CN106177999B - 一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法及用途 - Google Patents
一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法及用途 Download PDFInfo
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Abstract
本发明涉及生物技术领域，具体涉及一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法。本发明采用的条件性敲除技术通过在小鼠T淋巴细胞上特异性敲除Ptpn11基因（编码SHP‑2蛋白），建立了一种脊柱关节炎的小鼠病理模型。该条件性敲除小鼠自发形成了脊柱关节炎的“竹节样脊柱”病理特征，是研究脊柱关节炎发病机理和筛选改善脊柱关节炎候选药物的理想动物模型。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法及用途。
背景技术
脊柱关节炎(spondyloarthritis)，既往又称脊柱关节病(spondyloarthropathies)，这是一组慢性炎症性风湿性疾病，具有特定的病理生理、临床、放射学和遗传特征，炎性腰背痛伴或不伴外周关节炎，加之一定特征的关节外表现是这类疾病特有的症状和体征。这一类疾病包括：强直性脊柱炎(ankylosing spondylitis)、银屑病关节炎(psoriatic arthritis)、反应性关节炎(reactive arthritis)、炎症性肠病性关节炎(arthropathy of inflammatory bowel disease)、未分化脊柱关节炎和幼年慢性关节炎。
脊柱关节炎的炎症过程发生在韧带附着于骨的起止点位置，这不同于有许多临床和放射学证据的类风湿关节炎。脊柱关节炎最初的炎症过程的主要靶点出现在起止点、软骨和较小范围的滑膜，随着新骨在纤维瘢痕组织上的形成，炎症过程有自愈的倾向，导致关节强直和中轴与外周关节不可逆的骨化。
强直性脊柱炎作为最常见的一种脊柱关节炎疾病，是一种以累及脊柱和骶髂关节为主的慢性自身免疫性疾病，严重影响患者生活质量。强直性脊柱炎发病起始阶段无明显症状，到骶髂关节、肌腱端或脊柱部位炎症引起的背痛等症状明显时才会引起患者注意。发病几个月甚至几年后，患者骶髂关节处会最早出现软骨下骨的皮质边缘模糊、侵蚀和硬化。之后随着骨侵蚀、骨质增生和韧带骨赘形成等过程，关节完全变形至骨融合，使关节完全失去活动性。此外，强直性脊柱炎患者血液中IgA增加，50％～70％的患者发病伴随C反应蛋白和红细胞沉降率上调。骶髂关节处放射学诊断是强直性脊柱炎确诊的有力证据。若患者早期无法得到明确诊断，直至骨关节变形明显，严重影响生活质量时才确诊，则将错过最佳的前期治疗窗口。目前强直性脊柱炎发病的具体机制仍不清楚，缺少明确的早期诊断方法，基于病因的干预手段缺乏，只能针对患者炎症和骨关节病变进行对症治疗。
非甾体抗炎药如氟比洛芬、双氯芬酸、吲哚美辛等，能够有效改善脊柱关节炎患者的炎症，减轻患者痛苦，是目前治疗强直性脊柱炎的一线用药。虽然这类药物都具备抗炎作用，但脊柱关节炎患者对于这些药的反应性不尽相同，即在某些患者身上只有特定的一种或几种非甾体抗炎药具有疗效，因此需要对每位患者的用药进行不同的尝试。鉴于脊柱关节炎炎患者需要长期服用非甾体抗炎药，这些药物在胃肠道的副作用亦不容忽视。综合考虑疗效及副作用因素，糖皮质激素和改善病情抗风湿药(DMARD)也不适合脊柱关节炎炎患者长期服用。近年研制的TNF-α单克隆抗体，包括英夫利昔单抗、依那西普、阿达木单抗等均已应用于临床，可有效减轻脊柱关节炎患者的炎症，为不适用非甾体抗炎药的患者找到了新的曙光。虽然新一代选择性更强的非甾体抗炎药和TNF-α单抗的上市，能在一定程度上改善脊柱关节炎患者的症状，减轻患者痛苦，但目前为止还没有药物能够改善或者阻滞骨关节处病变，所以脊柱关节炎尤其是强直性脊柱炎患者最终还是无法摆脱盆骨关节变形和脊柱骨融合的命运。
在动物整体水平建立真实模拟人类疾病的模型，对探索疾病发生发展机理、发现药物新靶点以及临床前药效学评价等生物医学研究均具有十分重要的科学意义和临床意义。因此，理想的动物模型是人们寻找疾病诱因、研究发病机制和设计验证靶向药物的重要工具。鉴于脊柱关节炎炎发病机制仍不清楚，特异性的干预药物十分匮乏，建立与临床强直性脊柱炎相似性高、稳定性强的疾病动物模型尤为迫切。
现将已有的脊柱关节炎动物模型逐一进行简单介绍，并分析其优缺点。
(1)HLA-B27转基因模型：HLA-B抗原是由α链与β2微球蛋白非共价结合的糖蛋白，基本表达在机体所有有核细胞上。HLA-B27抗原的表达与强直性脊柱炎、银屑病关节炎等脊柱关节炎疾病具备高度相关性，超过90％的强直性脊柱炎患者其HLA-B27抗原表达为阳性。HLA-B27转基因大鼠(B27Tgβ2-microglobulin-/-)可诱发后肢关节炎、关节强直、指甲变形，但该病变仅局限在外周，不会累及脊柱，因此该模型与临床强直性脊柱炎的脊柱病变相差较远。
(2)TNF-α转基因小鼠模型：脊柱关节炎患者血清中炎症因子TNF-α的水平显著上调。此外，TNF-α的单克隆抗体在脊柱关节炎的治疗中亦有疗效。这些研究均提示TNF-α与脊柱关节炎的发病进展密切相关。因此，有研究者构建了TNF-α转基因小鼠，用于模拟脊柱关节炎。过表达TNF-α的小鼠会出现一系列的炎症反应，包括肠炎和骶髂关节炎，但并不会自发出现关节强直症状。这也与TNF-α单克隆抗体虽可显著改善脊柱关节炎尤其强直性脊柱炎的炎症症状，但并不能阻止骨关节处强直症状的进展相符合。
(3)蛋白聚糖诱导的小鼠脊柱炎模型：关节有两种主要的蛋白多糖aggrecan和versican。在一些敏感的小鼠品系，例如BALB/c和C3H中，以人关节中酶解出的蛋白聚糖aggrecan或versican免疫小鼠，前期会诱发小鼠关节炎，后期一些小鼠在脊柱处发生炎症，之后骶髂关节腐蚀、软骨增生。脊柱椎骨两端骨质增生，严重时会压迫脊髓，影响神经传导。关节处起止点的纤维结构消失，直接影响关节的活动性。但该模型诱导的关节炎症属毁坏型，包括血管翳的形成和破骨细胞的活化，这一特征明显不同于临床强直性脊柱炎患者的关节病变，后者关节炎症的特点是新的软骨和骨组织的形成如韧带骨赘的形成带来关节僵直等病变。此外，该模型的另一不足是造模成功率偏低，大约只有60％的造模小鼠发病。
(4)DBA/1小鼠自发强直性肌腱病模型：DBA/1小鼠老年后会自发强直性肌腱端病，出现关节炎症和关节强直等症状，但是这些症状局限在外周关节而不影响轴关节，与临床脊柱关节炎相去甚远。
综上所述，现有的脊柱关节炎动物模型离真实模拟临床脊柱关节炎还存在一定的差距。因此，建立一种崭新的、更贴近临床脊柱关节炎病理特征的动物疾病模型迫在眉睫。
发明内容
本发明主要是解决现有技术所存在的造模不稳定、病理特征与临床疾病相差较大等问题，提供了一种新的采用条件性敲除技术建立的遗传背景清楚、更接近疾病真实症状的脊柱关节炎病理模型的方法。
本发明的上述技术问题主要是通过下述技术方案得以解决的：
一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法，其特征在于，所述的一种基于条件性敲除技术的脊柱关节炎小鼠，其基因型为SHP-2flox/flox:CD4-Cre+。
一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法，其特征在于所述的模型小鼠由如下步骤实现，将SHP-2flox/flox小鼠和CD4-Cre转基因小鼠杂交得到F1代小鼠，将F1代小鼠自交，并鉴定，得到基因型为SHP-2flox/flox:CD4-Cre+的小鼠，以下称条件性敲除鼠或SHP-2CD4-/-小鼠。
具体来说：SHP-2flox/flox转基因小鼠：loxp插入位置为四号外显子两端，即第111–176位氨基酸(rwfhghlsgkeaeklltekgkhgsflvresqshpgdfvlsvrtgddkgesndgkskvthvmircqe)两端，DNA序列为：
aggtggttccatggtcacttgtctggaaaagaagcagagaagctgctgacggagaagggcaagcatggcagcttcctcgttcgagagagccagagccaccccggagacttcgttctctccgtgcgcactggtgacgacaaaggggagagcaacgacggcaagtccaaagtgacccacgtcatgatccgctgtcaggag)；
CD4-Cre转基因小鼠：CD4启动子下游插入Cre序列；
小鼠具体杂交示意图详见附图1。
条件性敲除原理为：基于Cre-loxP重组原理，通过SHP-2flox/flox转基因小鼠与CD4-Cre转基因小鼠交配，得到的F2代小鼠出现SHP-2flox/flox:CD4-Cre+表型，该表型的小鼠其CD4+T细胞上表达的Cre重组酶可将SHP-2基因从该细胞上特异性敲除。与传统基因敲除不同，该体系只有特定细胞缺失目标分子，能更真实地反映目标分子在特定细胞中的作用。野生型小鼠，以WT表示，基因型为SHP-2flox/flox:CD4-Cre-；条件性敲除鼠，以SHP-2CD4-/-小鼠表示，基因型为SHP-2flox/flox:CD4-Cre+。
按上述一种方法形成的小鼠模型的应用，其特征在于，该模型用于改善脊柱关节炎候选药物的筛选与评价。
所述的应用，其特征在于，所述的脊柱关节炎包括：强直性脊柱炎、银屑病关节炎、反应性关节炎、炎症性肠病性关节炎、未分化脊柱关节炎和幼年慢性关节炎。
因此，本发明具有如下优点：发病率接近100％，模型重现性好，操作简单，更接近疾病真实症状，可作为研究脊柱关节炎发病机制和筛选脊柱关节炎防治药物的有利工具。
附图说明
图1为SHP-2flox/flox:CD4-Cre+条件性敲除小鼠构建过程示意图；基于Cre-loxP重组原理，通过SHP-2flox/flox小鼠与CD4-Cre小鼠交配，得到的F2代小鼠出现SHP-2flox/flox:CD4-Cre+表型，该表型的小鼠其CD4+T细胞上表达的Cre重组酶可将SHP-2基因从该细胞上特异性敲除。
图2为条件性敲除鼠的基因型鉴定结果；通过分离敲除鼠和野生鼠的脾细胞，利用商品化CD4磁珠分离柱纯化出CD4+T细胞，PCR进行基因型鉴定，最后一列为T细胞上成功敲除SHP-2小鼠的样本。
图3为条件性敲除鼠的蛋白水平鉴定结果，通过分离敲除鼠和野生鼠的脾细胞，利用商品化CD4磁珠分离柱纯化出CD4+T细胞，Western blot进行蛋白的免疫印迹分析，第二列清楚显示CD4阳性T细胞上成功敲除了SHP-2。
图4为野生型和条件性敲除鼠外观对比图；12月龄条件性敲除鼠尾巴明显出现结节，脊柱严重变形，小鼠行动能力显著下降。
图5野生型和条件性敲除鼠脊柱对比图；从脊柱外观可以看出，12月龄条件性敲除鼠脊柱较野生型鼠相比明显变形。
图6为野生型和条件性敲除鼠X光分析对比图；从图中可清晰地显示12月龄条件性敲除鼠脊柱明显变形，出现“竹节样”脊柱的病理特征。
图7为野生型和条件性敲除鼠髋关节CT图；从图中可明显看出12月龄条件性敲除鼠髋关节出现赘生的不规则的骨质，显示出典型的异位骨增生。
具体实施方式
野生型小鼠(以WT表示，基因型为SHP-2flox/flox:CD4-Cre-)和条件敲除鼠(以SHP-2CD4-/-小鼠表示，基因型为SHP-2flox/flox:CD4-Cre+)分别购自南京大学模式动物研究所。
实施例1
一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法，按以下步骤实现：
步骤1，小鼠配繁：将SHP-2flox/flox转基因小鼠和CD4-Cre转基因小鼠杂交得到F1代小鼠，将F1代小鼠自交，并鉴定，基因型为SHP-2flox/flox:CD4-Cre+即为条件性敲除鼠，也即本发明中的脊柱关节炎模型鼠，具体由以下步骤获得：
通过SHP-2flox/flox小鼠(loxp插入位置为四号外显子即第111–176位氨基酸(rwfhghlsgkeaeklltekgkhgsflvresqshpgdfvlsvrtgddkgesndgkskvthv mircqe，见SEQ NO.1)两端，DNA序列为：aggtggttccatggtcacttgtctggaaaagaagcagagaagctgctgacggagaagggcaagcatggcagcttcctcgttcgagagagccagagccaccccggagacttcgttctctccgtgcgcactggtgacgacaaaggggagagcaacgacggcaagtccaaagtgacccacgtcatgatccgctgtcaggag,见SEQ NO.2)和CD4-Cre(CD4启动子下游插入Cre序列)转基因小鼠杂交繁殖获得；将SHP-2flox/flox小鼠和CD4-Cre转基因小鼠杂交得到F1代小鼠，将F1代小鼠自交，并鉴定，得到SHP-2CD4-/-小鼠，其基因型为SHP-2flox/flox:CD4-Cre+。
步骤2，小鼠基因型鉴定：
1)基因组DNA提取：第一天剪取0.5cm小鼠尾巴(同样适用于脚趾)，放入1.5mlEppendorf管，加入500μl digestion buffer(1M Tris-HCl(pH 8.0)25ml，0.5M EDTA(pH8.0)100ml，NaCl 2.92g，10％SDS 50ml，和5μl 1％蛋白酶K溶液放入55℃的水浴中，消化过夜。第二天将消化好的组织彻底打散，加入250μl(1/2消化液的体积)饱和氯化钠溶液，12000rpm离心10min，将400μl上清转移到新的标记好的1.5ml Eppendorf管内，加入800μl无水乙醇，上下颠倒混和均匀，此时应可看到白色丝状的DNA，12000rpm离心5min，吸去上清，加入800μl 70％乙醇，于12000rpm离心5min，吸去上清，晾干至透明，加入200μl TE溶解DNA。短期4℃保存，长期-20℃保存。
2)PCR鉴定：100μl体系加尾巴DNA(300-500ng)约1.2μl样品。引物信息及PCR程序如下：cre-forward，5’-CCTGATCCTGGCAATTTCGG-3’，见SEQNO.3；
cre-reverse，5’-CCCAACCAACAAGAGCTC-3’，见SEQ NO.4；
PCR条件：94℃30s，59℃45s，72℃60s，共30个循环。
shp-2Ex4flox-forward，5’-ACGTCATGATCCGCTGTCAG-3’，见SEQ NO.5；
shp-2Ex4flox-reverse，5’-ATGGGAGGGACAGTGCAGTG-3’，见SEQ NO.6；
PCR条件：94℃30s，59℃45s，72℃60s，共40个循环。
步骤3，PCR的产物进行琼脂糖凝胶电泳：称取琼脂糖溶解在电泳缓冲液中(1％的凝胶)，置微波炉或沸水浴中加热至完全溶化(不要加热至沸腾)，取出摇匀。将冷却到60℃的琼脂糖溶液轻轻倒入电泳槽水平板上。待琼脂糖胶凝固后，在电泳槽内加入电泳缓冲液，然后拔出梳子。将DNA样品与加样缓冲液按4：1混匀后，用微量移液器将混合液加到样品槽中，每槽加10-20μl，记录样品的点样次序和加样量。安装好电极导线，点样孔一端接负极，另一端接正极，打开电源，调电压至3-5V/cm，电泳1h，当溴酚蓝移到距凝胶前沿1-2cm时，停止电泳。取出凝胶，放在含有溴化乙锭的染色液中染色30min，即可在254nm的紫外灯下观察，有橙红色荧光条带的位置，即为DNA条带，在紫外灯下照相记录电泳图谱。
该模型的特点及与临床脊柱关节炎的内在相关性：相较于野生鼠，SHP-2条件敲除鼠在6月龄之后体重明显下降、活动能力降低，尾或后肢无力，部分小鼠尿道出现炎症。8至9月龄，当小鼠双后肢均出现无力时，其盆骨关节处已发生明显病变。后肢无力的症状持续2至3个月之后小鼠膝关节僵直，膝关节失去活动性，此时髋关节也失去活动性，导致小鼠整个后肢瘫痪(10至12月龄)。伴随着小鼠后肢由无力到僵直的过程，小鼠脊柱变形逐渐加重，表现为颈部脊柱下陷，腰部脊柱上凸，最终小鼠后肢、腰部脊柱完全僵直，腰部以下部位失去活动性。部分小鼠在脊柱变形过程中尾巴处关节同时受累，由单个关节凸出进展为整个尾巴成“竹节状”。由小鼠膝关节和脊柱骨组织切片观察到小鼠骨关节结构已完全破坏。软骨下骨组织骨密度减少、骨质疏松、骨垢线凌乱、骨侧向增殖、关节空间消失。小鼠发病后期骨组织经计算机断层扫描三维重构显示，骨侧向韧带骨赘明显，膝关节和脊柱腰部关节处均发生骨融合。以上这些病理变化与临床脊柱关节炎尤其是强直性脊柱炎患者的特征十分吻合。
SEQUENCE LISTING
<110> 南京大学
<120> 一种基于条件性敲除技术的脊柱关节炎小鼠模型的建立方法及用途
<130>
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<170> PatentIn version 3.3
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Arg Trp Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys Leu Leu
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Thr Glu Lys Gly Lys His Gly Ser Phe Leu Val Arg Glu Ser Gln Ser
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His Pro Gly Asp Phe Val Leu Ser Val Arg Thr Gly Asp Asp Lys Gly
35 40 45
Glu Ser Asn Asp Gly Lys Ser Lys Val Thr His Val Met Ile Arg Cys
50 55 60
Gln Glu
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Claims (1)
1.一种基于条件性敲除技术的脊柱关节炎小鼠模型在筛选与评价预防或者治疗脊柱关节炎的药物中应用，其特征在于，所述的应用是非疾病治疗为目的的，其中所述的模型小鼠由如下步骤实现，将SHP-2flox/flox转基因小鼠和CD4-Cre 转基因小鼠杂交得到F1代小鼠，将F1代小鼠自交，并鉴定，得到基因型为SHP-2flox/flox : CD4-Cre+的小鼠，以下称条件性敲除鼠或SHP-2CD4-/-小鼠，所述的SHP-2为SH2 domain-containing protein-tyrosinephosphatase-2，酪氨酸磷酸酶SHP-2。
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| CN109418230A (zh) * | 2017-08-25 | 2019-03-05 | 香港大学深圳医院 | 一种骶髂关节炎小鼠模型 |
| EP3860717A1 (en) | 2018-10-03 | 2021-08-11 | Gilead Sciences, Inc. | Imidozopyrimidine derivatives |
| CN110684736A (zh) * | 2019-08-15 | 2020-01-14 | 扬州大学 | 一种基于CRISPR-Cas9编辑技术的敲除鸡Shp-2基因的细胞系及其构建方法 |
| CN111264468B (zh) * | 2020-02-07 | 2022-01-28 | 广东省实验动物监测所 | 一种小鼠强直性脊柱炎模型的构建方法 |
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