CN114503953B - 一种椎间盘退变动物模型的构建方法及应用 - Google Patents
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Abstract
本发明公开了一种椎间盘退变动物模型的构建方法及应用，属于医学动物模型技术领域。本发明的椎间盘退变动物模型的构建方法如下：取实验动物，在其椎间盘中施用Piezo1激动剂，得所述椎间盘退变动物模型。本发明的椎间盘退变动物模型的构建方法，可在大鼠和小鼠椎间盘中执行，操作简单，诱导成功率高，且缩短了椎间盘退变所需要的时间，为后续的研究提供了基础，为人们进一步认识椎间盘退变的发生机制奠定了基础，进而为解决椎间盘退变提供新的策略。
Description
技术领域
本发明属于医学动物模型技术领域，具体涉及一种椎间盘退变动物模型的构建方法及应用。
背景技术
下腰痛已成为全球健康问题，每五人就有四人一生中会经历下腰痛，而椎间盘退变是下腰痛的常见原因。目前为止，还没有发现能够针对发病元的治疗方法，临床上主要以缓解症状为主。所以建立可以模拟人椎间盘退变的动物模型有助于椎间盘退行性疾病的发生发展、病理过程以及治疗方法的深入研究。目前椎间盘退行性病变模型主要包括椎间盘细胞培养模型和动物体内椎间盘模型，其中动物体内模型包括自发性椎间盘退行性变模型和诱发性椎间盘退行性变模型，临床上的研究常用的是诱发性椎间盘退变模型，如针刺模型、动静态应力改变模型、脊柱失稳模型、化学法构建模型等，其中应力改变模型被认为是最接近临床的椎间盘退行性变的病理机制，但机械应力作用不能标准化，致使诱发的动物椎间盘退行性变程度无法标准化，故在应用中受到限制。
研究表明，机械力负荷是导致椎间盘退变的重要诱因之一。目前模拟机械力负荷诱导的椎间盘退变模型手术操作繁琐、容易感染、且大鼠尾部所带的压力装置会因大鼠的活动而受到一定程度的影响，同时手术所带来的疼痛持续到实验结束，并且压力装置模型在小鼠中操作难度系数高，不易把控。
发明内容
本发明的目的在于克服现有技术的不足，提供一种椎间盘退变动物模型的构建方法，本发明的椎间盘退变动物模型的构建方法，可在大鼠和小鼠椎间盘中执行，操作简单，诱导成功率高，且缩短了椎间盘退变所需要的时间，为后续的研究提供了基础，为人们进一步认识椎间盘退变的发生机制奠定了基础，进而为解决椎间盘退变提供新的策略。
为实现上述目的，本发明采取的技术方案为：一种椎间盘退变动物模型的构建方法：取实验动物，在其椎间盘中施用Piezo1激动剂，得所述椎间盘退变动物模型。
机械感受对生物有机体进行正常的生命活动具有非常重要的意义。机械门控离子通道作为重要的分子受体在机械感受过程中承担着重要的感受器的作用，它能够将机械刺激快速的转化成电化学信号，在低等到高等生物的生命活动中起着至关重要的作用。Piezo蛋白家族近来已经被证实是哺乳动物中长期寻找的首类真正意义上的机械门控阳离子通道。Piezo1通道作为哺乳动物中机械敏感阳离子通道的原型，已被证实在众多机械传导过程中具有重要的生理学功能。本申请发明人经过大量的研究发现，在实验动物的椎间盘中注射Piezo1激动剂，能够得到椎间盘退变动物模型，且操作简单，诱导成功率高。
作为本发明所述的椎间盘退变动物模型的构建方法的优选实施方式，所述Piezo1激动剂为Yoda1。Yoda1是第一个被发现能够特异性激活Piezo1通道的化学激活剂，而且只对Piezo1有特异性作用，对于Piezo2无明显作用。本申请发明人经过实验筛选和验证，发现选择Yoda1对动物实验进行注射，可成功诱导得到椎间盘退变动物模型。
作为本发明所述的椎间盘退变动物模型的构建方法的优选实施方式，所述Yoda1的浓度为4～6uM。
作为本发明所述的椎间盘退变动物模型的构建方法的优选实施方式，所述Yoda1的浓度为5uM。本申请发明人研究发现，Yoda1的浓度对椎间盘退变动物模型的诱导成功率有影响，选择浓度为4～6uM的Yoda1可成功诱导得到椎间盘退变动物模型。
作为本发明所述的椎间盘退变动物模型的构建方法的优选实施方式，所述Piezo1激动剂施用两次，每次施用时间间隔为3天。本申请发明人研究发现，采用Piezo1激动剂诱导制备椎间盘退变动物模型时，当Piezo1激动剂施用两次，每次施用时间间隔为3天时，诱导成功率高且诱导所需要的时间短。
作为本发明所述的椎间盘退变动物模型的构建方法的优选实施方式，所述Piezo1激动剂的施用量为45～50ul。
作为本发明所述的椎间盘退变动物模型的构建方法的优选实施方式，所述实验动物为大鼠或小鼠。
本发明还提供一种由上述的椎间盘退变动物模型的构建方法构建的椎间盘退变动物模型。
本发明还提供上述椎间盘退变动物模型在椎间盘退变研究中的应用。
本发明的有益效果：本发明提供一种椎间盘退变动物模型的构建方法，本发明的椎间盘退变动物模型的构建方法通过在实验动物椎间盘施用Piezo1激动剂成功构建椎间盘退变动物模型。本发明的椎间盘退变动物模型的构建方法操作简单，诱导的动物椎间盘退行性变程度可实现标准化，诱导成功率高，缩短了椎间盘退变所需要的时间。
附图说明
图1为实施例1～3中动物模型构建实验分组及其实验流程图。
图2为实验组和对照组中CDKN1A、IL1A、IL1B、IL6、CDK4、CDK6、COL2A1、MMP3和MMP13的表达水平变化。
图3为实验组和对照组髓核细胞的periostin蛋白、p65蛋白、p-p65蛋白和β-actin蛋白电泳图。
图4为实验组和对照组大鼠的椎间盘磁共振影像图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例1 Yoda1对髓核细胞的影响
髓核是乳白色半透明胶状体，富于弹性，为椎间盘结构的一部分，位于两软骨板与纤维环之间。由纵横交错的纤维网状结构即软骨细胞和蛋白多糖黏液样基质构成的弹性胶冻物质。髓核细胞的过早衰老与凋亡是导致椎间盘退行性变过程的主要原因之一，主要表现为退变椎间盘内髓核细胞功能降低和数量减少，使得其Ⅱ型胶蛋白聚糖等基质分泌量下降，最终导致椎间盘维持脊柱高度、承受各方应力等生物力学功能丧失。NF-κB是一种常见的转录因子，可以被炎症因子、生长因子或趋化因子等激活。NF-κB由两类亚基形成同源或异源二聚体。一类亚基包括p65(也称RelA)、RelB和C-Rel；另一类亚基包括p50和p52。最常见的NF-κB亚基组成形式为p65/p50或p65/p65。NF-κB未被激活时和IκB-α形成一个复合物，分布在细胞浆中。在炎症因子、生长因子或趋化因子等可以激活NF-κB的刺激存在的情况下，IκB-α会在Ser32和Ser36被磷酸化，随后被泛素-蛋白酶体途径降解。NF-κB和IκB-α解聚后，其核定位序列被暴露，从而被转运到细胞核内促进NF-κB依赖的基因转录。通过免疫染色检测NF-κB的主要亚基p65是否被转移到细胞核内，就可以判断NF-κB是否被激活。本实施例采用Yoda1处理髓核细胞，研究Yoda1对髓核细胞的影响，从而说明Yoda1用于诱导椎间盘退变动物模型的机制。
细胞周期蛋白依赖性激酶抑制剂1A(CDKN1A)编码的蛋白质结合并抑制细胞周期蛋白依赖性激酶2(CDK2)或细胞周期蛋白依赖性激酶6(CDK6)复合物的活性，因此在细胞周期G1期进程中起到调节剂的作用。IL1A、IL1B和IL6均为白细胞介素细胞因子家族的成员，也是细胞衰老分泌相关表型的重要分子,参与各种免疫反应、炎症过程和造血。在炎症过程中可以诱导细胞凋亡。细胞周期蛋白依赖性激酶4(CDK4)是蛋白激酶复合物的催化亚基，对细胞周期G1期进展很重要，该激酶的活性仅限于G1-S期，由调节亚基D型细胞周期蛋白和CDK抑制剂p16(INK4a)控制。细胞周期蛋白依赖性激酶6(CDK6)是蛋白激酶复合物的催化亚基，对细胞周期G1期进展和G1/S转变很重要。这种激酶的活性首先出现在G1中期，受调节亚基的控制，包括D型细胞周期蛋白和CDK抑制剂家族的成员。COL2A1基因编码II型胶原蛋白的alpha-1链，这是一种在软骨中发现的纤维状胶原蛋白,合成细胞外基质，主要表达在正常的软骨组织和髓核组织。基质金属肽酶3(MMP3)，基质金属肽酶13(MMP13)参与疾病过程(如关节炎和椎间盘退行性病变)中参与细胞外基质分解，加速疾病进展。
实验方法：(1)培养髓核细胞，将其分为两组，实验组采用5uM Yoda1处理24小时，对照组采用生理盐水处理。采用β-半乳糖苷酶活性染色法对处理后的髓核细胞进行染色，并置于光学纤维镜下观察髓核细胞；(2)培养髓核细胞，将其分为两组，实验组采用5uMYoda1处理24小时，对照组采用生理盐水处理。提取两组髓核细胞的RNA，进行qPCR检测基质分解代谢以及CDKN1A、IL1A、IL1B、IL6、CDK4、CDK6、COL2A1、MMP3和MMP13的表达水平变化；(3)培养髓核细胞，将其分为两组，实验组采用5uM Yoda1处理48小时，对照组采用生理盐水处理。提取两组髓核细胞的蛋白进行western blot检测。
实验结果如图1～3所示。由图1可知，采用Yoda1处理后的实验组髓核细胞的体积增大，且细胞多呈现蓝色，表明Yoda1处理后的实验组髓核细胞衰老增加。由图2可知，采用Yoda1处理后的实验组髓核细胞中衰老分泌相关分子CDKN1A、IL1A、IL1B、IL6的表达增加，而细胞周期相关分子CDK4、CDK6的表达降低，基质合成分子COL2A1的表达下降，基质金属蛋白酶MMP3和MMP13的表达量明显升高。由图3可知，采用Yoda1处理后的实验组髓核细胞的periostin和p-p65蛋白表达明显升高。综上，采用Yoda1处理髓核细胞可诱导髓核细胞的衰老。
实施例2椎间盘退变动物模型的构建
本实施例的一种椎间盘退变动物模型的构建方法：选取10只健康成年大鼠，分为两组，一组为实验组，一组为对照组。实验组分别在每只大鼠的椎间盘中注射50ul浓度为5uM的Yoda1，每隔3天注射1次，一共注射2次。对照组分别在每只大鼠的椎间盘中注射50ul生理盐水，每隔3天注射1次，一共注射2次。利用磁共振成像(magnetic resonanceimaging,MRI)通过Pfirrmann分级法观察椎间盘退变的程度。对上述两组大鼠进行椎间盘磁共振检查，椎间盘磁共振影像结果如图4所示。
由图4可知，对照组的椎间盘高度无明显变化，髓核组织的水含量高而透亮，而实验组椎间盘高度有稍些降低，且髓核组织的水含量显著流失，髓核和纤维环的边界有些模糊，Pfirrmann分级在对照组中无明显改变，而在实验组中随着时间的延长，Pfirrmann分级越高，退变程度逐渐加深，表明该椎间盘退变动物模型诱导成功。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (4)
1.一种椎间盘退变动物模型的构建方法，其特征在于，取实验动物，在其椎间盘中施用Piezo1激动剂，得所述椎间盘退变动物模型；所述Piezo1激动剂为Yoda1；所述Piezo1激动剂的施用量为45~50ul；所述实验动物为大鼠或小鼠。
2.根据权利要求1所述的椎间盘退变动物模型的构建方法，其特征在于，所述Yoda1的浓度为4~6uM。
3.根据权利要求2所述的椎间盘退变动物模型的构建方法，其特征在于，所述Yoda1的浓度为5uM。
4.根据权利要求1所述的椎间盘退变动物模型的构建方法，其特征在于，所述Piezo1激动剂施用两次，每次施用时间间隔为3天。
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