CN112501175A - 一种银屑病抑制基因及其应用 - Google Patents
一种银屑病抑制基因及其应用 Download PDFInfo
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Abstract
本发明提供了一种银屑病抑制基因，为N4BP1基因。同时提供了基因N4BP1在制备治疗银屑病药物中的应用及在制备检测银屑病试剂盒中的应用。N4BP1在皮肤的角质细胞和中性粒细胞中高表达。N4BP1缺失后，角质细胞增殖加快，中性粒细胞数量和反映增强。因此N4BP1是银屑病发展的关键基因，N4BP1的功能缺失会加重疾病的进展。本发明通过过表达N4BP1基因，或用药物稳定、促进N4BP1的功能，为银屑病的发展研究及治疗药物的制备提供理论依据。
Description
技术领域
本发明属于医药技术领域，具体涉及一种银屑病抑制基因及其应用。
背景技术
银屑病是一种慢性的免疫细胞和角质细胞异常激活的炎症性皮肤病，其与遗传和环境密切相关。银屑病的临床表现多样，但主要以红色斑块上附银白色鳞屑为主。临床上，银屑病可以分为寻常型银屑病、关节型银屑病、红皮病型银屑病及脓疱型银屑病。
银屑病，俗称“牛皮癣”，发病机制复杂，其确切的细胞分子机制依然没有完全阐明。目前认为银屑病是具有较强的遗传性的自身免疫性疾病，多见于青状年。银屑病在人群中的发病率大约为2%，是皮肤科的常见疾病。
银屑病的发病主要有两大类细胞参与其中：角质形成细胞和免疫细胞。角质形成细胞是人皮肤表皮的主要细胞，在表皮基底细胞层至角质层的分化过程中，角质形成细胞最后失去细胞核及细胞器。角质形成细胞的分化过程，也就是角化的过程，可以认为是一种特殊的细胞凋亡过程。角化过程中，产生一系列特征性形态学变化，角质蛋白从基底层开始积累，最后充满整个角质细胞。在正常的上皮新生及稳态维持的过程中，角化及角质形成细胞的分化过程必须受到严密的调控，使角化过程和表皮的更新维持在一个正常的稳态。当银屑病发病时，角质形成细胞增殖和分化发生异常。角质形成细胞过度增殖，使表皮层明显增厚。角质形成细胞分化显著加快，形成大量的角质层。角质形成细胞的这些异常，一部分原因是由于遗传，环境及别的因素导致角质形成细胞在分子水平发生异常改变，另外一个部分原因是由于受到异常的免疫细胞刺激。
银屑病的最典型皮肤病变是具有银白色鳞屑的凸起红斑，其主要细胞包括中性粒细胞、T淋巴细胞、角质生成细胞、树突状细胞、巨噬细胞及肥大细胞。除了角质生成细胞外，这些异常活跃的免疫细胞会分泌大量的炎症因子如TNFa、IL-17和IL-23等。这些细胞因子会刺激角质形成细胞的异常增生和分化。异常增生和分化的角质形成细胞会进一步释放趋化和激活免疫细胞的因子形成正反馈。因此，银屑病的主要原因是，始动因素激活角质形成细胞或免疫细胞或同时激活这两类细胞，激活的细胞相关活化形成不受控制的正反馈，导致银屑病的发生。其中细胞因子在引起角质形成细胞和免疫细胞异常活化中发挥了极为重要的作用。目前为止，TNFa、IL-17和IL-23这3种细胞因子是临床和基础研究较充分的在银屑病中发挥核心作用的分子。针对这3种细胞因子的抗体在临床银屑病治疗中起到了较好的效果。但，目前所有的临床治疗只能控制和减缓银屑病的发病，都不能根治银屑病。因此，寻找新的核心基因，开发更有效的方法和药物依然是银屑病研究中的重点。
传统治疗银屑病的方法主要包括物理治疗、局部外用治疗及系统治疗。局部外用药物目前主要包括类维生素A、维生素D衍生物、皮质激素和钙剂抑制剂等。物理治疗包括光线和激光治疗等。系统用药主要是甲氨蝶呤、阿维A 和环孢素等。
近年来，银屑病治疗领域取得了根本性突破，针对单个分子的生物制剂取得了较好的临床治疗效果。如针对TNF-a的抗体有依那西普、英夫利昔单抗、阿达木单抗、赛妥珠单抗及戈利木单抗。针对IL-23 的抗体有乌司奴单抗。针对IL-17的抗体有司库奇尤单抗和依奇珠单抗等。这些生物制剂在临床治疗中效果较好，但依然有较大部分的病人对上述药物反应不佳，或治疗后短时间内出现复发。更为重要的是，这些生物制剂也不能根治银屑病，只能控制和减缓症状。
银屑病的治疗药物有了较大的进步，但目前的所有治疗方法都只能控制或缓解疾病，不能根除银屑病。上述表明，我们对于银屑病的发病机制有待深入研究，寻找银屑病中的未知重要基因，进而开发新的治疗方法，能从根本上进一步提高银屑病的治疗效果，或实现根除银屑病的目的。
如图1所示，在人的所有组织中，N4BP1在皮肤中高表达。图2所示，在所有的免疫细胞亚类中，中性粒细胞中N4BP1的表达最高。但N4BP1基因仍是一个研究较少的基因，对其功能的研究还较少。有研究表明，N4BP1可以通过降解HIV病毒的RNA来抑制病毒的复制。对于N4BP1的其它功能几乎未知。目前用于治疗银屑病的生物制剂的靶点主要和T细胞的功能相关，虽然有效实现了对疾病的控制和缓解，但没有实现对疾病的根治。表明除了T细胞外，在银屑病的发病中，还有其它因子或其它细胞参与了银屑病的始动机制。为此，本发明旨在提供一种银屑病抑制基因及其在制备治疗银屑病药物中的应用。
发明内容
本发明的发明目的是为了解决上述技术问题，提供一种银屑病抑制基因及其应用。
为解决上述技术问题，本发明的实施例提供一种银屑病抑制基因，为N4BP1基因。
本发明还提供一种银屑病抑制基因N4BP1在制备治疗银屑病药物中的应用。
本发明还提供一种银屑病抑制基因N4BP1在制备检测银屑病试剂盒中的应用。
本发明还提供一种基因N4BP1抑制银屑病的检测方法，包括如下步骤：
（1）4BP1敲除细胞系和N4BP1敲除小鼠
（1-1）HeLa细胞及其遗传学改造后的N4BP1过表达Hela细胞，用改良的DMEM高血糖培养基(含10%胎牛血清和1%抗生素)在37℃和5% CO2条件下培养；用脂质体2000分别转染过表达空载和过表达N4BP1质粒，转染6h后更换培养基；转染48h后，用嘌呤霉素筛选转染成功的细胞，培养2周；
（1-2）为了获得N4BP1敲除小鼠，我们设计了特异性识别第二外显子两端的两个sgRNA, N4BP1基因敲除小鼠的制备程序由上海模式生物中心有限公司完成。将N4BP1+/-小鼠杂交产生N4BP1-/-小鼠，并在C57BL/6遗传背景上回交10代以上；
（2）建立银屑病小鼠模型
用电动剃毛刀将8-10周龄小鼠背部的毛发剃掉，取咪喹莫特乳膏1.875mg，分别在N4BP1敲除小鼠和野生型小鼠背部连续涂抹11天，利用游标卡尺每天测量背部皮肤厚度，在预先设定好的时间内将小鼠断颈处死，取患处皮肤进行后续实验；
（3）苏木精-伊红染色
（3-1）将患处皮肤组织切成1cm*1cm大小的小块，浸在4%的多聚甲醛中24小时，第二天取出，进行酒精梯度脱水，石蜡包埋成组织块；
（3-2）组织块切成5um厚的切片，脱蜡、水化，苏木精染色30min，伊红染色5s，将染好的切片酒精脱水、二甲苯透明、树脂封片后镜下观察；
（4）制备小鼠胚胎成纤维细胞
（4-1）将N4BP1杂合子小鼠合笼13-15天后，取出孕鼠，断颈处死，取出胎鼠，在事先加有抗生素的PBS中漂洗两次后，置于直经为10cm的培养皿中；
（4-2）剪下胎鼠头部，放入1.5ml离心管用作后续基因型鉴定，将其余部分用剪刀剪碎后，直接向培养皿中加入改良的DMEM高血糖培养基（含10%胎牛血清和1%抗生素）；
（4-3）将MEF细胞置于37℃和5% CO2 中培养，每两天换液一次；
（5）RNA提取与实时荧光定量PCR
（5-1）取小鼠皮肤组织约30mg，放入1.5ml离心管中，向里加入1ml Trizol，用匀浆机匀浆后进行提取；
（5-2）将要提取RNA的细胞进行消化，离心收集，同样加入1ml Trizol，室温静置五分钟；加氯仿，离心取上层RNA；加异丙醇，萃取RNA；离心，75%酒精洗两次，溶于DEPC水；
（5-3）用Nano Drop 1000分光光度计(Thermo Fisher Science)测定总RNA的纯度和浓度；
（5-4）用HiScript II一步法RT-PCR试剂盒对1μg总RNA进行逆转录；CDNA稀释5倍，SYBR Green PCR试剂盒(#Q112-02/03，Vazyme)定量检测基因表达；
（6）RNA结合蛋白免疫沉淀
（6-1）用预冷的PBS洗涤两次除去培养基，然后加入PBS 10ml，在上述溶液中加入甲醛(37%原液)至1%的最终溶液浓度，室温下缓慢旋转孵化10分钟；再加入甘氨酸溶液至0.25M的最终浓度使交联反应(pH=7.0)终止，然后室温孵育5分钟，轻轻摇晃；加入5-10ml预冷PBS清洗细胞，并在2ml预冷的PBS中刮除细胞；
（6-2）离心收集细胞，用预冷过的PBS洗涤两次；收集细胞，加入含PMSF的RIPA裂解缓冲液，超声裂解；旋转1h后，以4℃、13000rpm离心；取上清液，用蛋白A预处理，离心后与抗FLAG抗体（#F3165，Sigma）孵育2h；之后，收集蛋白A珠子并清洗；免疫沉淀后的样品在70℃的去交联剂中重新悬浮45min，进行反向交联；反向交联后，用Trizol提取RNA。
本发明的上述技术方案的有益效果如下：本发明发现，N4BP1在皮肤的角质细胞和中性粒细胞中高表达，N4BP1缺失后，角质细胞增殖加快，中性粒细胞数量和反映增强。因此N4BP1是银屑病发展的关键基因，N4BP1的功能缺失会加重疾病的进展。本发明通过过表达N4BP1基因，或用药物稳定、促进N4BP1的功能，为银屑病的发展研究及治疗药物的制备提供理论依据。
附图说明
图1为在人的所有组织中，N4BP1在皮肤中高表达的示意图；
图2为在所有的免疫细胞亚类中，中性粒细胞中N4BP1的表达最高的示意图；
图3 本发明中，N4BP1缺陷小鼠，在IMQ药物诱导下，发展成更严重的银屑病的示意图；
图4为本发明中，N4BP1缺陷小鼠，在IMQ药物诱导下，银屑病相关基因表达增加的示意图；
图5为本发明中，过表达N4BP1后，显著下调银屑病相关基因CXCL1的表达的示意图；
图6为本发明中，过表达N4BP1后，显著下调银屑病相关基因JunB和FosB的表达的示意图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
本发明提供了一种银屑病抑制基因，为N4BP1基因。同时提供了基因N4BP1在制备治疗银屑病药物中的应用及在制备检测银屑病试剂盒中的应用。
下面通过具体实验步骤阐述通过过表达N4BP1基因，或用药物稳定、促进N4BP1的功能，可抑制银屑病的发展。
一种基因N4BP1抑制银屑病的检测方法，其特征在于，包括如下步骤：
（1）N4BP1敲除细胞系(研究N4BP1抑制银屑病的分子机制)和N4BP1敲除小鼠 （研究N4BP1抑制银屑病的体内作用和分子机制）
（1-1）HeLa细胞用改良的DMEM高血糖培养基(含10%胎牛血清和1%抗生素)在37℃和5% CO2条件下培养；用脂质体2000分别转染过表达空载和过表达N4BP1质粒，转染6h后更换培养基；转染48h后，用嘌呤霉素筛选转染成功的细胞，培养2周；
（1-2）为了获得N4BP1敲除小鼠，我们设计了特异性识别第二外显子两端的两个sgRNA, 当N4BP1基因的第2外显子敲除后，N4BP1基因会发生移码突变，导致不能产生有功能的N4BP1蛋白。N4BP1基因敲除小鼠的制备程序由上海模式生物中心有限公司完成。将N4BP1+/-小鼠杂交产生N4BP1-/-小鼠，并在C57BL/6遗传背景上回交10代以上。
（2）建立银屑病小鼠模型
用电动剃毛刀将8-10周龄小鼠背部的毛发剃掉，取咪喹莫特乳膏1.875mg，分别在N4BP1敲除小鼠和野生型小鼠背部连续涂抹11天，利用游标卡尺每天测量背部皮肤厚度，在预先设定好的时间内将小鼠断颈处死，取患处皮肤进行后续实验。
（3）苏木精-伊红染色
（3-1）将患处皮肤组织切成1cm*1cm大小的小块，浸在4%的多聚甲醛中24小时，第二天取出，进行酒精梯度脱水，石蜡包埋成组织块；
（3-2）组织块切成5um厚的切片，脱蜡、水化，苏木精染色30min，伊红染色5s，将染好的切片酒精脱水、二甲苯透明、树脂封片后镜下观察；
（4）制备小鼠胚胎成纤维细胞
（4-1）将N4BP1杂合子小鼠合笼13-15天后，取出孕鼠，断颈处死，取出胎鼠，在事先加有抗生素的PBS中漂洗两次后，置于直经为10cm的培养皿中；
（4-2）剪下胎鼠头部，放入1.5ml离心管用作后续基因型鉴定，将其余部分用剪刀剪碎后，将胎鼠的组织变成单细胞。在含有单细胞的培养皿中加入改良的DMEM高血糖培养基（含10%胎牛血清和1%抗生素），细胞成长扩增后变成MEFs细胞；
（4-3）将MEF细胞置于37℃和5% CO2 中培养，每两天换液一次；
（5）RNA提取与实时荧光定量PCR
（5-1）取小鼠皮肤组织约30mg或培养的MEFs细胞，放入1.5ml离心管中，向里加入1ml Trizol，用匀浆机匀浆后进行提取；
（5-2）将要提取RNA的细胞进行消化，离心收集，同样加入1ml Trizol，室温静置五分钟；加氯仿，离心取上层RNA；加异丙醇，萃取RNA；离心，75%酒精洗两次，溶于DEPC水；
（5-3）用Nano Drop 1000分光光度计(Thermo Fisher Science)测定总RNA的纯度和浓度；
（5-4）用HiScript II一步法RT-PCR试剂盒对1μg总RNA进行逆转录；CDNA稀释5倍，SYBR Green PCR试剂盒(#Q112-02/03，Vazyme)定量检测基因表达；
mRNA的水平以18S作为参照，各引物序列如下：
| Name | Oligo sequences (5’ – 3’) |
| Human/Mouse 18S, forward primer: | GAACGAGACTCTGGCATGCTA |
| Human/Mouse 18S, reverse primer: | CACGCTGAGCCAGTCAGTGTA |
| Human JUNB, forward primer: | CAGGAGGGCTTCGCCGACGGC |
| Human JUNB, reverse primer: | AGTAGCTGCTGAGGTTGGTGT |
| Human JUNC, forward primer: | GAACGTGACAGATGAGCAGGA |
| Human JUNC, reverse primer: | CCGGCGGCTCGCTGTGCAGGC |
| Human FOSB, forward primer: | GAGGAGAAGCGAAGGGTGCGC |
| Human FOSB, reverse primer: | CCCGGTTTGTGGGCCACCAGC |
| Human FOSC, forward primer: | AGCTGACTGATACACTCCAAG |
| Human FOSC, reverse primer: | CAGGCAGGTCGGTGAGCTGCC |
| Human CXCL1, forward primer: | CAAAGTGTGAACGTGAAGTCCC |
| Human CXCL1, reverse primer: | GCTTTCCGCCCATTCTTGAG |
| Human TNF-α, forward primer: | TCTGGGCAGGTCTACTTTGG |
| Human TNF-α, reverse primer: | TGAGCCAGAAGAGGTTGAGG |
| Mouse CXCL1, forward primer: | CCTATCGCCAATGAGCTGC |
| Mouse CXCL1, reverse primer: | CTCGCGACCATTCTTGAGTG |
| Mouse CCL20, forward primer: | CGTCTGCTCTTCCTTGCTTT |
| Mouse CCL20, reverse primer: | ACAGTCGTAGTTGCTTGCTG |
| Mouse S100A8, forward primer: | GTTGTCTCCATAGCCCGAGG |
| Mouse S100A8, reverse primer: | TCCAGTTCAGACGGCATTGTC |
| Mouse S100A9, forward primer: | ACTGGGCTTACACTGCTCTT |
| Mouse S100A9, reverse primer: | AGGTGTCGATGATGGTGGTT |
（6）RNA结合蛋白免疫沉淀
（6-1）用预冷的PBS洗涤培养的细胞或组织两次除去培养基，然后加入PBS 10ml，在上述溶液中加入甲醛(37%原液)至1%的最终溶液浓度，室温下缓慢旋转孵化10分钟；再加入甘氨酸溶液至0.25M的最终浓度使交联反应(pH=7.0)终止，然后室温孵育5分钟，轻轻摇晃；加入5-10ml预冷PBS清洗小鼠胚胎成纤维细胞并在2ml预冷的PBS中刮除细胞；
（6-2）离心收集小鼠胚胎成纤维细胞用预冷过的PBS洗涤两次；收集细胞，加入含PMSF的RIPA裂解缓冲液，超声裂解；旋转1h后，以4℃、13000rpm离心；取上清液，用蛋白A预处理，离心后与抗FLAG抗体（#F3165，Sigma）孵育2h；之后，收集蛋白A珠子并清洗；免疫沉淀后的样品在70℃的去交联剂中重新悬浮45min，进行反向交联；反向交联后，用Trizol提取RNA。
图3所示为N4BP1缺陷小鼠，在IMQ药物诱导下，发展成更严重的银屑病。其中，图3A所示为N4BP1野生型和缺陷型小鼠，IMQ诱导的皮炎照片；图3B所示为N4BP1野生型和缺陷型小鼠，IMQ诱导的皮炎厚度；图3C所示为N4BP1野生型和缺陷型小鼠，IMQ诱导的皮炎, 组化显示角质细胞增生。
图4所示为 N4BP1缺陷小鼠，在IMQ药物诱导下，银屑病相关基因表达增加。其中，图4A所示为N4BP1野生型和缺陷型小鼠，IMQ诱导的皮炎后，CXCL1, CCL20, S100A8,S100A9基因在N4BP1缺陷型小鼠中表达显著增加；图4B所示为N4BP1野生型和缺陷型MEF细胞，IL-17处理后，CXCL1, CCL20, S100A8,基因在N4BP1缺陷型MEF细胞中表达显著增加；图4C所示为N4BP1野生型和缺陷型MEF细胞，R848处理后，CXCL1, CCL20, S100A8,基因在N4BP1缺陷型MEF细胞中表达显著增加。
图5所示为过表达N4BP1后，显著下调银屑病相关基因CXCL1的表达。其中，图5A所示为Hela细胞稳定过表达N4BP1后，CXCL1显著下调；图5B所示为Hela细胞稳定过表达N4BP1后，CXCL1 mRNA的稳定性显著下调；图5C和图5 D所示为Hela细胞稳定过表达Flag-N4BP1后，FLAG-N4BP1和CXCL1 mRNA有结合。
图6所示为过表达N4BP1后，显著下调银屑病相关基因JunB和FosB的表达。其中，图6A所示为Hela细胞稳定过表达N4BP1后，JunB和FosB显著下调；图6B所示为Hela细胞稳定过表达N4BP1后，JunB和FosB的 mRNA的稳定性显著下调；图6C和图6D所示为Hela细胞稳定过表达Flag-N4BP1后，FLAG-N4BP1和JunB和FosB mRNA有结合。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南通大学
<120> 一种银屑病抑制基因及其应用
<141> 2020-12-14
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Claims (4)
1.一种银屑病抑制基因，其特征在于，为N4BP1基因。
2.一种银屑病抑制基因N4BP1在制备治疗银屑病药物中的应用。
3.一种银屑病抑制基因N4BP1在制备检测银屑病试剂盒中的应用。
4.一种基因N4BP1抑制银屑病的检测方法，其特征在于，包括如下步骤：
（1）4BP1敲除细胞系和N4BP1敲除小鼠
（1-1）HeLa细胞用改良的DMEM高血糖培养基在37℃和5% CO2条件下培养；用脂质体2000分别转染过表达空载和过表达N4BP1质粒，转染6h后更换培养基；转染48h后，用嘌呤霉素筛选转染成功的细胞，培养2周；
（1-2）将N4BP1+/-小鼠杂交产生N4BP1-/-小鼠，并在C57BL/6遗传背景上回交10代以上；
（2）建立银屑病小鼠模型
用电动剃毛刀将8-10周龄小鼠背部的毛发剃掉，取咪喹莫特乳膏1.875mg，分别在N4BP1敲除小鼠和野生型小鼠背部连续涂抹11天，利用游标卡尺每天测量背部皮肤厚度，在预先设定好的时间内将小鼠断颈处死，取患处皮肤进行后续实验；
（3）苏木精-伊红染色
（3-1）将患处皮肤组织切成1cm*1cm大小的小块，浸在4%的多聚甲醛中24小时，第二天取出，进行酒精梯度脱水，石蜡包埋成组织块；
（3-2）组织块切成5um厚的切片，脱蜡、水化，苏木精染色30min，伊红染色5s，将染好的切片酒精脱水、二甲苯透明、树脂封片后镜下观察；
（4）制备小鼠胚胎成纤维细胞
（4-1）将N4BP1杂合子小鼠合笼13-15天后，取出孕鼠，断颈处死，取出胎鼠，在事先加有抗生素的PBS中漂洗两次后，置于直经为10cm的培养皿中；
（4-2）剪下胎鼠头部，放入1.5ml离心管用作后续基因型鉴定，将其余部分用剪刀剪碎后，直接向培养皿中加入改良的DMEM高血糖培养基；
（4-3）将MEF细胞置于37℃和5% CO2 中培养，每两天换液一次；
（5）RNA提取与实时荧光定量PCR
（5-1）取小鼠皮肤组织30mg，放入1.5ml离心管中，向里加入1ml Trizol，用匀浆机匀浆后进行提取；
（5-2）将要提取RNA的细胞进行消化，离心收集，同样加入1ml Trizol，室温静置五分钟；加氯仿，离心取上层RNA；加异丙醇，萃取RNA；离心，75%酒精洗两次，溶于DEPC水；
（5-3）用分光光度计测定总RNA的纯度和浓度；
（5-4）用HiScript II一步法RT-PCR试剂盒对1μg总RNA进行逆转录；CDNA稀释5倍， PCR试剂盒定量检测基因表达；
（6）RNA结合蛋白免疫沉淀
（6-1）用预冷的PBS洗涤两次除去培养基，然后加入PBS 10ml，在上述溶液中加入甲醛至1%的最终溶液浓度，室温下缓慢旋转孵化10分钟；再加入甘氨酸溶液至0.25M的最终浓度使交联反应终止，然后室温孵育5分钟，轻轻摇晃；加入5-10ml预冷PBS清洗细胞，并在2ml预冷的PBS中刮除细胞；
（6-2）离心收集细胞，用预冷过的PBS洗涤两次；收集细胞，加入含PMSF的RIPA裂解缓冲液，超声裂解；旋转1h后，以4℃、13000rpm离心；取上清液，用蛋白A预处理，离心后与抗FLAG抗体孵育2h；之后，收集蛋白A珠子并清洗；免疫沉淀后的样品在70℃的去交联剂中重新悬浮45min，进行反向交联；反向交联后，用Trizol提取RNA。
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