香叶木素取代物调控静脉血栓蛋白质组及靶蛋白的探索
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摘要
蓬子菜是中国东北的一种野生植物,这种植物以它是维生素的优良的食物来源而著称,它还包含绿原酸,水杨酸和黄酮类等物质。报道蓬子菜的抗静脉血栓活性值比其它植物特别是黄酮丰富的植物要高,通过高效液相色谱法(反相液相色谱)从蓬子菜中分离得到的香叶木素取代物已被鉴定为抗血栓的药效物质。本研究通过分析蓬子菜中香叶木素取代物作用于静脉血栓的血浆蛋白质组的变化,以期寻找香叶木素取代物作用于静脉血栓的生物标记和靶蛋白,为将其作为先导化合物提供理论依据。
实验选取Wistar大鼠随机分为对照组,模型组和预防组三个组,其中对照组不进行任何处理,模型组只建立了体外静脉血栓模型而预防组是腹腔注射香叶木素取代物后,然后建立体外静脉血栓模型,采用病理学检查进行验证,对亚洲人常见的蛋白c进行了分析,结果表明在血栓形成过程中,蛋白c起到了灭活活化的凝血因子,促进纤维蛋白溶解,减轻内皮细胞的损伤的作用,这与电镜分析的结果一致,验证了血栓动物模型建立成功。采用双向电泳与高效液相色谱法相结合的方法进行蛋白质组分析,血浆蛋白通过高速离心分离提取,采用Sephadex柱层析和低丰度蛋白富集试剂盒进行分离,以去除免疫球蛋白等的高丰度蛋白质。对低丰度蛋白质进行双向聚丙烯酰胺凝胶电泳,用PDquest软件进行图像分析,寻找差异表达的蛋白质。
分别对空白组,模型组和预防组的2-DE图像进行比较和分析,模型组中一共有147个蛋白点,预防中一共有99个蛋白点;和模型组比较,预防组共有49个差异蛋白点表达,其中表达上调2倍的蛋白点有28个,表达下降2倍的蛋白点有21个;在模型组中差异表达的有56个蛋白点,在预防组中差异表达的有有8个蛋白点。经过基质辅助激光解析飞行时间质谱分析后,成功鉴定了13个靶蛋白(10个已知蛋白,3个未知蛋白),其中匹配率较高的靶蛋白是Full=Serum albumin; Flags: Precursor,serum albumin precursor, glutathione S-transferase pi,albumin isoform CRA_a。它们的检索比对得分分别是287,278,109,104,匹配率分别为25%,23%,40%,37%。
Galium verum L. is a wild plant from Northeast China, this grass is well known as an excellent food source of vitamin, and it also contains phytochemicals such as Chlorogenic Acid, Salicylic Acid and flavone. Anti-venous thrombosis activity values obtained from Galium were higher than other plant particuLarly rich in flavones. Diosmetin substituent compounds by high performance liquid chromatography (RPLC), as main factor responsible for anti-venous thrombosis activity,were determined. This study was to analyze the proteome changes of diosmetin substituent in venous thrombosis of plasma ,in order to search for biomarkers and target proteins in venous thrombosis, as will as a lead compound and provide a theoretical basis.
To investigation the proteome changes of the plasma, Wistar rats were selected to establish the model and verificated by pathologic diagnosis and protein C according to it’s key role in thrombosis. The control group did not undergo any processing, the model group and the prevention group were established thrombosis model in vitro and the difference between them was the prevention group had been given diosmetin substituent before model was established. Dimensional gel electrophoresis and high performance liquid chromatography method were combined to analysis proteome. Plasma proteins were extracted by high velocity centrifugation and separated by Sephadex column to remove high-abundance proteins such as immunoglobuLin. Low-abundance proteins were separated by dimensional gel electrophoresis and the images were analyzed by PDquest software.
Comparative and analysis dimensional gel electrophoresis gel images of the control group, the model group and the prevention group, there are 147 protein spots in model group,while 99 protein spots in prevention group. Comparing with the model group there are 49 expressed differential protein spots in the prevention group ,among them there 28 protein spots expressing increased while 21 decreased ; 56 protein spots were identified in model group only, 8 differently proteins spots were in prevention group only.
By Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry, 13 kinds (10named , 3unnamed )proteins were identified successfuLly , the higher rate matched target protein are Full = Serum albumin; Flags: Precursor, serum albumin precursor, glutathione S-transferase pi, albumin isoform CRA_a. The retrieval scores are 287,278,109,104, and the matching rate are 25%, 23%, 40%, 37% respectly.
实验选取Wistar大鼠随机分为对照组,模型组和预防组三个组,其中对照组不进行任何处理,模型组只建立了体外静脉血栓模型而预防组是腹腔注射香叶木素取代物后,然后建立体外静脉血栓模型,采用病理学检查进行验证,对亚洲人常见的蛋白c进行了分析,结果表明在血栓形成过程中,蛋白c起到了灭活活化的凝血因子,促进纤维蛋白溶解,减轻内皮细胞的损伤的作用,这与电镜分析的结果一致,验证了血栓动物模型建立成功。采用双向电泳与高效液相色谱法相结合的方法进行蛋白质组分析,血浆蛋白通过高速离心分离提取,采用Sephadex柱层析和低丰度蛋白富集试剂盒进行分离,以去除免疫球蛋白等的高丰度蛋白质。对低丰度蛋白质进行双向聚丙烯酰胺凝胶电泳,用PDquest软件进行图像分析,寻找差异表达的蛋白质。
分别对空白组,模型组和预防组的2-DE图像进行比较和分析,模型组中一共有147个蛋白点,预防中一共有99个蛋白点;和模型组比较,预防组共有49个差异蛋白点表达,其中表达上调2倍的蛋白点有28个,表达下降2倍的蛋白点有21个;在模型组中差异表达的有56个蛋白点,在预防组中差异表达的有有8个蛋白点。经过基质辅助激光解析飞行时间质谱分析后,成功鉴定了13个靶蛋白(10个已知蛋白,3个未知蛋白),其中匹配率较高的靶蛋白是Full=Serum albumin; Flags: Precursor,serum albumin precursor, glutathione S-transferase pi,albumin isoform CRA_a。它们的检索比对得分分别是287,278,109,104,匹配率分别为25%,23%,40%,37%。
Galium verum L. is a wild plant from Northeast China, this grass is well known as an excellent food source of vitamin, and it also contains phytochemicals such as Chlorogenic Acid, Salicylic Acid and flavone. Anti-venous thrombosis activity values obtained from Galium were higher than other plant particuLarly rich in flavones. Diosmetin substituent compounds by high performance liquid chromatography (RPLC), as main factor responsible for anti-venous thrombosis activity,were determined. This study was to analyze the proteome changes of diosmetin substituent in venous thrombosis of plasma ,in order to search for biomarkers and target proteins in venous thrombosis, as will as a lead compound and provide a theoretical basis.
To investigation the proteome changes of the plasma, Wistar rats were selected to establish the model and verificated by pathologic diagnosis and protein C according to it’s key role in thrombosis. The control group did not undergo any processing, the model group and the prevention group were established thrombosis model in vitro and the difference between them was the prevention group had been given diosmetin substituent before model was established. Dimensional gel electrophoresis and high performance liquid chromatography method were combined to analysis proteome. Plasma proteins were extracted by high velocity centrifugation and separated by Sephadex column to remove high-abundance proteins such as immunoglobuLin. Low-abundance proteins were separated by dimensional gel electrophoresis and the images were analyzed by PDquest software.
Comparative and analysis dimensional gel electrophoresis gel images of the control group, the model group and the prevention group, there are 147 protein spots in model group,while 99 protein spots in prevention group. Comparing with the model group there are 49 expressed differential protein spots in the prevention group ,among them there 28 protein spots expressing increased while 21 decreased ; 56 protein spots were identified in model group only, 8 differently proteins spots were in prevention group only.
By Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry, 13 kinds (10named , 3unnamed )proteins were identified successfuLly , the higher rate matched target protein are Full = Serum albumin; Flags: Precursor, serum albumin precursor, glutathione S-transferase pi, albumin isoform CRA_a. The retrieval scores are 287,278,109,104, and the matching rate are 25%, 23%, 40%, 37% respectly.
引文
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