CN110218740A - 一种用于快速检测经典Wnt信号通路活性的脑血管内皮细胞系 - Google Patents

一种用于快速检测经典Wnt信号通路活性的脑血管内皮细胞系 Download PDF

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CN110218740A
CN110218740A CN201910412805.XA CN201910412805A CN110218740A CN 110218740 A CN110218740 A CN 110218740A CN 201910412805 A CN201910412805 A CN 201910412805A CN 110218740 A CN110218740 A CN 110218740A
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畅君雷
王田喜
尹美芳
丘林辉
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Abstract

本发明提供了一种稳定表达TOP‑Flash/Renilla双荧光素酶报告基因的脑血管内皮细胞系的制备方法，其通过以下方法步骤获得：1)选择脑微血管内皮细胞，并以带有Renilla基因的慢病毒感染脑微血管内皮细胞，筛选获得稳定表达Renilla的细胞系；2)以带有TOP‑Flash基因的慢病毒感染步骤1)获得的细胞系，筛选获得通知稳定表达Renilla和TOP‑Flash双荧光素酶报告基因的脑血管内皮细胞系。本发明的细胞系，能够用于筛选激活脑血管内皮细胞内Wnt信号通路活性的活性物质，提高了筛选效率和准确性，制备方法简单。

Description

一种用于快速检测经典Wnt信号通路活性的脑血管内皮细 胞系
技术领域
本发明涉及生物技术领域，具体涉及一种用于快速检测经典Wnt信号通路活性的药物筛选方法。
背景技术
一、脑卒中的现状
脑卒中，也称脑中风，是由于脑内血管突然破裂或者堵塞导致局部大脑血液供应障碍，脑组织缺氧缺血性病变坏死的一种急性脑血管疾病，其中超过75％为缺血性脑卒中(1)。脑卒中具有发病率高，致死致残率高和复发率高的特点。研究表明，脑卒中是我国居民死亡和残疾的首要原因，每年大约有270万新发患者，且呈逐年上升趋势(2,3)。脑卒中给整个社会和患者家庭带来沉重的经济负担。临床上仍缺少有效的治疗手段，以预防为主。
血脑屏障高度选择性的输送血液中有益的物质进入大脑，同时严格限制血液中有害的物质通过，从而保持大脑内环境的稳态及维持中枢神经系统的稳定(4)。研究表明，在脑卒中发生时脑血管遭受到缺血再灌注的损伤，血脑屏障被破坏，血液中有害成分，例如炎性因子和免疫细胞，进入脑组织中加剧脑组织损伤(5)。近年来，抑制缺血再灌注导致的血脑屏障损伤已成为治疗脑卒中的新思路。此外，卒中后脑组织缺氧和损伤诱导的脑血管生成也对神经功能恢复和患者预后有重要的提高和改善作用。深入探索缺血性脑卒中之后血脑屏障损伤和血管生成的分子调控机制，开发具有应用前景的新颖蛋白或分子药物，有利于临床治疗脑卒中。
二、脑卒中的治疗
药物溶栓是治疗缺血性脑卒中的主要方案。迄今为止，美国食品药品管理局(FDA)批准用于缺血性脑卒中的唯一溶栓药物是重组组织型纤溶酶原激活物(recombinanttissue-type plasminogen activator,rtPA)。rtPA的缺点是有效治疗窗狭窄，仅有4.5h。在治疗时间窗之外使用溶栓药物极易破坏血脑屏障，从而导致脑出血等严重后果(6)。由于大多数患者到达医院的时间都超过了药物溶栓治疗时间窗，我国大约仅有1.6％的缺血性脑卒中患者符合溶栓治疗的标准。迄今为止，在临床研究中，神经保护剂(7,8)、抗氧化应激(9-11)、钙通道阻滞剂(12,13)和抑制炎症反应(14,15)等治疗方法都没有效果。筛选新的治疗脑卒中的小分子或生物大分子药物具有重要临床意义。
缺血性脑卒中疏通血管之后，血流再灌注使缺血缺氧的脑组织重新获得供血、供氧的同时也会造成急性脑血管损伤。再灌注加剧破坏血脑屏障功能与结构，进一步伤害脑组织，并增加脑出血和脑水肿的风险。研究表明，闭塞的脑血管在血管疏通之后，再灌注在短时间内就能激活一系列的生化反应，引起神经兴奋毒性、氧化应激水平上升、炎性因子分泌增多等(16-18)。这些炎症因子进一步激活脑血管内皮细胞，细胞表面的细胞粘附因子表达升高(19,20)。促进淋巴细胞穿透血脑屏障，进入脑组织的淋巴细胞加剧脑内炎症反应，从而引起大脑内神经细胞大量死亡(21-23)。
三、经典Wnt信号通路对脑血管生成和血脑屏障功能的重要作用
经典Wnt信号通路是指在Wnt蛋白存在时，激活膜受体Fzd4和Lrp5/6，可以稳定细胞质内β-连环蛋白(β-catenin)，引起β-连环蛋白在细胞质中积累并最终进入细胞核中，在细胞核内作为转录因子TCF/LEF家族的转录共激活因子，上调靶标基因的转录水平。当没有Wnt蛋白时，β-连环蛋白不会在细胞质中积累，因为破坏复合物通常会把它迅速降解。利用β-连环蛋白激活的β-半乳糖苷酶报告基因(BAT-Gal)转基因小鼠，检测脑血管细胞中经典Wnt信号通路的表达。在胚胎期9.5天，脑血管内皮中LacZ-报告基因活性显著增强。在整个脑血管生成过程中，大部分脑血管内皮细胞是LacZ阳性的(24)。利用另一个Wnt报告基因报告小鼠TOP-GAL也确认经经典Wnt信号通路在脑血管发育过程中被激活(25)。
经典Wnt信号通路中的配体(Wnt7a、Wnt 7b和Norrin)，受体(Fzd4)，共同受体(Lrp5/6)，共激活因子(Tspan12、Gpr124和Reck)以及Ctnnb1(β-连环蛋白)本身在整个脑血管生成以及维持血脑屏障功能的过程中非常重要。内皮细胞中特异性敲除Ctnnb1基因导致小鼠在胚胎期12.5天死亡，主要表型有脑血管缺陷，前脑和脊髓中没有毛细血管形成(25)。条件性敲除脑血管内皮细胞中Gpr124基因导致小鼠胚胎期致死，主要表现为前脑出血和中枢神经系统血管停止生成(26,27)。条件性敲除脑血管内皮细胞中Reck，导致胚胎期13.5天前脑和脊髓出血(28)。条件性敲除脑血管内皮细胞中Fzd4基因不会导致胚胎期致死，但导致成年小脑和嗅球血脑屏障完整性被破坏(29)。在正常生理条件下，成年小鼠脑血管内皮细胞中敲除Gpr124基因对血脑屏障功能没有显著性改变(30)。但在缺血性脑卒中和脑胶质瘤等病理条件下，成年小鼠脑血管内皮细胞中敲除Gpr124基因加剧血脑屏障的破坏及脑损伤(30)。通过激活脑血管内皮细胞中的经典Wnt信号通路，可增强血脑屏障的完整性及减少肿瘤出血等。成年小鼠中内皮细胞条件性缺失Ctnnb1诱导血脑屏障破坏和脑瘀点出血(31)。综上所述，经典Wnt信号通路对于脑血管发育及维持血脑屏障功能至关重要。通过靶向脑血管内皮细胞中经典Wnt信号通路，筛选出能显著调节脑血管内皮细胞中经典Wnt信号通路的药物，将有望用于治疗缺血性脑卒中。
四、现有检测经典Wnt信号通路活性的实验体系
现有检测经典Wnt信号通路活性的实验体系有两种：1)体内小鼠体系：利用β-catenin依赖性激活的报告基因转基因小鼠，目前有BAT-Gal小鼠(报告基因为β-半乳糖苷酶)、TOP-GAL小鼠(报告基因为β-半乳糖苷酶)、Axin2-LacZ(报告基因为β-半乳糖苷酶)和Tcf/Lef-H2B-EGFP小鼠(报告基因为绿色荧光蛋白)；2)体外细胞体系：稳定转染β-连环蛋白依赖性的TOP Flash/Renilla双荧光素酶报告基因的HEK 293细胞系和小鼠L细胞系。
如上所述，在临床上缺血性脑卒中仍然缺乏有效的治疗药物。鉴于经典Wnt信号通路对脑血管生成和血脑屏障的重要调节作用，本发明拟通过构建一种细胞系从而实现快速筛选能够靶向激活脑血管内皮细胞中经典Wnt信号通路的药物，用于治疗缺血性脑卒中。
现有的检测经典Wnt信号通路活性的体内和体外实验体系各具有显著的局限性：1)体内小鼠体系成本高、耗时长、检测方法技术难度大且可重复性差，需要进行免疫荧光染色或免疫组化，容易产生假阴性或假阳性，也无法实现对大数量的候选药物库的高通量筛选；2)体外细胞体系可以避免上述体内小鼠体系的缺点，成本低、灵敏度高、操作方便、可高通量筛选，然而现有的体外细胞筛选体系均为非脑血管内皮细胞，基于这些细胞体系筛选的药物无法反映它们对脑血管内皮细胞的作用。而由于脑血管内皮细胞中高表达其他细胞中并不具有的经典Wnt信号通路膜受体和调节蛋白(Gpr124、Reck等)，只有使用脑血管内皮细胞作为实验体系才能筛选到真正特异性激活此类细胞中经典Wnt信号通路的药物。制备过程中，虽然现有技术中存在HEK293细胞系的转染实验，但现有的HEK 293细胞系和小鼠L细胞系容易转染，很容易导入外源型DNA质粒，而脑血管内皮细胞难以利用普通的转染试剂转染，必须使用慢病毒手段，技术要求高。同时脑血管内皮细胞对多种抗生素具有抗性，选择抗生素种类或浓度较为困难。
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发明内容
本发明公开了稳定表达TOP-Flash/Renilla双荧光素酶报告基因的脑血管内皮细胞系。
本发明一个方面提供了一种稳定表达TOP-Flash/Renilla双荧光素酶报告基因的脑血管内皮细胞系的制备方法，其通过以下方法步骤获得：
1)选择脑微血管内皮细胞，并以带有Renilla基因的慢病毒感染脑微血管内皮细胞，筛选获得稳定表达Renilla的细胞系；
2)以带有TOP-Flash基因的慢病毒感染步骤1)获得的细胞系，筛选获得稳定表达Renilla和TOP-Flash双荧光素酶报告基因的脑血管内皮细胞系。
在本发明的技术方案中，所述步骤1)中带有Renilla基因的慢病毒为具有潮霉素抗性的慢病毒，优选为Lenti CMV Renilla病毒。
在本发明的技术方案中，所述步骤2)中带有TOP-Flash基因的慢病毒为具有嘌呤霉素抗性的慢病毒，优选为Lenti TCF/LEF Reporter病毒。
在本发明的技术方案中，所述步骤1)中筛选的方法为在转染后，在细胞培养基中加入的潮霉素后进行筛选，优选地，潮霉素的浓度为100μg/ml以上。
在本发明的技术方案中，所述步骤2)中筛选的方法为在转染后，在细胞培养基中加入的嘌呤霉素和潮霉素后进行筛选，优选地，先以4μg/ml嘌呤霉素和50μg/ml潮霉素筛选1-5天，再以1μg/ml嘌呤霉素和50μg/ml潮霉素的浓度筛选5-15天。
在本发明的技术方案中，步骤1)中脑血管内皮细胞系为人或动物的脑血管内皮细胞系，优选为bEnd.3细胞系。
本发明另一个方面提供了由所述稳定表达TOP-Flash/Renilla双荧光素酶报告基因的脑血管内皮细胞系的制备方法获得的脑血管内皮细胞系。
本发明再一个方面提供了所述脑血管内皮细胞系用于筛选激活脑血管内皮细胞内Wnt信号通路活性的物质的用途。
本发明再一个方面提供了一种用于筛选激活脑血管内皮细胞内Wnt信号通路活性物质的试剂盒，其中包含了一种稳定表达TOP-Flash/Renilla双荧光素酶报告基因的脑血管内皮细胞系。
本发明再一个方面提供了筛选激活脑血管内皮细胞内Wnt信号通路活性的物质的方法，其包括使用权利要求？所述的脑血管内皮细胞系与待检测物质进行培养，当仅检测到Renilla信号时，证明待检测物质不能激活Wnt信号通路；当检测到TOP-Flash/Renilla双信号时，证明待检测物质能激活Wnt信号通路；当检测到检测不到Renilla信号时，需重新检测。
有益效果
本发明提供了能够用于筛选激活脑血管内皮细胞内Wnt信号通路活性的细胞系，能够用于筛选激活脑血管内皮细胞内Wnt信号通路活性的活性物质，提高了筛选效率和准确性，制备方法简单。
附图说明
图1.不同浓度的潮霉素处理bEnd.3细胞7天。
图2.不同浓度的嘌呤霉素处理bEnd.3细胞4天。
图3.Renilla bEnd.3稳转细胞系的筛选。Lenti-Renilla病毒感染bEnd.3细胞72h，然后在细胞培养基中加入潮霉素(100μg/ml)持续筛选bEnd.3细胞12天。直到对照组细胞全部被潮霉素杀死。
图4.TOP-Flash/Renilla bEnd.3稳转细胞系的筛选。Lenti-TOP-Flash病毒感染稳转Renilla bEnd.3细胞系72h，然后在细胞培养基中加入嘌呤霉素(4μg/ml)和潮霉素(50μg/ml)持续筛选3天。最后在细胞培养基中加入嘌呤霉素(1μg/ml)和潮霉素(50μg/ml)持续筛选10天。直到对照组细胞全部被嘌呤霉素杀死。
图5.验证稳转细胞系TOP-Flash/Renilla bEnd.3的表达效率。在TOP-Flash/Renilla bEnd.3的细胞培养基中加入Wnt3a(100ng/ml)，继续培养24h。通过检测荧光强度来验证细胞系的表达效率。A，仪器检测细胞内的荧光信号强度。B，A的柱状图。**P<0.01。
图6.利用稳转细胞系TOP-Flash/Renilla bEnd.3细胞内检测经典Wnt信号通路。将5千个TOP-Flash/Renilla bEnd.3细胞接种在96孔板中培养过夜。再进行氧糖剥夺再复氧模型(OGD/R)，氧糖剥夺的时间为6h，再恢复糖氧继续培养18h。检测细胞内经典Wnt信号通路的变化。检测有或无TNFα时，TOP-Flash/Renilla bEnd.3细胞内经典Wnt信号通路的变化。在培养基中分别加入Wnt3a(100ng/ml)和TNFα(100ng/ml)，继续培养24h。检测经典Wnt信号通路的变化。A，氧糖剥夺再复氧细胞模型。B，TNFα处理稳转细胞系。*P<0.05，***P<0.001。
图7.实验方案流程图，首先构建稳定表达Renilla的bEnd.3细胞系，然后在此基础上再构建TOP-Flash/Renilla bEnd.3的细胞系，最后验证该稳转细胞系的表达效率。当细胞内的经典Wnt信号通路被激活时，稳转细胞系大量表达TOP-Flash。通过检测细胞内的荧光信号强度的变化，可以判断经典Wnt信号通路的变化。当不同的药物处理该稳转细胞系时，筛出能够激活经典Wnt信号通路的药物，进一步用于缺血性脑卒中治疗的动物实验或临床实验。
具体实施方式
实施例1准备工作和预实验
从美国模式培养物集存库(ATCC)购买bEnd.3细胞系。从Qiagen公司购买CignalLenti CMV Renilla Control(Hygro)和Cignal Lenti TCF/LEF Reporter(Luciferase；Puro)慢病毒。从Sigma公司购买潮霉素(Hygromycin)和嘌呤霉素(Puromycin)。从Promega公司购买Dual-Reporter Assay System，用于检测荧光素酶的表达量。扩大培养bEnd.3细胞，同时冻存一些bEnd.3细胞用来保种。不同浓度的潮霉素(图1)处理bEnd.3细胞一周，在普通光学显微镜下观察bEnd.3细胞的形态。
将10万个bEnd.3细胞均匀接种到六孔板中，培养过夜并观察细胞的生长状态。当细胞的密度达到80％的时候加入潮霉素。潮霉素的浓度分别为0μg/ml、50μg/ml、100μg/ml和200μg/ml。每天对bEnd.3细胞进行换液培养，观察细胞的生长状态并拍照保存。连续培养bEnd.3细胞一周，选择死亡率超过90％以上的药物浓度。如果不同的浓度均能导致bEnd.3细胞死亡率超过90％以上，选择其中最低的浓度，浓度过高影响细胞的生长速度。在实验中发现，100μg/ml和200μg/ml的潮霉素导致bEnd.3细胞死亡率超过90％，因此，选择100μg/ml作为筛选的浓度。
同样的原理，不同浓度的嘌呤霉素(图2)处理bEnd.3细胞四天，在普通光学显微镜下观察bEnd.3细胞的形态，选择最适合的浓度用于细胞系的筛选。将10万个bEnd.3细胞均匀接种到六孔板中，培养过夜并观察细胞的生长状态。当细胞的密度达到80％的时候加入嘌呤霉素。嘌呤霉素的浓度分别为0μg/ml、0.5μg/ml、1μg/ml和2μg/ml。每天对bEnd.3细胞进行换液培养，观察细胞的生长状态并拍照保存。连续培养bEnd.3细胞一周，选择死亡率超过90％以上的药物浓度。如果不同的浓度均能导致bEnd.3细胞死亡率超过90％以上，选择其中最低的浓度，浓度过高影响细胞的生长速度。在实验中发现，1μg/ml和2μg/ml的嘌呤霉素导致bEnd.3细胞死亡率超过90％。因此，选择1μg/ml作为筛选浓度。如果细胞的生长密度过大，会发生明显的接触抑制。因此，当细胞密度过高时，可适当提高药物筛选的浓度，加快杀死细胞。
实施例2构建稳定表达Renilla的bEnd.3细胞系
Cignal Lenti CMV Renilla Control(Hygro)的启动子是CMV，带有潮霉素的抗性。利用Lenti CMV Renilla病毒感染bEnd.3细胞72h。然后在细胞培养基中加入100μg/ml的潮霉素持续筛选12天，最后筛出稳定表达Renilla的细胞系(图3)。稳定表达Renilla的bEnd.3细胞系在双荧光素酶报告基因实验中用做内参荧光。用含有潮霉素(50μg/ml)的培养基继续培养稳定表达Renilla的bEnd.3细胞系Renilla bEnd.3细胞系，扩大培养细胞，并冻存一部分细胞用于保种。
实施例3构建稳定表达Renilla/TOP flash的bEnd.3细胞系
在Renilla bEnd.3细胞系中加入Cignal Lenti TCF/LEF Reporter(Puro)病毒，并持续感染细胞72h。Lenti TCF/LEF Reporter(Puro)病毒利用TCF/LEF启动子，带有嘌呤霉素的抗性，表达TOP-Flash荧光素酶。然后在细胞培养基中加入嘌呤霉素(4μg/ml)和潮霉素(50μg/ml)筛选Renilla bEnd.3稳转细胞系3天。再降低抗生素的浓度，在细胞培养基中加入嘌呤霉素(1μg/ml)和潮霉素(50μg/ml)筛选Renilla bEnd.3细胞10天，筛选出稳定表达TOP-Flash/Renilla的细胞系(图4)。继续扩大培养稳转细胞系，并冻存一些细胞用于保种。
5.2效果实例
利用Wnt3a激活经典Wnt信号通路，验证稳转TOP-Flash/Renilla的bEnd.3细胞系。实验表明，浓度为100ng/ml的Wnt3a显著增强TOP-Flash/Renilla bEnd.3细胞中的TOP-Flash信号(图5)。表明该稳转细胞系切实有效，可以用于针对经典Wnt信号通路的药物筛选。与此同时，发明人进行了药物筛选实验。通过不同的药物或方法处理稳转TOP-Flash/Renilla bEnd.3细胞，检测经典Wnt信号通路。申请人发现氧糖剥夺再复氧模型(OGD/R)和炎症因子(TNFα)能够显著抑制经典Wnt信号通路的活性(图6)。脑血管内皮细胞中经典Wnt信号通路对于维持血脑屏障功能至关重要。找出影响经典Wnt信号通路的分子或药物对于保护血脑屏障功能至关重要。过表达激活经典Wnt信号通路的分子或抑制干扰经典Wnt信号通路的分子，将有利于改善血脑屏障的功能。
5.3实验方案流程图
详细的实验方案流程图见附图与说明(图7)。

Claims (10)

1.一种稳定表达TOP-Flash/Renilla双荧光素酶报告基因的脑血管内皮细胞系的制备方法，其通过以下方法步骤获得：
1)选择脑微血管内皮细胞，并以带有Renilla基因的慢病毒感染脑微血管内皮细胞，筛选获得稳定表达Renilla的细胞系；
2)以带有TOP-Flash基因的慢病毒感染步骤1)获得的细胞系，筛选获得稳定表达Renilla和TOP-Flash双荧光素酶报告基因的脑血管内皮细胞系。
2.根据权利要求1所述的制备方法，所述步骤1)中带有Renilla基因的慢病毒为具有潮霉素抗性的慢病毒，优选为Lenti CMV Renilla病毒。
3.根据权利要求1-2任一项所述的制备方法，所述步骤2)中带有TOP-Flash基因的慢病毒为具有嘌呤霉素抗性的慢病毒，优选为Lenti TCF/LEF Reporter病毒。
4.根据权利要求1-3任一项所述的制备方法，所述步骤1)中筛选的方法为在转染后，在细胞培养基中加入的潮霉素后进行筛选，优选地，潮霉素的浓度为100μg/ml以上。
5.根据权利要求1-4任一项所述的制备方法，所述步骤2)中筛选的方法为在转染后，在细胞培养基中加入的嘌呤霉素和潮霉素后进行筛选，优选地，先以4μg/ml嘌呤霉素和50μg/ml潮霉素筛选1-5天，再以1μg/ml嘌呤霉素和50μg/ml潮霉素的浓度筛选5-15天。
6.根据权利要求1-5任一项所述的制备方法，步骤1)中脑血管内皮细胞系为人或动物的脑血管内皮细胞系，优选为bEnd.3细胞系。
7.根据权利要求1-6任一项所述的制备方法获得的脑血管内皮细胞系。
8.根据权利要求7所述的脑血管内皮细胞系用于筛选激活脑血管内皮细胞内Wnt信号通路活性物质的用途。
9.一种用于筛选激活脑血管内皮细胞内Wnt信号通路活性物质的试剂盒，其中包含了权利要求7的脑血管内皮细胞系。
10.筛选激活脑血管内皮细胞内Wnt信号通路活性的物质的方法，其包括使用权利要求7所述的脑血管内皮细胞系与待检测物质进行培养，当仅检测到Renilla信号时，证明待检测物质不能激活Wnt信号通路；当检测到TOP-Flash/Renilla双信号时，证明待检测物质能激活Wnt信号通路；当检测到检测不到Renilla信号时，需重新检测。
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