养液,以后每隔2d换液1次;
培养基为含20%FBS的高糖型DMEM,并在其中添加浓度为100ug/ml的ECGS,20ug/ml的Plasmocin,100u/ml的青霉素,100ug/ml的链霉素,3ug/ml的两性霉素。
当原代细胞融合80%以上后,加入0.05%胰蛋白酶0.02%EDTA溶液1mL消化,倒置显微镜下观察,当细胞皱缩变圆、彼此分离时弃消化液,加入细胞培养液终止消化,收集细胞悬液1 000r/min离心10min,弃上清,制成单细胞悬液;此时可用0.5%台盼蓝染色,计算活细胞百分率,并以血细胞计数板计数;调整细胞浓度,接种于培养瓶中继续培养;待细胞长满,彼此融合成片时依上述方法继续传代培养;
3免疫荧光鉴定乳腺上皮细胞和内皮细胞
分别将长满上皮细胞和内皮细胞的盖玻片置于2.5%多聚甲醛固定10min;室温下1×PBS漂洗3次,每次5min;10%羊血清37℃封闭30min后,去掉正常血清直接加羊抗牛K18抗体和CD31抗体孵育,4℃过夜,抗体用0.3%TritonX-100/PBS稀释;室温下1×PBS漂洗3次,每次5min;FITC-兔抗羊IgG 37℃孵育30min;室温下1×PBS漂洗3次,每次5min;PI复染5min后,室温下1×PBS漂洗3次,每次5min;DABCO封片液封片,激光扫描共聚焦显微镜观察、照像;阴性对照片不加一抗孵育,直接进行下步操作;
激光扫描共聚焦显微镜(laser scanning confocal microscopy,LSCM)使用双通道(PMT)检测,激发谱线分别为488nm(激发FITC标记的绿色荧光,染K18、波形蛋白和CD31)和543nm(激发PI标记的红色荧光,染核);扫描模式为xyz,以0.4μm的Z轴步距逐层扫描,点平均2次,面平均4次,两个通道同时扫描成像,overlay处理后采图保存;激光功率和PMT增益恒定;重复3次,每张切片选取5个视野观察;所有图像分辨率为1024×1024,存储格式为Tif;
4 Transwell共培养
(1)1mol/L氢氧化钠和10×磷酸盐缓冲液中和胶原,然后1:1的比例和Matrigel混合制成Matrigel-胶原凝胶,胶原保持1mg/mL。共培养体系中第三代上皮细胞和成纤维细胞以3:1的比例悬浮在3mLMatrigel-胶原混合液中(模拟体内的比例300 000:100 000),接种transwell下室,再将培养第三代的静脉内皮细胞3×104的接种于transwell上室Matrigel-胶原凝胶表面,让上下室的培养液相互通融,从而建立起内皮细胞、成纤维细胞和乳腺上皮细胞的共培养体系,在联合培养基上培养12天;
(2)联合培养基的配制
①培养基中添加激素和细胞因子的选择
以氢化可的松、胰岛素、催乳素、牛生长激素、转铁蛋白、孕酮,雌激素、血管内皮生长因子、成纤维细胞生长因子、表皮生长因子、胰岛素样生长因子-1,作为与乳腺上皮细胞、成纤维细胞和内皮细胞生长相关的11种激素和细胞因子,设计正交试验L12(211)在三维培养模型中依次添加或不添加上述激素或细胞因子,并检测细胞活力;实验设计和细胞活力检测结果如表1所示;
利用CASY细胞活力分析仪测定细胞的活力值,以未添加因子培养的细胞为对照,消化消化三维共培养的细胞模型,制成单细胞悬液,盛有10mlCASY-TON的CASY-CUP中稀释摇匀,运行一次START检测;将测量结果保存到电脑备用;取200ul样品加入600ul酒精(酒精终浓度75%)放置5min;将酒精杀死的细胞再次放入10mlCASY-TON中稀释,摇匀后运行START检测;将死细胞的测量结果传入电脑;并保存在与活细胞相同的DATA内以便图形叠加;运行OVERLINE ON,将死活细胞图形叠加;将鼠标移至两条曲线交叉的地方即可读取相应数值;取100μl细胞悬液加入装有10ml ton的cup,细胞充分混合均匀;将混合物置于外部电极处,运行控制面板上的START,连续进行3次测量,输出数据;
表1是十一因素两水平的正交表重复3次的实验设计和细胞活力结果,各字母代表如下激素或细胞因子A:催乳素;B:胰岛素C:氢化可的松;D:雌激素;E:表皮生长因子;F:牛生长激素G:胰岛素样生长因子-1;H:孕酮;I:血管内皮生长因子;J成纤维细胞生长因子;K转铁蛋白,两水平分别是添加和不添加。
表1添加激素和细胞因子选择实验方案
应用SPSS16.0提供的一般线形模型的"Univariate"过程,对实验所得数据进行方差分析,结果如表2所示。结果显示,A(催乳素)、B(胰岛素)、E(表皮生长因子)、F(牛生长激素)、I(血管内皮生长因子)、J(成纤维细胞生长因子)差异显著,后续实验将添加这6种激素和细胞因子。
表2方差分析结果
②培养基中添加激素和细胞因子最佳浓度的选择
为了确定上述选择添加的激素和细胞因子的最佳添加浓度,设计了正交试验L25(56),因素水平表如表3所示。
表3激素和细胞因子添加浓度筛选实验因素水平表
表4显示了激素和细胞因子添加浓度筛选实验方案,并且通过极差分析我们找到了最佳添加浓度组合A4B4C1D4E3F3,即胰岛素0.3ug/ml、催乳素3ug/ml、牛生长激素8ng/ml、血管皮生长因子2.5ug/ml、成纤维细胞生长因子1.5ug/ml、表皮生长因子5ug/ml。
表4激素和细胞因子添加浓度筛选实验方案及结果分析
③联合培养基成分
含10%FBS的DMEM/F12,并在其中添加浓度为8ug/ml的牛生长激素,0.3ug/ml胰岛素,3ug/ml催乳素,血管内皮生长因子2.5ug/ml、成纤维细胞生长因子1.5ug/ml、表皮生长因子(EGF)5ug/ml。
5体外构建奶牛血乳屏障模型
通过图5所示在transwell小室接种上皮细胞、成纤维细胞和内皮细胞,采用本方法确定的联合培养基成功构建了奶牛血乳屏障模型。通过免疫荧光染色的方法对乳腺上皮细胞屏障中紧密连接蛋白ZO-1的表达情况进行检测,激光共聚焦显微镜观察结果如图6所示,结果显示蓝色荧光部位为DAPI染色后的细胞核,绿色荧光部位是紧密连接蛋白ZO-1的表达,说明上皮细胞形成紧密连接,模型构建成功。
Claims (2)
1.一种体外构建奶牛血乳屏障三维模型的方法,其特征在于包括以下步骤:
1奶牛乳腺上皮细胞和成纤维细胞培养
无菌切取约2g乳腺组织,置于无菌并含的D-Hanks’液的50ml三角瓶中,温盒带回实验室;超净工作台中,将乳腺组织置于50ml无菌烧杯中,以含10×双抗的D-Hanks’液冲洗5次,洗净组织附着的乳汁、血液等杂质;将洗净组织置于10ml无菌烧杯中,以灭菌手术剪刀剪成1mm3的小组织块;将所有小组织块转移至另一无菌三角瓶中,加入20ml消化液,封盖后置于37℃摇床中,200r/min振荡消化4h,至无肉眼可见明显组织块;将三角瓶中的消化液通过灭菌的200目金属网过滤到50ml烧杯中,弃掉未消化组织块,收集小烧杯中消化液;将滤出液等分至2个无菌10ml玻璃离心管中,封口后室温,1 500r/min,离心8min,收集细胞沉淀;分别以8ml含1×双抗的D-Hanks液洗涤细胞沉淀4次,室温,1 500r/min,离心5min;以总体积5ml,DF-12完全培养液重悬细胞沉淀,并分别铺于2个25cm2细胞培养瓶中,以DF-12完全培养液将2离心管中液体补至5.5ml,封口后置于5%CO2、37℃恒温培养箱中培养,并观察细胞生长情况;每隔2d更换一次培养液,至细胞生长密度达80%~90%时,0.05%胰蛋白酶0.02%EDTA溶液1mL消化10s,成纤维细胞对胰酶较敏感,此时消化下来的全是成纤维细;加入5mL完全培养基终止消化后,将培养液吸出接种到培养瓶中进行成纤维细胞培养;在原瓶中加入0.05%胰蛋白酶0.02%EDTA溶液1mL消化3min,加入10mL完全培养基终止消化,吸出5mL培养液接种到新培养瓶中进行乳腺上皮细胞传代培养;
2奶牛静脉内皮细胞培养
在无菌条件下取奶牛静脉,长度>20cm,挤干净血管内血液,用生理盐水冲洗动脉表面至无血色,动脉脉中灌入0.1%I型胶原酶溶液,使之充盈,37℃水浴中孵育20min,其间轻轻挤压并旋转,以使酶溶液充分接触血管内壁,然后将细胞酶溶液收集于预先装有温培养液三角瓶中;用2倍于消化液的温PBS冲洗血管,并收集于小三角瓶中,将细胞悬液装入10ml离心管,1 000r/min离心10min,弃上清,吹打悬浮,再次离心10min,重悬细胞并接种于铺有明胶的25cm2培养瓶中,置于37℃5%CO2饱和湿度培养箱中培养,24h后更换培养液,以后每隔2d换液1次;
当原代细胞融合80%以上后,加入0.05%胰蛋白酶0.02%EDTA溶液1mL消化,倒置显微镜下观察,当细胞皱缩变圆、彼此分离时弃消化液,加入细胞培养液终止消化,收集细胞悬液1 000r/min离心10min,弃上清,制成单细胞悬液;此时可用0.5%台盼蓝染色,计算活细胞百分率,并以血细胞计数板计数;调整细胞浓度,接种于培养瓶中继续培养;待细胞长满,彼此融合成片时依上述方法继续传代培养;
3免疫荧光鉴定乳腺上皮细胞和内皮细胞
分别将长满上皮细胞和内皮细胞的盖玻片置于2.5%多聚甲醛固定10min;室温下1×PBS漂洗3次,每次5min;10%羊血清37℃封闭30min后,去掉正常血清直接加羊抗牛K18抗体和CD31抗体孵育,4℃过夜,抗体用0.3%TritonX-100/PBS稀释;室温下1×PBS漂洗3次,每次5min;FITC-兔抗羊IgG 37℃孵育30min;室温下1×PBS漂洗3次,每次5min;PI复染5min后,室温下1×PBS漂洗3次,每次5min;DABCO封片液封片,激光扫描共聚焦显微镜观察、照像;阴性对照片不加一抗孵育,直接进行下步操作;
激光扫描共聚焦显微镜(laser scanning confocal microscopy,LSCM)使用双通道(PMT)检测,激发谱线分别为488nm(激发FITC标记的绿色荧光,染K18、波形蛋白和CD31)和543nm(激发PI标记的红色荧光,染核);扫描模式为xyz,以0.4μm的Z轴步距逐层扫描,点平均2次,面平均4次,两个通道同时扫描成像,overlay处理后采图保存;激光功率和PMT增益恒定;每张切片选取5个视野观察;所有图像分辨率为1024×1024,存储格式为Tif;
4 Transwell共培养
(1)1mol/L氢氧化钠和10×磷酸盐缓冲液中和胶原,然后1:1的比例和Matrigel混合制成Matrigel-胶原凝胶,胶原保持1mg/mL。共培养体系中第三代上皮细胞和成纤维细胞以3:1的比例悬浮在3mLMatrigel-胶原混合液中(模拟体内的比例300 000:100 000),接种transwell下室,再将培养第三代的静脉内皮细胞3×104的接种于transwell上室Matrigel-胶原凝胶表面,让上下室的培养液相互通融,从而建立起内皮细胞、成纤维细胞和乳腺上皮细胞的共培养体系,在联合培养基上培养12天;
(2)联合培养基的配制
①培养基中添加激素和细胞因子的选择
以A(催乳素)、B(胰岛素)、E(表皮生长因子)、F(牛生长激素)、I(血管内皮生长因子)、J(成纤维细胞生长因子)作为与乳腺上皮细胞、成纤维细胞和内皮细胞生长相关的激素和细胞因子,后续实验将添加这6种激素和细胞因子;
②培养基中添加激素和细胞因子最佳浓度的选择
设计了正交试验L25(56),以确定上述选择添加的激素和细胞因子的最佳添加浓度;并通过极差分析我们找到了最佳添加浓度组合A4B4C1D4E3F3,即胰岛素0.3ug/ml、催乳素3ug/ml、牛生长激素8ng/ml、血管皮生长因子2.5ug/ml、成纤维细胞生长因子1.5ug/ml、表皮生长因子5ug/ml;
③联合培养基成分
含10%FBS的DMEM/F12,并在其中添加浓度为8ug/ml的牛生长激素,0.3ug/ml胰岛素,3ug/ml催乳素,血管内皮生长因子2.5ug/ml、成纤维细胞生长因子1.5ug/ml、表皮生长因子(EGF)5ug/ml;
5体外构建奶牛血乳屏障模型
在transwell小室接种上皮细胞、成纤维细胞和内皮细胞,从而成功构建了奶牛血乳屏障模型。
2.如权利要求1所述的一种体外构建奶牛血乳屏障三维模型的方法,其特征在于步骤2中的重悬细胞的培养基为含20%FBS的高糖型DMEM,并在其中添加浓度为100ug/ml的ECGS,20ug/ml的Plasmocin,100u/ml的青霉素,100ug/ml的链霉素,3ug/ml的两性霉素。
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CN109136171A (zh) * | 2018-09-26 | 2019-01-04 | 广东省农业科学院动物科学研究所 | 一种3d培养猪乳腺上皮细胞的方法 |
CN111378614A (zh) * | 2020-03-18 | 2020-07-07 | 中国农业大学 | 单层致密奶牛乳腺上皮细胞培养方法 |
CN112129345A (zh) * | 2020-09-14 | 2020-12-25 | 安徽军松现代农业科技有限公司 | 一种基于数据采集的土壤监测的环境监测系统 |
CN113564114A (zh) * | 2021-01-16 | 2021-10-29 | 浙江工商大学 | 一种cmt93-dc-t细胞的三维细胞模型的构建方法和应用 |
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CN101857852A (zh) * | 2010-05-31 | 2010-10-13 | 东北农业大学 | 奶牛乳腺腺泡泌乳模型的体外构建方法 |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109136171A (zh) * | 2018-09-26 | 2019-01-04 | 广东省农业科学院动物科学研究所 | 一种3d培养猪乳腺上皮细胞的方法 |
CN111378614A (zh) * | 2020-03-18 | 2020-07-07 | 中国农业大学 | 单层致密奶牛乳腺上皮细胞培养方法 |
CN112129345A (zh) * | 2020-09-14 | 2020-12-25 | 安徽军松现代农业科技有限公司 | 一种基于数据采集的土壤监测的环境监测系统 |
CN113564114A (zh) * | 2021-01-16 | 2021-10-29 | 浙江工商大学 | 一种cmt93-dc-t细胞的三维细胞模型的构建方法和应用 |
CN113564114B (zh) * | 2021-01-16 | 2023-04-07 | 浙江工商大学 | 一种cmt93-dc-t细胞的三维细胞模型的构建方法和应用 |
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