CN114847223B - 一种建立oa食蟹猴模型的方法 - Google Patents
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Abstract
建立OA食蟹猴模型的诱导剂，其制法和使用方法，属生物技术领域。诱导剂为碘乙酸钠水溶液，其浓度为0.2‑0.3mg/μL。将诱导剂用于食蟹猴，施用剂量为15‑20mg/Kg/关节，将诱导剂通过影像设备介导注射到食蟹猴关节腔内，配置好的碘乙酸钠溶液在30分钟内完成注射；正常饲养食蟹猴不短于三个月时间，得到OA食蟹猴模型。本发明建立的OA食蟹猴模型可用于进行啮齿类、家兔和仔猪动物模型无法完成的生物技术新药的评价和干细胞移植治疗技术的评价。
Description
技术领域
本发明属生物技术领域，具体涉及一种建立食蟹猴骨关节炎动物模型的诱导剂和其使用方法。
背景技术
膝关节骨关节炎(OA)是世界范围的一个严重健康问题。由于人口老龄化，该病的负担持续增加。主要临床表现为膝关节移动时疼痛、活动受限、晨僵、摩擦音、骨性膨大、“交锁”感等。OA在55岁以上的人群中，发病率为80％。我国目前大约有1.1亿膝关节骨关节炎患者。其高患病率、高致残率的特点，已成为国家沉重的医疗和经济负担。由于关节软骨再生修复能力十分有限，目前的药物只能缓解疼痛而无法根治。OA发展到末期，只能选择行关节置换手术。在OA的发病中首先受到损伤的是关节软骨，正常的关节软骨包含大量含水的细胞外基质，基质的主要成分是胶原和蛋白聚糖。蛋白聚糖是含有一个与糖胺聚糖相连的蛋白核心的大分子复合物，糖胺聚糖主要是硫酸软骨素和硫酸角质蛋白，这些在溶液中带负电荷的氨基葡聚糖对维持组织的水合作用和巨大膨胀力有重要作用。金属蛋白酶(MMPs)在软骨基质巨分子(包括二型胶原和蛋白聚糖聚合体)的裂解过程中起主要作用。随着年龄的增长，这种改变更加明显。
目前OA研究最常用的动物模型是啮齿类。由于啮齿类在进化关系上与人类相距甚远，以其为研究对象所获得的实验数据对临床指导意义不大。食蟹猴的解剖特点、生理功能，免疫系统和代谢系统以及对疾病和治疗药物的反应性与人类最为接近，是进行干细胞移植和异种移植临床前有效性和安全性评价的最合适的动物。利用食蟹猴模型评价新的OA治疗药物，研究干细胞移植治疗机制，这都是国际公认的临床前实验体系，对未来这些治疗方案的临床应用具有重要的指导意义，因此建立非人灵长类骨关节炎模型是干细胞移植治疗研究和临床前药物评价的迫切需要。
现在常用的OA动物模型有实验性骨关节炎动物模型和自发性骨关节炎动物模型。自发性模型应用价值较高，但因发病率低，价格昂贵，饲养和繁殖条件要求严格而不能得到广泛应用。实验性模型则应用比较广泛，常用的诱导方法包括关节腔内注射化合物诱发OA，注射的化合物包括胰蛋白酶，透明质酸酶和木瓜蛋白酶等。手术致关节失稳的OA动物模型，包括前交叉韧带切断，半月板切除和半月板损伤致失稳的模型，遗传修饰的动物OA模型。其中木瓜蛋白酶，胰蛋白酶，透明质酸酶，胶原酶制备OA模型是选择性降解软骨细胞外基质，注射这些蛋白质可以诱发急性炎症反应，这种炎症就可能导致软骨的破坏，且与类风湿性关节炎有一定的病程重合。半月板切除和半月板损伤致失稳的OA模型主要是关节内干预,手术过程中的出血及炎症易影响OA早期软骨，滑膜的生化代谢,不能排除创伤性滑膜炎对实验干扰,不宜用于探讨药物对早期OA的生化影响及观察OA早期病变。碘乙酸钠是一种细胞糖酵解抑制剂。在小鼠，家兔，犬类上进行碘乙酸钠注射后软骨轻度变薄,软骨下骨出现回声带。组织学证实软骨细胞减少和退变,基质蛋白多糖减少,软骨下骨板纤维血管性结缔组织增生。这与人类的骨关节炎的发病机制和致病机理非常接近。在众多骨关节炎造模的药物中，碘乙酸钠制备OA的原理与人类骨关节炎的发病机制最为接近，但是这种制备方法并未在灵长类动物上开展过。
目前碘乙酸钠常用在大鼠，家兔和仔猪的骨关节炎模型制备中。在大鼠的关节腔单次注射3mg的碘乙酸钠，用X光和MRI扫描发现关节软骨变薄，骨质受损，符合骨关节炎的病理特征。给仔猪关节腔单次注射40mg的碘乙酸钠，X光和MRI检查同样发现软骨变薄，骨质受损，病理染色发现软骨磨损严重，而且不管是大鼠还是仔猪皆没有发现机体严重的炎症反应。因此关节腔注射碘乙酸钠的造模方法是比较符合人类OA的病理特征。
碘乙酸钠制备食蟹猴骨关节炎模型目前还没有相关报道，所以将碘乙酸钠施用于灵长类动物需要进行严格的实验研究。
发明内容
本发明的目的是提供一种建立OA食蟹猴模型的诱导剂，其制法和使用方法。
本发明建立OA食蟹猴模型的诱导剂的特征是为碘乙酸钠水溶液，其浓度为0.2-0.3mg/μL。
本发明建立OA食蟹猴模型的诱导剂的制法是：将碘乙酸钠溶解在0.9％的生理盐水中，使其浓度为0.2-0.3mg/μL，轻轻震荡充分混匀，配置好的碘乙酸钠溶液在30分钟内完成注射。
本发明建立OA食蟹猴模型的诱导剂的使用方法是：
将本发明诱导剂用于食蟹猴，施用剂量为15-20mg/Kg/关节，最佳施用剂量为18mg/kg/关节；
用直径为430～460nm的注射器将诱导剂通过B超介导注射到食蟹猴关节腔内。
拔出注射器消毒食蟹猴关节；
正常饲养食蟹猴不短于三个月时间，得到OA食蟹猴模型。
施用剂量中的Kg为食蟹猴体重单位，表示施用剂量与体重相关。
本发明的积极效果：提供了能可靠建立OA食蟹猴模型的诱导剂，该诱导剂的制法，以及使用该诱导剂建立OA食蟹猴模型的方法，建立的OA食蟹猴模型可用于进行啮齿类、家兔和仔猪动物模型无法完成的生物技术新药的评价和干细胞移植治疗技术的评价。
本发明的骨关节炎模型的评价指标主要有：关节的弯曲度，关节B超检查，关节MRI扫描。本发明动物模型可以用于啮齿类、家兔和仔猪动物模型无法完成的生物技术新药的评价和干细胞移植治疗技术的评价。
附图说明
图1为实施例正常食蟹猴左后肢关节弯曲度和肿胀度测量情况。
图2为实施例正常食蟹猴右后肢关节弯曲度和肿胀度测量情况。
图3为实施例正常食蟹猴左后肢膝关节髌骨B超检查情况。
图4为实施例正常食蟹猴左后肢膝关节股骨B超检查情况。
图5为实施例正常食蟹猴右后肢膝关节髌骨B超检查情况。
图6为实施例正常食蟹猴右后肢膝关节股骨B超检查情况。
图7为实施例正常食蟹猴左后肢膝关节MRI扫描情况。
图8为实施例正常食蟹猴右后肢膝关节MRI扫描情况。
图9为实施例给予碘乙酸钠后食蟹猴的左后肢膝关节弯曲度测量情况。
图10为实施例给予碘乙酸钠后食蟹猴的右后肢膝关节弯曲度测量情况。
图11为实施例给予碘乙酸钠后食蟹猴的左后肢膝关节肿胀度测量情况。
图12为实施例给予碘乙酸钠后食蟹猴的右后肢膝关节肿胀度测量情况。
图13为实施例给予碘乙酸钠后食蟹猴的左后肢膝关节髌骨B超检查情况。
图14为实施例给予碘乙酸钠后食蟹猴的左后肢膝关节股骨B超检查情况。
图15为实施例给予碘乙酸钠后食蟹猴的右后肢膝关节髌骨B超检查情况。
图16为实施例给予碘乙酸钠后食蟹猴的右后肢膝关节股骨B超检查情况。
图17为实施例给予碘乙酸钠后食蟹猴的左后肢膝关节MRI扫描pd_fs图像。
图18为实施例给予碘乙酸钠后食蟹猴的左后肢膝关节MRI扫描T1图像。
图19为实施例给予碘乙酸钠后食蟹猴的右后肢膝关节MRI扫描pd_fs图像。
图20为实施例给予碘乙酸钠后食蟹猴的右后肢膝关节MRI扫描T1图像。
具体实施方式
下面结合具体实施例对本发明作进一步详细说明，但本发明的保护范围并不限于所述内容。
1、材料与方法
1.1实验材料
10-12岁雌性和雄性食蟹猴7只，无B疱疹病毒、猴逆转录病毒、猴白血病病毒和免疫缺陷病毒感染；来自昆明理工大学灵长类转化医学研究院。
1.2主要试剂
碘乙酸钠：购自Thermo Fisher Scientific旗下的ACROS ORGANICS，
0.9％的生理盐水：购自广西裕源药业有点公司。
1.3主要仪器
电子分析天平(奥豪斯仪器)；游标卡尺(得力，DL91150)，大三角尺；B超机(西门子，德国)，核磁共振(MRI，西门子，德国)
1.3实验方法
1.3.1预实验
为了验证碘乙酸钠的施用剂量，我们先挑选了一只食蟹猴用于药物剂量的验证，我们的结果证明施用剂量为15-20mg/Kg/次/关节的碘乙酸钠施用剂量可以构建食蟹猴的骨关节炎模型。
1.3.2食蟹猴挑选
根据动物体重，按15mg/Kg肌肉注射氯胺酮(50mg/ml)，待动物麻醉后，用三角尺和游标卡尺测量食蟹猴双下肢膝关节弯曲度和肿胀度，关节弯曲度大于170°，后肢无肿胀的食蟹猴为备选动物(图1，图2)。关节B超扫描双后肢膝关节，股骨和髌骨光滑，无骨赘，软骨明显的动物纳入实验(图3，图4，图5，图6)。麻醉后的动物进行双后肢的关节MRI扫描，MRI扫描未发现骨质水肿，股骨平台光滑，软骨明显存在(图7，图8)。动物信息见下表1。
| 猴号 | 性别 | 年龄 | 体重 |
| 1# | 雌性 | 13 | 6.6 |
| 2# | 雌性 | 13 | 4.23 |
| 3# | 雌性 | 13 | 4.06 |
| 4# | 雌性 | 12 | 5.7 |
| 5# | 雌性 | 13 | 4.1 |
| 6# | 雌性 | 14 | 4.6 |
| 7# | 雄性 | 8 | 7.15 |
1.3.3碘乙酸溶液配置
根据动物体重用分析天平称取碘乙酸钠，剂量为18mg/kg/关节。将碘乙酸钠溶解在0.9％的生理盐水中，使其浓度为0.25/μL。轻轻震荡充分混匀。配置好的碘乙酸溶液在30分钟内完成注射。
1.3.4碘乙酸钠注射到膝关节腔
用氯胺酮麻醉动物，氯胺酮使用剂量为15mg/Kg。麻醉后的动物用碘伏对膝关节进行消毒。在B超机介导用直径为450nm的注射器将配置后的碘乙酸钠注射到膝关节腔内，注射完成后使用酒精棉球按压针口。
1.3.5食蟹猴骨关节炎模型评估
碘乙酸钠注射后正常饲养观察动物三个月，观察食蟹猴骨关节炎进展情况和自愈情况。在药物注射后的1周，两周，三周，1个月，2个月，3个月进行膝关节弯曲度和肿胀度测量。在药物注射后1个月，2个月，3个月进行关节B超扫描。在造模后3个月，半年对动物膝关节进行MRI扫描。关节弯曲度和肿胀度测量发现碘乙酸钠注射后关节肿胀度增加，但是在一个月内又逐渐消退，关节弯曲度逐渐减小(图9，图10，图11，图12)。关节B超检查发现髌骨粗糙，有的动物还伴有骨赘发生，股骨变粗糙，软骨变薄(图13，图14，图15，图16)。关节MRI扫描发现在碘乙酸钠注射后三个月，骨质水肿严重，骨质粗糙，关节间隙变窄，软骨变薄(图17，图18，图19，图20)。以上结果都表明在碘乙酸钠注射后动物双后肢关节不能完全伸直，髌骨粗糙并伴有骨赘产生，股骨粗糙，关节间隙变窄，软骨变薄甚至完全消退。并且碘乙酸钠的造模方法并未产生剧烈的炎症反应，食蟹猴双后肢的肿胀度在一个月内几乎完全消退，这些症状与临床骨关节炎患者的症状非常接近。
Claims (3)
1.一种建立OA食蟹猴模型的方法，其特征在于：
将碘乙酸钠溶解在0.9％的生理盐水中，使其浓度为0.2-0.3mg/μL，得到诱导剂；
用直径为430～460nm的注射器将诱导剂通过影像设备介导注射到食蟹猴关节腔内，施用剂量为15-20mg/Kg/关节，配置好的碘乙酸钠溶液在30分钟内完成注射；
拔出注射器消毒食蟹猴关节；
正常饲养食蟹猴不短于三个月时间，得到OA食蟹猴模型。
2.如权利要求1所述的建立OA食蟹猴模型的方法，其特征在于施用剂量为18mg/kg/关节。
3.如权利要求1所述的建立OA食蟹猴模型的方法，其特征在于所述影像设备是B超机。
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| CN112538456A (zh) * | 2019-09-20 | 2021-03-23 | 北京干细胞与再生医学研究院 | 一种多能干细胞、药物组合物及其制备方法与用途 |
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| CN112538456A (zh) * | 2019-09-20 | 2021-03-23 | 北京干细胞与再生医学研究院 | 一种多能干细胞、药物组合物及其制备方法与用途 |
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