CN114438167A - 一种检测枸杞氨基酸对心血管功能改变程度的方法 - Google Patents
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Abstract
本发明提供了一种检测枸杞氨基酸对心血管功能改变程度的方法，将实验动物随机分组，灌胃给予枸杞氨基酸，每天一次，连续灌胃给药数周后，完成血管取材；将待测血管放入盛有生理盐水的解剖皿中，冲洗掉血液并在体视镜下剥离掉血管外部的脂肪组织及结缔组织；对血管一氧化氮受体鸟苷酸环化酶(sGC)含量，血管收缩功能、血管中糖萼成分、糖胺聚糖含量的检测，得到枸杞氨基酸对心血管功能改变程度，为全面评估枸杞氨基酸对心血管功能的影响提供定量检测方法。
Description
技术领域
本发明涉及枸杞氨基酸在心血管疾病保护领域，尤其是对于血管功能改变程度的检测方法。
背景技术
心血管疾病(CVD)是目前引起心脏病的最大原因。世界卫生组织数据表明，2019年三分之一(1710万)的死亡是由于CVD[1]。其中大多数与冠状动脉病变有关。动脉疾病、中风、高血压和/或外周动脉疾病，这些疾病的共同特征是血管结构和/或功能的改变，这些因素改变是心血管疾病风险的主要因素[2]。因此，这一系列改变的需要及时评估，对于治疗心血管疾病至关重要[3]。目前对于心血管疾病的治疗方法与药物虽然有了一定的疗效，但是不尽让人满意，所以对于心血管的预防是非常必要。
枸杞氨基酸靶向作用点为人体血管，具体靶标为血管内皮细胞。根据大数据统计分析枸杞氨基酸的活性成分L-精氨酸/L-瓜氨酸具有软化和扩张血管、清除血液垃圾、消除血管斑块的功效，可以预防和逆转心脑血管病变，但是枸杞氨基酸对心血管功能改变程度如何，目前没有报道相关检测方法。
参考文献
1.Clifton P(2009)High protein diets and weight control.NutrMetabCardiovasc Dis 19:379–382.
2.Bedarida T,Baron S,Vessieres E et al(2014) High-protein-lowcarbohydrate diet:deleterious metabolic and cardiovascular effects
3.depend on age.Am J Physiol Heart Circ Physiol 307:H649–H657.
4.Burcelin R,Crivelli V,Dacosta A et al(2002)Heterogeneous metabolicadaptation of C57BL/6J mice to high-fat diet.Am J PhysiolEndocrinolMetab282:E834–E842.
发明内容
本发明的目的是提供一种检测枸杞氨基酸对心血管功能改变程度的方法，定量检测了枸杞氨基酸对心血管结构和/或功能的改变程度，阐述了枸杞氨基酸在预防心血管疾病中的作用。
为了实现上述目的，本发明采用的技术手段为：
一种检测枸杞氨基酸对心血管功能改变程度的方法，包括如下步骤：
1)、动物分组：将实验动物随机分组，灌胃给予枸杞氨基酸，每天一次，连续灌胃给药数周后，完成血管取材；
2)、血管预处理：将待测血管放入盛有生理盐水的解剖皿中，冲洗掉血液并在体视镜下剥离掉血管外部的脂肪组织及结缔组织；
3)鸟苷酸环化酶测定：将步骤2)处理后的血管在冰上迅速的匀浆成单细胞悬液，检测单细胞悬液中蛋白含量、sGC含量；
4)血管收缩功能测定：将步骤2)处理后的血管切为长度为 2-3mm的血管环，采用不同浓度的乙酰胆碱刺激血管环，记录绘制曲线；
5)糖萼成分测定：将步骤2)处理后的血管采用生理盐水1：9 匀浆，制备单细胞悬液，按ELISA试剂说明方法检测血管中糖萼成分；
6)糖胺聚糖含量测定：提取血管壁的sGAG，使用酶标仪测量吸光值，得到标准曲线后计算得到总sGAG含量值。
步骤1)所述实验动物为体重180-220g的实验大鼠。
步骤2)所述待测血管为主动脉与颈动脉血管。
步骤3)中采用BCA检测血管匀浆单细胞匀浆液蛋白含量，采用ELISA法测定血管单细胞悬液sGC含量。
步骤4)中所述乙酰胆碱浓度为0.1～2.5mg/mL。
步骤6)中提取血管壁的sGAG方法为：血管样本中加染料试剂，振荡以形成GAG-dye沉淀，离心，去除上清液并用滤纸吸出离心管底部残留液体，每个离心管中加0.5ml解离液，混合均匀，沉淀溶解后，离心去除泡沫后即得。
步骤6)中酶标仪测量656nm处的吸光值。
有益效果：
本发明提供了一种检测枸杞氨基酸对心血管功能改变程度的方法，通过对血管一氧化氮受体鸟苷酸环化酶(sGC)含量，血管收缩功能、血管中糖萼成分、糖胺聚糖含量的检测，得到枸杞氨基酸对心血管功能改变程度，为全面评估枸杞氨基酸对心血管功能的影响提供定量检测方法，可以更好的开发枸杞氨基酸产品。
附图说明
图1枸杞氨基酸对血管(主动脉)收缩功能的影响；
图2枸杞氨基酸对大鼠血管(主动脉与颈动脉)中糖萼成分影响： (A：主动脉)，(B：颈动脉)注：与空白组比较：##P<0.01；
图3枸杞氨基酸对大鼠血管(主动脉与颈动脉)中糖胺聚糖含量： (A：主动脉)，(B：颈动脉)注：与空白组比较：##P<0.01。
具体实施方式
以下实施例中采用的雄性SD大鼠(180-220g)购自卡文斯实验动物有限公司，合格证号：202111743，许可证号： SCXK(苏)02016-0010。所有动物实验操作均按照《药理实验动物操作指南》规范进行。
所用试剂及其来源厂家如表1所示。
表1
对照氨基酸是按照美国穆拉德博士编写的《一氧化氮让你远离心脑血管疾病》(北京大学医学出版社)第六章一氧化氮养生法保健品的作用中给出的配方，并参照美国有关一氧化氮补充剂生产厂家的配方生产的主要成分为麦芽糊精、L-精氨酸、牛磺酸、叶酸、VC为对照氨基酸。
实施例1：
采用SD雄性大鼠(SPF级，180-200g左右)，15只，随机分为 5组。分别为：空白组：生理盐水；实验组1：灌胃给予对照氨基酸(1.35g/kg·bw/d)组；实验组11：枸杞氨基酸(2.7/kg·bw/d) 组，实验组2：对照氨基酸(1.35g/kg·bw/d)组，实验组21：对照氨基酸(2.7/kg·bw/d)组。连续6周，完成主动脉、颈动脉、静脉血取材。枸杞氨基酸给药剂量：按人体最大推荐摄入量 0.3333g/kg·bw/d。检测大鼠血管反应性、血管功能生化指标等。
实施例2：血管一氧化氮(NO)受体鸟苷酸环化酶(sGC)检测
将待测血管(主动脉与颈动脉)放入盛有生理盐水的解剖皿中，冲洗掉血液并在体视镜下剥离掉血管外部的脂肪组织，在冰上迅速的匀浆成单细胞悬液。采用BCA检测血管(主动脉与颈动脉)匀浆单细胞匀浆液蛋白含量。采用ELISA法测定血管(主动脉与颈动脉)单细胞悬液sGC含量，按照说明书方法进行。
计算血管(主动脉与颈动脉)sGC含量，计算公式：血管(主动脉与颈动脉)sGC含量＝血管(主动脉与颈动脉)单细胞悬液sGC含量/ 血管(主动脉与颈动脉)匀浆单细胞匀浆液蛋白含量。结果如表2所示。
与空白组比较，对照氨基酸与枸杞氨基酸组血管NO含量显著升高，说明对照氨基酸与枸杞氨基酸均能显著增加血管弹性与舒张力。与对照氨基酸组比较，枸杞氨基酸组血管NO显著升高，说明枸杞氨基酸比氨基酸更能增加血管弹性与舒张力。
表2枸杞氨基酸对大鼠颈动脉与主动脉血管NO受体-鸟苷酸环化酶(sGC)的影响
注：与空白组比较：##P<0.01,与对照氨基酸组比较：**p<0.01
实施例3枸杞氨基酸对血管收缩功能的影响研究
将对照组与实验组大鼠取下的一段动脉(主动脉与颈动脉)立即放入盛有预冷的PBS的解剖皿中，于解剖镜下小心清除血管表面的脂肪及结缔组织。在显微镜下使用手术刀将其切为长度为2-3mm的血管环，链接仪器。并采用0～2.5mg/mL不同的浓度的乙酰胆碱刺激血管环，绘制曲线。结果如图1所示，与空白组比较，枸杞氨基酸组血管对乙酰胆碱反应性显著升高。与对照氨基酸组比较，枸杞氨基酸组血管对乙酰胆碱反应性显著升高，说明枸杞氨基酸比氨基酸更能增加血管收缩力。
实施例4ELISA法检测各组大鼠血管中糖萼成分
将对照组与实验组大鼠取下的一段动脉(主动脉与颈动脉)立即放入盛有预冷的PSS的解剖皿中，于解剖镜下小心清除血管表面的脂肪及结缔组织，采用生理盐水1：9匀浆，制备单细胞悬液。按ELISA 试剂说明方法加入标准品和实验样品于微孔板上，密封反应孔,室温下摇床上孵育2h。用洗涤液清洗微孔板，取出洗涤液，每孔加入酶标检测抗体,密封反应孔。室温下在摇床孵育30min。重复洗板 5次，每孔加入显色底物,室温孵育30min。孵育后，每孔加入终止液在450nm处立即读取OD值。结果如图2所示，与空白组比较，对照氨基酸与枸杞氨基酸组血管(主动脉与颈动脉)对sydecan-1、HA 和HS释放增多。
实施例5糖胺聚糖含量的测定
配制木瓜蛋白酶提取液，提取血管(主动脉与颈动脉)壁的sGAG。具体来讲，将得到的样本中分别加1ml染料试剂，振荡30min以形成GAG-dye沉淀，12000rpm离心10min，去除上清液并用滤纸吸出离心管底部残留液体。每个离心管中加0.5ml解离液使用涡旋振荡器混合均匀，沉淀溶解后，12000rpm离心5min以去除泡沫。缓慢吸取200μl混合试剂加到96孔板中，每个样本设置两个平行样，使用酶标仪测量656nm处的吸光值，得到标准曲线后计算得到各个样本总sGAG含量值。结果如如图3所示，与空白组比较，对照氨基酸与枸杞氨基酸组血管(主动脉与颈动脉)糖胺聚糖含量增多。与对照氨基酸组比较，枸杞氨基酸组血管糖胺聚糖含量显著增多。
Claims (7)
1.一种检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，包括如下步骤：
1）、动物分组：将实验动物随机分组，灌胃给予枸杞氨基酸，每天一次，连续灌胃给药数周后，完成血管取材；
2）、血管预处理：将待测血管放入盛有生理盐水的解剖皿中，冲洗掉血液并在体视镜下剥离掉血管外部的脂肪组织及结缔组织；
3）鸟苷酸环化酶测定：将步骤2）处理后的血管在冰上迅速的匀浆成单细胞悬液，检测单细胞悬液中蛋白含量、sGC含量；
4）血管收缩功能测定：将步骤2）处理后的血管切为长度为 2-3mm 的血管环，采用不同浓度的乙酰胆碱刺激血管环，记录绘制曲线；
5）糖萼成分测定：将步骤2）处理后的血管采用生理盐水1：9匀浆，制备单细胞悬液，按ELISA试剂说明方法检测血管中糖萼成分；
6）糖胺聚糖含量测定：提取血管壁的sGAG，使用酶标仪测量吸光值，得到标准曲线后计算得到总 sGAG 含量值。
2.根据权利要求1所述的检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，步骤1）所述实验动物为体重180-220g的实验大鼠。
3.根据权利要求1所述的检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，步骤2）所述待测血管为主动脉与颈动脉血管。
4.根据权利要求1所述的检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，步骤3）中采用BCA检测血管匀浆单细胞匀浆液蛋白含量，采用ELISA法测定血管单细胞悬液sGC含量。
5.根据权利要求1所述的检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，步骤4）中所述乙酰胆碱浓度为0.1~2.5mg/mL。
6.根据权利要求1所述的检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，步骤6）中提取血管壁的sGAG方法为：血管样本中加染料试剂，振荡以形成 GAG-dye 沉淀，离心，去除上清液并用滤纸吸出离心管底部残留液体，每个离心管中加 0.5ml 解离液，混合均匀，沉淀溶解后，离心去除泡沫后即得。
7.根据权利要求1所述的检测枸杞氨基酸对心血管功能改变程度的方法，其特征在于，步骤6）中酶标仪测量 656nm 处的吸光值。
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