CN114384185A - 用于检测γ-氨基丁酸含量的试剂在制备诊断静脉流出障碍性疾病的试剂盒中的应用 - Google Patents
用于检测γ-氨基丁酸含量的试剂在制备诊断静脉流出障碍性疾病的试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开了用于检测γ‑氨基丁酸含量的试剂在制备诊断静脉流出障碍性疾病的试剂盒中的应用,涉及生物医学技术领域,本发明首次公开了差异代谢物γ‑氨基丁酸可以用于判断静脉流出障碍性疾病是否发生以及发病程度,相对于传统的影像学检查而言,要更加的快速和易于操作,为静脉流出障碍性疾病的诊断、治疗和研究提供了新的途径。
Description
技术领域
本发明涉及生物医学技术领域,具体而言,涉及用于检测γ-氨基丁酸含量的试剂在制备诊断静脉流出障碍性疾病的试剂盒中的应用。
背景技术
血管疾病已成为威胁人群健康的头号杀手,严重影响患者的生活质量,同时给患者家庭以及社会带来巨大经济负担。脑静脉引流系统包括脑静脉、静脉窦以及负责脑静脉引流的颈部静脉结构,这些结构的异常可以导致脑静脉流出障碍。越来越多的证据表明,脑静脉系统结构异常或脑静脉流出障碍可导致多种神经功能损害症状,如头痛/头昏、耳鸣/脑鸣、视物模糊/视野缺损/复视、睡眠障碍、焦虑抑郁等,并在多种退行性神经系统疾病(如帕金森和包括阿尔兹海默症在内的各种痴呆)的发病和进展中发挥了重要作用。与动脉系统疾病的研究相比,当前对脑静脉系统结构和功能异常在中枢神经系统疾病过程中的病理生理作用尚需进一步研究。
静脉流出障碍可以引起颅内静脉血液回流障碍,头颈静脉侧枝循环形成,如果侧枝循环不能代偿静脉回流,会引起脑静脉血管压力增高和颅内压升高。由于解剖变异和侧支循环代偿能力不同,脑静脉流出障碍的临床表现可能因患者而异。静脉的解剖结构不同于动脉,静脉壁比动脉薄,缺少平滑肌层和弹性纤维;而且静脉内压力偏低,所以静脉在外源性压迫下更容易塌陷。因此,除了明确脑静脉流出障碍的诊断外,还需要明确脑静脉流出障碍的病因,因为病因决定治疗方案的选择。
现有的颈静脉彩色多普勒超声、血管内超声(IVUS)、头颈一体CE-MRV、头颈一体强化CTV检查等是用于评估静脉流出障碍的主要筛查工具,但由于上述筛查项目依赖大型医疗设备,在广大基层及偏远地区医疗单位尚未普及,严重制约了对脑静脉流出障碍的诊断。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供用于检测γ-氨基丁酸含量的试剂在制备诊断静脉流出障碍性疾病的试剂盒中的应用。
本发明是这样实现的:
第一方面,本发明实施例提供了用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用。
第二方面,本发明实施例提供了γ-氨基丁酸和/或用于促进GABA合成的试剂在制备用于预防或治疗静脉流出障碍性疾病的药物中的应用。
第三方面,本发明实施例还提供了用于检测γ-氨基丁酸含量的试剂在构建或训练静脉流出障碍性疾病预测模型中的应用。
本发明具有以下有益效果:
本发明首次公开了差异代谢物γ-氨基丁酸(GABA)可以用于判断静脉流出障碍性疾病是否发生以及发病程度,相对于传统的影像学检查而言,要更加的快速和易于操作,为静脉流出障碍性疾病的诊断、治疗和研究提供了新的途径。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例1中大鼠脑静脉流出障碍模型手术示意图;其中,A为脑静脉流出障碍大鼠模型,即同时结扎盂后静脉及颈内静脉(红色对向箭头所示);B为盂后静脉,其是颅外横窦的延续,主要引流上矢状窦和直窦的静脉血,与颞浅静脉吻合,后汇入面后静脉,与面前静脉汇合形成颈外静脉,最终经颈外静脉流出入右心房;C为盂后静脉分离暴露过程图;
图2为实施例1中假手术组与脑静脉流出障碍模型组动物血管结构和功能的比较;其中,A为两组动物颅内静脉影像学检测结果;B为两组动物血脑屏障破坏程度——EB渗漏情况;C为两组动物血脑屏障破坏程度——NaF渗漏情况;D为两组动物颅内压变化比例;
图3为实施例1中大鼠脑皮层、海马组织以及脑脊液中γ-氨基丁酸(GABA)的含量检测;其中,A为海马组织的GABA的含量,B为皮层组织的GABA的含量,C为脑脊液的GABA的含量;
图4为实施例1中人脑脊液中GABA的含量检测。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
首先,本发明实施例提供了用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用。
目前关于诊断静脉狭窄尚无金标准且没有可以诊断静脉流出障碍的标记物。本发明通过设计研究性实验筛选出的差异代谢物γ-氨基丁酸可以用于判断静脉流出障碍性疾病是否发生以及发病程度,为静脉流出障碍性疾病的诊断、治疗和研究提供了新的途径。
优选地,所述静脉流出障碍性疾病包括静脉流出障碍或由静脉流出障碍引发的临床症状、病理损害或相关疾病。
优选地,所述临床症状包括:头痛、头昏、耳鸣、脑鸣、视物模糊、视野缺损、复视、睡眠障碍和焦虑抑郁中的至少一种。
优选地,所述病理损害包括:血脑屏障破坏、神经退行性改变、静脉源性脑微出血、脑脊液产生异常、类淋巴系统功能受损和脑血流动力学紊乱中的至少一种,脑静脉流出障碍可引发这些病理损害。
优选地,所述相关疾病包括血管性、血栓性、炎症性和退行性疾病中的至少一种。
优选地,所述静脉流出障碍由静脉窦血栓和静脉窦狭窄中的至少一种引发。所述静脉流出障碍性包括脑静脉流出障碍和颈内静脉流出障碍中的至少一种。当静脉流出障碍为脑静脉流出障碍时,所述脑静脉流出障碍由脑静脉窦血栓、脑静脉窦狭窄和颈静脉狭窄中的至少一种引发,即颈内静脉障碍也会同样导致脑静脉流出障碍进而引发脑静脉流出障碍性疾病。
在可选的实施例中,所述用于诊断或辅助诊断静脉流出障碍性疾病包括:检测静脉流出障碍性疾病是否发生以及检测静脉流出障碍性疾病的发病程度。
在可选的实施例中,所述检测的方式包括液相色谱、纸层析法和Berthelot比色法。只要是将能够用于检测γ-氨基丁酸的试剂用于制备诊断或辅助诊断样本静脉流出障碍性疾病的试剂盒的,均属于本申请的保护范围内。
当静脉流出障碍为脑静脉流出障碍时,所述样本为动物的脑组织样本或脑脊液样本。可选地,所述动物为哺乳动物,优选为非人哺乳动物。
本发明实施例还提供了γ-氨基丁酸和/或用于促进γ-氨基丁酸合成的试剂在制备用于预防或治疗静脉流出障碍性疾病的药物中的应用。
静脉流出障碍性疾病同前述实施例所述,不再赘述。
在可选的实施例中,所述药物还包括医学上可接受的载体。
所述医学上可接受的载体包括:赋性剂、稀释剂、抗腐蚀剂和抗凝剂中的至少一种。稀释剂可以为水。
在可选的实施例中,所述药物的剂型选自粉剂、片剂、胶囊剂、乳液剂、丸剂、口服液剂、颗粒剂和注射液剂中的任意一种。
在可选的实施例中,所述药物还可以包括医学上可接受的载体,如赋性剂。
此外,本发明实施例还提供了用于检测γ-氨基丁酸含量的试剂在构建或训练静脉流出障碍性疾病预测模型中的应用。
可以理解的是,所述静脉流出障碍性疾病可以同前述任意实施例所述,不再赘述。
在优选的实施方式中,所述静脉流出障碍性疾病预测模型根据样本γ-氨基丁酸含量的检测结果,判断样本静脉流出障碍性疾病是否发生或发病程度。在构建的特征数据(样本γ-氨基丁酸含量的)清楚的情况下,本领域技术人员可以基于现有的分类器或神经网络的算法进行预测模型的构建。
构建好预测模型后,模型的训练方法可以包括以下步骤:
获取训练样本的γ-氨基丁酸含量的检测结果以及对应的标注结果;标注结果可以为疾病是否患病或发病的程度;
将训练样本的γ-氨基丁酸含量的检测结果属于预先构建好的预测模型中,获得样本的预测结果;
基于样本的预测结果和对应的标注结果,对预测模型进行参数更新,获得训练好的预测模型。
当静脉流出障碍为脑静脉流出障碍时,所述样本为动物的脑组织样本或脑脊液样本。
实施例1
1、脑静脉流出障碍动物模型建立;
选择成年(6-8周龄)雌/雄SD大鼠,将SD大鼠分为两组:
(1)假手术对照组(Sham组)。
(2)根据啮齿类动物头颈静脉解剖特点设计符合人脑静脉回流障碍发病特点的动物模型,即盂后静脉及脑静脉同时结扎手术方案(JVL组),具体过程如下:
用4%的异氟烷诱导大鼠麻醉,手术过程中将异氟烷浓度保持在1.5%和2%之间。将每只大鼠放在保温垫上,用剃毛器除去颈部以及耳前毛发。在手术显微镜下,调整显微镜视野以及焦距,常规消毒,首先在颈部中线处做一个纵向皮肤切口。用血管钳钝性分离皮下组织以及唾液腺,分离唾液腺背外侧并依次分离并暴露位于左右两侧的颈动脉鞘内的脑静脉(脑动脉上方,迷走神经旁),使用8-0非吸收手术缝合线结扎脑静脉。常规缝合伤口,在伤口处涂抹罗红霉素抗生素软膏,然后,常规消毒大鼠耳部及耳至眼睛之间的区域皮肤,近耳前5mm处做斜行约1.5cm切口,仔细分离面后静脉、颞浅静脉及盂后静脉(如图1),使用8-0非吸收手术缝合线结扎盂后静脉,终止麻醉,常规缝合伤口,在伤口处涂抹罗红霉素抗生素软膏,每天1次,连续5天常规饲养。
本实施例建立的脑静脉流出障碍模型组动物与假手术对照组动物血管结构和功能的比较参照图2,图2中,A显示模型组动物颅内静脉窦扩张更为明显,B-C显示模型组血脑屏障破坏程度较假手术对照组显著改变,D显示模型组颅内压较假手术对照组颅内压变化比例显著升高。通过对比试验,模型组动物在行为学表现、血脑屏障完整性、颅内血管形态学变化方面都表现出显著的功能/结构异常,符合脑静脉流出障碍基础研究的需要。
2、非靶向代谢组学筛选差异代谢物(表1)。
表1差异代谢物GABA相关信息
3、大鼠脑组织及脑脊液中GABA含量检测;
(1)分别选取术后1月JVL组及其对应假手术对照组大鼠各6只,4%水合氯醛麻醉,采集0.2ml脑脊液后生理盐水灌注,冲去血液。快速取出脑组织,分离海马、皮层等亚结构。
(2)样本做如下处理:
脑脊液:4℃、1000g离心20min,取上清检测;
脑组织:用预冷的PBS(0.01M,PH7.4)冲洗脑组织,去除残留血液,称重并剪碎研磨(按组织:PBS=1:9的质量体积比配置体系,即1g组织中加入9ml PBS缓冲液,并加入一定量蛋白酶抑制剂PK),匀浆液进行超声破碎,4℃、5000g离心10min,取上清检测;
(3)按照江莱生物GABA大鼠ELISA检测试剂盒(JL12343)所提供检测说明书依次进行各步骤操作,最终于酶标仪OD450nm处读取各孔的OD值并进行浓度计算(图3)。
4、人脑脊液中GABA含量检测;
(1)分别收集诊断为脑静脉流出障碍以及非中枢神经系统损害患者脑脊液,使用普通脑脊液采集管按照常规采集操作,留取脑脊液1mL左右,于-80℃冰箱中保存;
非中枢神经系统损害患者脑脊液标本留取采取以下采样标准。
纳入标准:
①年龄、性别无特殊限制;
②非脑、脊髓等神经系统损害的患者(如疝气,骨折等患者)。
排除标准:
①存在帕金森、老年痴呆、癫痫、任何系统的恶性肿瘤;
②存在静脉窦血栓、颅内感染、血管炎以及其他风湿免疫性疾病;
③存在严重糖尿病、肝肾功能异常。
(2)样本做如下处理:
4℃、1000g离心20min,取上清检测;
(3)按照江莱生物GABA人ELISA检测试剂盒(JL13377)所提供检测说明书依次进行各步骤操作,最终于酶标仪OD450nm处读取各孔的OD值并进行浓度计算(图4)。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用。
2.根据权利要求1所述的用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用,其特征在于,所述用于诊断或辅助诊断静脉流出障碍性疾病包括:检测静脉流出障碍性疾病是否发生以及检测静脉流出障碍性疾病的发病程度。
3.根据权利要求1所述的用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用,其特征在于,所述静脉流出障碍性疾病包括静脉流出障碍或由静脉流出障碍引发的临床症状、病理损害或相关疾病;
优选地,所述临床症状包括:头痛、头昏、耳鸣、脑鸣、视物模糊、视野缺损、复视、睡眠障碍和焦虑抑郁中的至少一种;
优选地,所述病理损害包括:血脑屏障破坏、神经退行性改变、静脉源性脑微出血、脑脊液产生异常、类淋巴系统功能受损和脑血流动力学紊乱中的至少一种;
优选地,所述相关疾病包括血管性、血栓性、炎症性和退行性疾病中的至少一种。
4.根据权利要求1~3任一项所述的用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用,其特征在于,所述静脉流出障碍性包括脑静脉流出障碍和颈内静脉流出障碍中的至少一种;
优选地,所述静脉流出障碍由静脉窦血栓和静脉窦狭窄中的至少一种引发。
5.根据权利要求4所述的用于检测样本中γ-氨基丁酸含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用,其特征在于,当静脉流出障碍为脑静脉流出障碍时,所述样本为动物的脑组织样本或脑脊液样本。
6.根据权利要求1~3任一项所述的用于检测样本中γ-氨基丁酸(GABA)含量的试剂在制备用于诊断或辅助诊断静脉流出障碍性疾病的试剂盒中的应用,其特征在于,所述检测的方式包括液相色谱、纸层析法和Berthelot比色法。
7.γ-氨基丁酸和/或用于促进γ-氨基丁酸合成的试剂在制备用于预防或治疗静脉流出障碍性疾病的药物中的应用。
8.用于检测γ-氨基丁酸含量的试剂在构建或训练静脉流出障碍性疾病预测模型中的应用。
9.根据权利要求8所述的用于检测γ-氨基丁酸含量的试剂在构建或训练静脉流出障碍性疾病预测模型中的应用,其特征在于,所述静脉流出障碍性疾病预测模型根据样本γ-氨基丁酸含量的检测结果,判断样本静脉流出障碍性疾病是否发生或发病程度。
10.根据权利要求8或9所述的应用,其特征在于,所述静脉流出障碍性疾病包括静脉流出障碍或由静脉流出障碍引发的临床症状、病理损害或相关疾病;
优选地,所述静脉流出障碍包括脑静脉流出障碍和颈内静脉流出障碍中的至少一种。
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