CN116376842A - 可视化指示NF-κB信号的神经干细胞系及其构建方法与应用 - Google Patents
可视化指示NF-κB信号的神经干细胞系及其构建方法与应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，尤其是一种可视化指示NF‑κB信号的人源神经干细胞系及其构建方法与应用，所述人源神经干细胞系保藏在武汉大学保藏中心，保藏编号为CCTCCNO：C2022370。该细胞系使用人源神经干细胞，具有种属特异性，能够更加真实的反应人类脑类疾病的发生和发展过程，且关键炎症信号NF‑κB采用可视化表达策略，在没有NF‑κB激动剂存在的情况下，细胞于荧光显微镜下仅观察到微弱红色荧光，而在有NF‑κB激动剂(如TNF‑α或细胞处于炎症状态)存在的情况下，胞内NF‑κB信号通路被激活，于荧光显微镜下可观察到红色荧光蛋白表达强度显著呈时间梯度增加。这有益于及时监控细胞的炎症程度，为神经类药物抑制炎症反应效果做出准确判断，是神经类药物开发的有力工具。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种可视化指示NF-κB信号的神经干细胞系及其构建方法与应用。
技术背景
调查显示，全世界已有数千万的神经退行性疾病患者，包括阿尔茨海默病(AD)，帕金森病(PD)，运动神经元疾病(MND)，亨廷顿病(HD)，多发性硬化症(MS)等。中国人口众多，AD的发病率约为1％，年龄每增加5岁，AD的患病率约增加1倍，目前全球已有约5000万左右的阿尔茨海默病患者，约1/3病例发生在中国。随着老龄人口的快速增加和寿命的延长，此类疾病呈高发趋势，为家庭和国家医疗带来了极大的负担。
因此，随着老龄化社会的到来，能够预防或治疗阿尔兹海默症等神经退行性疾病的药物具有非常重大的社会意义。传统的检测药物抑制炎症效果的体外实验有免疫印迹、荧光定量PCR等，通过检测相关炎性因子的表达水平来进行判断，但这些手段费时费力费钱，且通常需要裂解细胞，不能在活体水平进行测试，当细胞样本比较珍贵时，这些手段显得有些不足。因此目前市面上急需一款能够模拟人神经系统，并快速对神经相关疾病进程进行反应的检测工具。
发明内容
为解决上述问题，本专利旨在构建一种可视化指示NF-κB信号的神经干细胞系，为针对以NF-κB信号通路为靶点的阿尔兹海默症等神经性疾病相关药物研发、筛选以及机理的研究提供一种人源的、简便的、可视化评估工具。
为达到上述目的，本专利采用具体以下技术方案予以实现：
本发明第一方面提供了一种可视化指示NF-κB信号的神经干细胞系，其微生物保藏编号是：C2022370，分类命名是：人源神经干细胞系NPC-NF-κB-mcherry，保藏日期是：2023年04月11日。保藏单位：湖北省武汉市武昌区武汉大学保藏中心，保藏地址：湖北省武汉市武昌区八一路299号武汉大学校内。
本发明第二方面提供了一种可视化指示NF-κB信号的神经干细胞系的构建方法，包括以下步骤：
(1)：构建lenti-NF-κB-mcherry真核细胞表达载体；将NF-κB结合序列作为启动子的组成部分克隆到mcherry报告基因载体中，引导红色荧光蛋白mcherry的转录，得到所述的真核表达载体；
(2)：将(1)所述的真核表达载体lenti-NF-κB-mcherry和病毒包装载体pCMV-VSV-G、pCAG-dR8.9共转至工具细胞HEK 293T进行慢病毒包装；
(3)：用慢病毒感染目的细胞H9-NPC，通过有限稀释法获取阳性克隆进行扩大培养，构建可视化指示NF-κB信号的人源神经干细胞系。
优选地，其特征在于，所述NF-κB结合序列的核苷酸序列如SEQ.ID.NO.1所示。
优选地，步骤(2)中pCMV-VSV-G载体、pCAG-dR8.9载体和目的基因载体lenti-NF-κB-mcherry三种质粒加入比为0.1：2：4。
本发明第三方面提供了一种第一方面所述的可视化指示NF-κB信号的神经干细胞系在药物筛选中的应用。
优选地，其特征在于，所述的药物以NF-κB信号通路为治疗靶点。
优选地，其特征在于，使用NF-κB信号通路激活剂处理细胞，引发细胞炎症反应，按时间梯度进行刺激，通过荧光显微镜和免疫印迹实验测试细胞NF-κB表达水平是否与红色荧光强度呈正相关。
优选地，其特征在于，所述的NF-κB信号通路激活剂包含但不限于TNF-α，使用浓度为1nM-1uM。
与现有技术相比，本发明的有益效果为：
(1)本发明提供的构建细胞系的技术简便快捷、成本较低；且相比于小鼠来源的细胞，使用人神经干细胞制备稳转细胞系来进行药物筛选，更接近人脑炎症反应的发生与发展的真实水平。
(2)本发明通过慢病毒感染的方式，将可视化指示细胞炎性水平的lenti-NF-κB-mcherry报告基因质粒导入细胞，可以保证该细胞的长久性和稳定性。
(3)该细胞在NF-κB信号通路激动剂的处理下，具有阿尔兹海默症等疾病存在的神经炎性特征，为以NF-κB信号通路为靶点的相关药物的研发与筛选以及机理研究提供体外细胞研究平台可能。
(4)该细胞是在活细胞水平对药物效果进行评价，评价完成后细胞可重复利用。
(5)该细胞的来源为人源神经干细胞，更能够真实的反应人脑疾病的发生和发展过程。
附图说明
图1lenti-NF-κB-mcherry质粒的载体图谱；
图2lenti-NF-κB-mcherry质粒的测序峰图结果；
图3倒置荧光显微镜下观察293T细胞的红色荧光蛋白表达，确认慢病毒的包装效率达到95％以上；其中(a)明场；(b)红色荧光通道；
图4倒置荧光显微镜下观察NPC细胞的红色荧光蛋白表达，确认目的质粒已成功导入NPC细胞；其中(a)明场；(b)红色荧光通道；
图5实时荧光监测NF-κB激动剂刺激后不同时间点的NPC稳转细胞系荧光强度的信号变化结果，其中(a)随着激动剂刺激时间的增长，荧光信号逐步被激发；(b)荧光强度的定量结果；
图6Western blot检测NPC-NF-κB-mcherry细胞的NF-κB蛋白表达量；其中，(a)目的蛋白NF-κB随时间表达量增加；(b)定量结果。
具体实施方式
下面对结合说明书附图本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所做的任何变换或替换，均属于本发明的保护范围。
实施例1慢病毒表达载体的构建及鉴定
1.1目的片段的合成
S1、为了可以更加灵敏的检测NF-κB的激活水平，设计4个NF-κB的结合位点，引入的酶切位点为5’的EcoR I和3’的KpnI。序列如表1所示，于生工生物工程有限公司合成。
S2、合成后将成对的引物干粉溶解于退火缓冲液中，90℃水浴15min，自然冷却至室温，退火产物即为目的片段。
1.2载体酶切
将载体pHR'-CMV-mcherry质粒进行酶切，酶切反应在37℃水浴反应2h。
反应体系如下表：
| 组分 | 体积 |
| pHR'-CMV-mcherry | 1ug |
| 10x Buffer | 2ul |
| KpnI | 1ul |
| EcoRI | 1ul |
| ddH2O | 补足至20ul |
1.3连接反应
质粒pHR'-CMV-mcherry用EcoRI+KpnI酶切后回收的大片段与目的片段进行连接，连接反应在16℃反应12小时。反应体系如下表:
| 组分 | 体积 |
| 目的片段 | 与载体摩尔比5:1 |
| 载体 | 50ng |
| 10x Ligation Buffer | 2ul |
| T4 DNA Ligase | 1ul |
| ddH2O | 补足至20ul |
1.4转化
S1、取10ul连接产物与50ul DH5a感受态细菌混匀；
S2、冰浴20min，42℃热激45s，立即置冰上放置2min；
S3、加入预热至室温的700ul LB培养基，37℃恒温摇床培养50min；
S4、取200ul的菌液，用移液器混匀后均匀涂布于氨苄抗性的LB平板上，37℃恒温培养箱倒置培养过夜。
1.5挑单克隆
挑单克隆小摇，摇混后按1：1000接种至10ml-15ml LB培养基(氨苄)过夜培养。
1.6质粒提取及验证
S1、取10-15mL的菌液，室温下，5,000xg离心10min收集细菌；
S2、倒弃培养基，加入500μl Solution I/RNase A混和液，漩涡振荡使细胞完全悬浮；
S3、将溶液转移到新的2mL离心管，加入500μl Solution II，轻轻颠倒混匀，将混合液室温放置2～3min；
S4、加入250μl预冷的N3 Buffer，轻柔上下颠倒离心管数次至形成白色絮狀沉淀；室温下，最大速度(≥13000xg)离心10min，转移上清液至新1.5mL离心管中；
S5、加入上清液0.1倍体积的ETR Solution，上下颠倒10次混匀，冰浴10min；
S6、42℃孵育5min，溶液再次变浑浊；
S7、25℃，12,000xg离心3min，将上层水相(含DNA)转移到新的1.5mL离心管中，加入0.5倍体积的无水乙醇，上下颠倒混匀6-7次，室温静置1-2min；
S8、转移混合液(一次最多转移700μl)至套有2mL收集管的
DNA结合柱中，室温，10000xg离心1min，弃滤液；
S9、重复步骤8，直至所有混合液转移过柱；
S10、把柱子重新装回收集管，加入500μl HBC Buffer，最大速度离心1min，弃滤液；
S11、把柱子重新装回收集管，加入700μl DNAWash Buffer，最大速度离心1min，弃滤液；
S12、重复步骤11；
S13、把柱子重新装回收集管，≥13000xg离心空柱2min干燥柱子基质；
S14、把柱子装在干净的1.5mL离心管上，加入80-100μl Elution Buffer到柱子基质中，静置1min，最大速度离心1min洗脱DNA即为目的产物，将其名为Lenti-NF-κB-mcherry，质粒图谱如图1所示，测序结果如图2所示，目的片段成功连入载体，且无突变。
实施例2病毒包装与感染
2.1HEK 293T细胞准备
S1、弃去旧培养基，加入2ml无菌PBS,清洗细胞生长面，然后弃去PBS溶液，重复一次。
S2、用1ml胰酶消化对数期生长的293T细胞3-5min。
S3、用含10％血清的培养基终止消化，调整细胞密度为1X106细胞/2ml，重新接种于六孔板中，每孔2ml，37℃，5％CO2继续培养，使转染时的细胞密度达到80％左右。
2.2质粒转染HEK 293T细胞
S1、在转染前，将培养有细胞的六孔板每孔更换2ml新鲜培养液(含有血清和抗生素的完全培养液)。
S2、脂质体-DNA复合物配制，取一个无菌无酶离心管，对待转染的六孔板中的每一孔的细胞，加入125ul opti-MEM，目的质粒4ug，慢病毒包装用质粒pCMV-VSV-G 2ug，pCAG-dR8.90.2ug,并用枪轻轻吹打混匀；再加入4ul lipo8000转染试剂，用枪轻轻吹打混匀，均匀滴加至293T细胞，继续培养，6h后更换新鲜的培养液。
2.3HEK 293T细胞转染效率确定
S1、转染有lenti-NF-κB-mcherry的HEK293T细胞，由于目的质粒含有红色荧光蛋白mcherry基因，在荧光倒置显微镜下观察可见红色荧光，未转染成功的细胞则不带荧光，因此可以以此初步判断病毒包装是否成功。若成功，方可进行后续系列操作。结果如图3所示，显微镜下可见293T细胞转染成功率很高，90％的细胞都携带红色荧光。
2.4病毒收集与目的细胞感染
S1、转染24h后，将293T细胞培养基(10％血清)更换成新鲜的H9NPC细胞培养基(H9NPC细胞培养基由湖南赛奥维生物技术有限公司提供)。
S2、转染后48h-72h，收集293T细胞上清，将收集的细胞上清1500rpm离心5min,并用0.45um的细胞滤膜进行过滤去除细胞碎片等杂质，收集含慢病毒的滤液进行后续感染用，过滤后的细胞上清如果暂时不进行进一步处理，则及时分装并于-70℃以下储存。
S3、取出待感染贴壁汇合度为70％-80％的目的细胞NPC，观察细胞状态。吸弃培养板内的原有液体，将收集的病毒进行NPC细胞感染，6h后适当补充新鲜NPC培养基。
S4、36h后，观察感染慢病毒的目的细胞的生长状态，以及荧光倒置显微镜下观察质粒是否成功导入，结果如图4所示，目的细胞NPC于荧光显微镜下可观察到微弱红色荧光，表明有细胞成功导入目的质粒。
实施例3目的细胞筛选
S1、用MG胶包被96孔板；
S2、制备待克隆的NPC稳转细胞悬液，用NPC培养基稀释到5、10、50个细胞/毫升。
S3、将上述三个浓度的细胞悬液加到96孔板中培养，100ul/孔，使相应的每孔分别含0.5、1和5个细胞。
S4、培养到第5天每孔补液50ul，第7-8天观察各孔内细胞的生长情况。于荧光显微镜下观察是否为阳性克隆。
S5、将阳性克隆传代扩繁。
实施例4NPC-NF-κB-mcherry稳转细胞系的应用
取对数生长期的NPC-NF-κB-mcherry细胞，PBS清洗细胞2次，用干细胞消化液Accutase对细胞进行消化，计数，取5x105个/孔密度接种于六孔培养板内，每孔体积2ml，37℃、5％CO2培养箱中培养，24h后待细胞贴壁，往细胞培养孔板中加入1nM TNFα，于加药后不同时间点，在荧光显微镜下，记录细胞荧光强度随时间的变化及相对荧光强度。同时将加药处理组分别于4h、8h、24h收取细胞裂解液进行Western blot检测炎性因子NF-κB的表达水平。本实验以不加TNFα处理的细胞作为阴性对照。
结果显示，对照组仅表达微弱的红色荧光，而加药处理组细胞能够被显著点亮，且随着加药处理时间的延长，红色荧光越来越亮，如图5所示。WB结果与之对应，指示细胞炎性水平的NF-κB蛋白表达水平随加药时间的延长呈现上升趋势，如图6所示，这表明NPC-NF-κB-mcherry稳转细胞系的炎性水平可以通过细胞的红色荧光强度反映，其可以作为细胞炎性高低的监测指标。
附序列表：
SEQ.ID.NO.1
ggactttccg ctgggacttt ccgctgggac tttccgctgg gactttccg 49
SEQ.ID.NO.2
aattccacct gcaacaggcc cgggactttc cgctgggact ttccgctggg actttccgct 60
gggactttcc ggtac 75
SEQ.ID.NO.3
cggaaagtcc cagcggaaag tcccagcgga aagtcccagc ggaaagtccc gggcctgttg 60
caggtgg 67
Claims (8)
1.一种可视化指示NF-κB信号的神经干细胞系，其特征在于，其保藏编号是：C2022370，分类命名是：人源神经干细胞系NPC-NF-κB-mcherry，保藏日期是：2023年04月11日。保藏单位：湖北省武汉市武昌区武汉大学保藏中心，保藏地址：湖北省武汉市武昌区八一路299号武汉大学校内。
2.一种可视化指示NF-κB信号的神经干细胞系的构建方法，其特征在于，包括以下步骤：
(1)：构建lenti-NF-κB-mcherry真核细胞表达载体；将NF-κB结合序列作为启动子的组成部分克隆到mcherry报告基因载体中，引导红色荧光蛋白mcherry的转录，得到所述的真核表达载体；
(2)：将(1)所述的真核表达载体lenti-NF-κB-mcherry和病毒包装载体pCMV-VSV-G、pCAG-dR8.9共转至工具细胞HEK 293T进行慢病毒包装；
(3)：用慢病毒感染目的细胞H9-NPC，通过有限稀释法获取阳性克隆进行扩大培养，构建可视化指示NF-κB信号的人源神经干细胞系。
3.根据权利要求2所述的方法，其特征在于，所述NF-κB结合序列的核苷酸序列如SEQ.ID.NO.1所示。
4.根据权利要求2所述的方法，其特征在于，步骤(2)中pCMV-VSV-G载体、pCAG-dR8.9载体和目的基因载体lenti-NF-κB-mcherry三种质粒加入比为0.1：2：4。
5.权利要求1所述的可视化指示NF-κB信号的神经干细胞系在药物筛选中的应用。
6.根据权利要求5所述的应用，其特征在于，所述的药物以NF-κB信号通路为治疗靶点。
7.根据权利要求6所述的应用，其特征在于，使用NF-κB信号通路激活剂处理细胞，引发细胞炎症反应，按时间梯度进行刺激，通过荧光显微镜和免疫印迹实验测试细胞NF-κB表达水平是否与红色荧光强度呈正相关。
8.根据权利要求7所述的应用，其特征在于，所述的NF-κB信号通路激活剂包含但不限于TNF-α，使用浓度为1nM-1uM。
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