螺旋藻激酶对损伤内皮细胞的影响
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摘要
目的提取发酵螺旋藻干粉中有生物活性的螺旋藻激酶(Spirulina kinase SPK)。一方面通过体内动物实验建立内皮损伤血栓模型研究螺旋藻激酶对活体内皮损伤模型大鼠血管内皮细胞抗凝、纤溶相关因子的影响。另一方面通过体外培养人脐静脉内皮细胞(HUVECs),探讨螺旋藻激酶对人脐静脉内皮细胞纤溶相关基因表达的影响,从而从内皮细胞抗凝、纤溶的功能来探讨螺旋藻激酶对心血管系统的保护作用。
方法1.建立动物血栓模型：将大鼠随机分为假手术组(Sham)、模型组(Model)、SPK低剂量(L-SPK+M)、SPK中剂量(M-SPK+M)、SPK高剂量(H-SPK+M)。不同剂量SPK干预后,麻醉动物,FeCl3诱导大鼠颈总动脉内皮损伤形成血栓,从大鼠腹主动脉取血,抗凝后分别用酶联免疫吸附法(ELISA)检测血浆纤维蛋白溶酶原(Plg)抗凝血酶Ⅲ (AT-Ⅲ),放射免疫法检测血栓素B2(TXB2)、6-酮-前列腺素F1α(6-Keto-PGF1α)。2.体外培养人脐静脉内皮细胞,建立肾上腺素(Adr)损伤模型,并加入不同浓度的SPK,以肝素(Hep)为阳性对照,空白组为阴性对照,经过培养12h,24h,48h后,逆转录多聚酶链式反应(RT-PCR),扩增产物经琼脂糖凝胶电泳分离后,凝胶图像分析仪照相和扫描,测定各组组织型纤溶酶原激活物信使RNA (t-PAmRNA)、纤溶酶原激活物抑制物信使RNA(PAI-1mRNA)与内参基因三磷酸甘油醛脱氢酶信使RNA(GAPDHmRNA)的相对表达量。
结果1.体内实验显示,动物模型构建成功后模型组与假手术组相比直接显著升高大鼠血液中AT-Ⅲ.TXB2、6-Keto-PGF1α的含量,差异有统计学意义(P<0.05),而Plg含量无明显影响(P>0.05)经不同浓度SPK液干预后,与损伤模型组比较,SPK中、高剂量组Plg明显升高(P<0.01),SPK中、高剂量组间差异没有统计学意义(P>0.05)；SPK各剂量组AT-Ⅲ含量明显升高(P<0.01),在中剂量组最为显著；TXB2在SPK各剂量组均呈下降趋势(P<0.01),低、高组间没有明显差异(P>0.05)；6-Keto-PGF1α含量在SPK低、中高剂量组均下调(P<0.01),SPK中、低剂量组最为显著。
2.体外培养HUVECs,SPK单独作用于HUVECs与空白对照组表达t-PAmRNA无明显变化(P>0.05)。在肾上腺素损伤组中t-PAmRNA表达量在48h中表现为下调趋势(P<0.01)；在12h内肝素组和SPK不同溶度组均表现出上调趋势,组间没有统计学差异(P>0.05)；SPK高剂量组在12h和48h时间段上调t-PAmRNA表达趋势明显优于肝素对照组影响效能(P<0.05)
3.培养人脐静脉内皮细胞,SPK单独作用于HUVECs表达PAI-1mRNA无明显影响(P>0.05)。肾上腺素干预后,对PAI-1mRNA表达均出现明显上调(P<0.05),不同浓度SPK组在各个时间段均表现出较损伤组溶度依赖性的PAI-1mRNA表达下调(P<0.05)。SPK对损伤HUVECs表达t-PAmRNA和PAI-1mRNA的影响总的来说提高t-PAmRNA-PAI-1mRNA比值。
结论：螺旋藻激酶在抗凝溶栓效应上，促进Plg、AT-Ⅲ释放,抑制TXB2、6-Keto-PGF1α分泌,同时提高t-PAmRNA/PAI-1mRNA比值,提高内皮细胞纤溶活性,从抗凝、纤溶发挥其抗血栓作用。
Objective Extract the biological activity of Spirulina kinase (SPK) from the fermented spirulina powder.On one hand, the endothrlial damage thrombosis model in vivo to study the impact of SPK on living rats'vascular endothelial cells anticoagulant fibrinolytic related factors. The other hand, by in vitro cultured human umbilical vein endothelial cells (HUVECs) to explore the influence of SPK on HUVECs fibrinolytic gene expression. From the antithrombotic function and fibrinolytic function of endothelial cells to explore the Spirulina kinase protection on the cardiovascular system.
Methods Establish animal endothelial model:The rats were randomly divided into sham operation group (Sham), model group (Model), Spirulina kinase low-dose group+model (L-SPK+M), Spirulina kinase meduim-dose group+model (M-SPK+M), Spirulina kinase low-dose group+model (H-SPK+M). After different doses of SPK intervene, animals were anesthetized and FeC13induced cephalic artery endothelial injury, blood was collected from the abdominal aorta in rats, anticoagulation after, detecte plasminogen (Pig), antithrombin Ⅲ (AT-Ⅲ) secreted from endothelial cells with ELISA.Besides thromboxane B2(TXB2) and6-keto-prostaglandin F1a (6-Keto-PGF1a) were determined by radioimmunoassay.2. Culture vitro HUVECs, establish adrenaline (Adr) damage model with different concentrations of SPK. And control with the blank group, heparin (Hep) as a positive control, after12h,24h,48h, reverse transcription polymerase chain reaction (RT-PCR), PCR products were gel electrophoresis, the gel image analyzer photography and scanning, determination of the t-PAmRNA, PAI-1mRNA and reference gene glyceraldehyde phosphate aldehyde dehydrogenation enzyme GAPDHmRNA relative expression levels.
Results1. In vivo experiments showed that thrombosis model was constructed successfully significantly higher AT-III,6-Keto-PGF1a, TXB2, in rat blood, with the sham group difference was statistically significant (P<0.05), while the Plg content had no significant effect (P>0.05). After the different concentrations SPK intervention,compare with model group, in the high dose group Plg significantly higher (P<0.01), differences between meduim-dose and high-dose group were not statistically significant (P>0.05); AT-Ⅲ were significantly increased, especially in the middle dose group (P<0.01); levels of TXB2decreased significantly (P<0.01), no significant difference between the low-dose group and high-dose group (P>0.05);6-Keto-PGF1a in content of high, medium and low dose groups were reduced (P<0.01), the most significant is in the low and middle dose group, and difference between two groups was not statistically significant (P>0.05)
2. Cultured in vitro human umbilical vein endothelial cells, studying the expression of t-PAmRNA at different solubility SPK:SPK alone to HUVECs with the blank control group have no significant change (P>0.05). The t-PAmRNA expression as a downward trend in the model group at48h (P<0.01); The t-PAmRNA expression of the heparin group and SPK groups all increase within12h between the two groups it was not statistically significant (P>0.05); SPK high dose group in the24h and48h time periods increase the expression of t-PAmRNA was significantly superior to heparin group (P<0.05).
3. Cultured in vitro human umbilical vein endothelial cells, studying the expression of PAI-1mRNA at different solubility SPK:SPK alone to HUVECs with the blank control group have no significant change (P>0.05). After adrenaline intervention expression of PAI-1mRNA significantly increase (P<0.05). Different concentrations of SPK group at each time periods showed the solubility dependence down at expression of PAI-1mRNA (P<0.05). The ratio of expression of t-PAmRNA and PAI-1mRNA was improved.
Conclusion the SPK promote secretion of Plg, AT-Ⅲ, inhibit the release of TXB2,6-Keto-PGF1a, while improving the ratio of expression of t-PAmRNA and PAI-1mRNA in the anticoagulant thrombolytic effect, increase the fibrinolytic activity of endothelial cells. SPK. play its antithrombotic effect from anticoagulation fibrinolysis.
方法1.建立动物血栓模型：将大鼠随机分为假手术组(Sham)、模型组(Model)、SPK低剂量(L-SPK+M)、SPK中剂量(M-SPK+M)、SPK高剂量(H-SPK+M)。不同剂量SPK干预后,麻醉动物,FeCl3诱导大鼠颈总动脉内皮损伤形成血栓,从大鼠腹主动脉取血,抗凝后分别用酶联免疫吸附法(ELISA)检测血浆纤维蛋白溶酶原(Plg)抗凝血酶Ⅲ (AT-Ⅲ),放射免疫法检测血栓素B2(TXB2)、6-酮-前列腺素F1α(6-Keto-PGF1α)。2.体外培养人脐静脉内皮细胞,建立肾上腺素(Adr)损伤模型,并加入不同浓度的SPK,以肝素(Hep)为阳性对照,空白组为阴性对照,经过培养12h,24h,48h后,逆转录多聚酶链式反应(RT-PCR),扩增产物经琼脂糖凝胶电泳分离后,凝胶图像分析仪照相和扫描,测定各组组织型纤溶酶原激活物信使RNA (t-PAmRNA)、纤溶酶原激活物抑制物信使RNA(PAI-1mRNA)与内参基因三磷酸甘油醛脱氢酶信使RNA(GAPDHmRNA)的相对表达量。
结果1.体内实验显示,动物模型构建成功后模型组与假手术组相比直接显著升高大鼠血液中AT-Ⅲ.TXB2、6-Keto-PGF1α的含量,差异有统计学意义(P<0.05),而Plg含量无明显影响(P>0.05)经不同浓度SPK液干预后,与损伤模型组比较,SPK中、高剂量组Plg明显升高(P<0.01),SPK中、高剂量组间差异没有统计学意义(P>0.05)；SPK各剂量组AT-Ⅲ含量明显升高(P<0.01),在中剂量组最为显著；TXB2在SPK各剂量组均呈下降趋势(P<0.01),低、高组间没有明显差异(P>0.05)；6-Keto-PGF1α含量在SPK低、中高剂量组均下调(P<0.01),SPK中、低剂量组最为显著。
2.体外培养HUVECs,SPK单独作用于HUVECs与空白对照组表达t-PAmRNA无明显变化(P>0.05)。在肾上腺素损伤组中t-PAmRNA表达量在48h中表现为下调趋势(P<0.01)；在12h内肝素组和SPK不同溶度组均表现出上调趋势,组间没有统计学差异(P>0.05)；SPK高剂量组在12h和48h时间段上调t-PAmRNA表达趋势明显优于肝素对照组影响效能(P<0.05)
3.培养人脐静脉内皮细胞,SPK单独作用于HUVECs表达PAI-1mRNA无明显影响(P>0.05)。肾上腺素干预后,对PAI-1mRNA表达均出现明显上调(P<0.05),不同浓度SPK组在各个时间段均表现出较损伤组溶度依赖性的PAI-1mRNA表达下调(P<0.05)。SPK对损伤HUVECs表达t-PAmRNA和PAI-1mRNA的影响总的来说提高t-PAmRNA-PAI-1mRNA比值。
结论：螺旋藻激酶在抗凝溶栓效应上，促进Plg、AT-Ⅲ释放,抑制TXB2、6-Keto-PGF1α分泌,同时提高t-PAmRNA/PAI-1mRNA比值,提高内皮细胞纤溶活性,从抗凝、纤溶发挥其抗血栓作用。
Objective Extract the biological activity of Spirulina kinase (SPK) from the fermented spirulina powder.On one hand, the endothrlial damage thrombosis model in vivo to study the impact of SPK on living rats'vascular endothelial cells anticoagulant fibrinolytic related factors. The other hand, by in vitro cultured human umbilical vein endothelial cells (HUVECs) to explore the influence of SPK on HUVECs fibrinolytic gene expression. From the antithrombotic function and fibrinolytic function of endothelial cells to explore the Spirulina kinase protection on the cardiovascular system.
Methods Establish animal endothelial model:The rats were randomly divided into sham operation group (Sham), model group (Model), Spirulina kinase low-dose group+model (L-SPK+M), Spirulina kinase meduim-dose group+model (M-SPK+M), Spirulina kinase low-dose group+model (H-SPK+M). After different doses of SPK intervene, animals were anesthetized and FeC13induced cephalic artery endothelial injury, blood was collected from the abdominal aorta in rats, anticoagulation after, detecte plasminogen (Pig), antithrombin Ⅲ (AT-Ⅲ) secreted from endothelial cells with ELISA.Besides thromboxane B2(TXB2) and6-keto-prostaglandin F1a (6-Keto-PGF1a) were determined by radioimmunoassay.2. Culture vitro HUVECs, establish adrenaline (Adr) damage model with different concentrations of SPK. And control with the blank group, heparin (Hep) as a positive control, after12h,24h,48h, reverse transcription polymerase chain reaction (RT-PCR), PCR products were gel electrophoresis, the gel image analyzer photography and scanning, determination of the t-PAmRNA, PAI-1mRNA and reference gene glyceraldehyde phosphate aldehyde dehydrogenation enzyme GAPDHmRNA relative expression levels.
Results1. In vivo experiments showed that thrombosis model was constructed successfully significantly higher AT-III,6-Keto-PGF1a, TXB2, in rat blood, with the sham group difference was statistically significant (P<0.05), while the Plg content had no significant effect (P>0.05). After the different concentrations SPK intervention,compare with model group, in the high dose group Plg significantly higher (P<0.01), differences between meduim-dose and high-dose group were not statistically significant (P>0.05); AT-Ⅲ were significantly increased, especially in the middle dose group (P<0.01); levels of TXB2decreased significantly (P<0.01), no significant difference between the low-dose group and high-dose group (P>0.05);6-Keto-PGF1a in content of high, medium and low dose groups were reduced (P<0.01), the most significant is in the low and middle dose group, and difference between two groups was not statistically significant (P>0.05)
2. Cultured in vitro human umbilical vein endothelial cells, studying the expression of t-PAmRNA at different solubility SPK:SPK alone to HUVECs with the blank control group have no significant change (P>0.05). The t-PAmRNA expression as a downward trend in the model group at48h (P<0.01); The t-PAmRNA expression of the heparin group and SPK groups all increase within12h between the two groups it was not statistically significant (P>0.05); SPK high dose group in the24h and48h time periods increase the expression of t-PAmRNA was significantly superior to heparin group (P<0.05).
3. Cultured in vitro human umbilical vein endothelial cells, studying the expression of PAI-1mRNA at different solubility SPK:SPK alone to HUVECs with the blank control group have no significant change (P>0.05). After adrenaline intervention expression of PAI-1mRNA significantly increase (P<0.05). Different concentrations of SPK group at each time periods showed the solubility dependence down at expression of PAI-1mRNA (P<0.05). The ratio of expression of t-PAmRNA and PAI-1mRNA was improved.
Conclusion the SPK promote secretion of Plg, AT-Ⅲ, inhibit the release of TXB2,6-Keto-PGF1a, while improving the ratio of expression of t-PAmRNA and PAI-1mRNA in the anticoagulant thrombolytic effect, increase the fibrinolytic activity of endothelial cells. SPK. play its antithrombotic effect from anticoagulation fibrinolysis.
引文
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[1]T·柯林·坎贝尔,托马斯·M·埃贝尔Ⅱ,吕奕欣,等.中国健康调查报告[M].中信出版社,2011,9：5-11.
[2]袁桂清.血栓性疾病已成为威胁人类健康和生命的重要疾病[J].中华检验医学杂志,2004,8(27)：487.
[3]徐秋霞主编.血栓栓塞性疾病[M].中国医药科技出版社,2004,6：120-132.
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[8]李春坚,黄峻,杨志健,等.国产r-SAK对兔股动脉血栓的溶栓效果[J].中国临床药学杂志,2007,16(1)：25-28.
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[12]王建明,许蕾,李令根,等.复方蓬子菜冲剂对大鼠血栓形成的影响[J].中国实验方剂学杂志,2006,12(1)：31-32.
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[1]T·柯林·坎贝尔,托马斯·M·埃贝尔Ⅱ,吕奕欣,等.中国健康调查报告[M].中信出版社,2011,9：5-11.
[2]袁桂清.血栓性疾病已成为威胁人类健康和生命的重要疾病[J].中华检验医学杂志,2004,8(27)：487.
[3]徐秋霞主编.血栓栓塞性疾病[M].中国医药科技出版社,2004,6：120-132.
[4]陈铁军,陈瑞芳,杨果杰.一种家兔股动脉血管内制作血栓模型的简便方法[J].实验动物科学与管理,1997,14(2)：33-34.
[5]孙靖辉,尚坤,苏宪英,等.复方红豆杉片抗血栓作用的实验研究[J].长春中医学院学报,2006,22(1)：57.
[6]金桂兰,陈超.香椿子水煎剂的抗血栓作用研究[J].中国药房杂志[J].2011,22(15)：1364-1366.
[7]Jiang Y, Yin H, Liu J, et al. Effects of propyl gallate on carotid artery thrombosis and coagulation/fibrinolysis system in rats[J]. Chinese Journal of Integrative Medicine,2008,14(1):42-45.
[8]李春坚,黄峻,杨志健,等.国产r-SAK对兔股动脉血栓的溶栓效果[J].中国临床药学杂志,2007,16(1)：25-28.
[9]陈奇.中药药理研究方法学[M].北京.人民卫生出版社,1993：508-511.
[10]赵炜明,许超千,何树庄,等.蚓激酶溶栓作用的实验研究[J].2002,36(3)：183-185.
[11]周先丽,雷丹青,雷卓青.尖吻蝮蛇毒抗凝与纤溶组分对动物血栓的溶栓效应[J].中国临床康复,2006,10(3)：77-79.
[12]王建明,许蕾,李令根,等.复方蓬子菜冲剂对大鼠血栓形成的影响[J].中国实验方剂学杂志,2006,12(1)：31-32.
[13]琳娜,杨坚,王超,等.大鼠溶栓模型建立及其应用[J].北京医科大学学报,2000,32(3)：283-284.
[14]陈洲,黄自强,许建华.葡萄球菌激酶对大鼠颈动脉血栓的溶栓作用[J].心血管康复医学杂志,2006,15(1)：33-35.
[15]Charlton PA, Faint RW, Bent F, et al. Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-Iactivity[J]. Thrombosis Haemost,1996,75(5):808-815.
[16]黄晓巍,张永和,周鸣,等.复心片对大鼠体内动脉血栓形成及家兔血小板聚集性的影响[J].中国中医药信息杂志,2006,1,3(3)：27-28.
[17]王宝玲.脉络灵片的药效学研究[J].时珍国医国药,2006,17(1)：51-52.
[18]夏红梅,高云华,卞爱娜,等.动脉血栓模型建立的方法学研究[J].中国医学影像技术,2007,23(1)：13-15,
[19]Kurz K D, Main B W, Sandusky G E. Rat model of arterial throm-bosis induced by ferric chloride[J]. Thrombosis research,1990, 60(4):269-280.
[20]唐映红,梁燕,杨静,等.FeCI3诱导的大鼠颈总动脉血.栓模型血浆TXA2、PGI2、抗凝和纤溶活性的变化[J].中国药理学通报,2006,22(11)：1353-1355.
[21]郭丹.中国黑眼镜蛇毒蛋白酶natrahagin抑制血小板凝集和动脉血栓形成的作用[J].中国药理学与毒理学杂志,2001,15(1)：27-30.
[22]Imura Y, Terashita Z, Nishikawa K. The role of thromboxane (TX)A2 in rabbit arterial thrombosis induced by endothelial damage[J]. Thrombosis Research,1990,59(1):195-205.
[23]谢协驹,谢富生,莫燕娜,等.降低红细胞压积对家兔颈总动脉血栓形成的影响[J].中国病理生理学杂志,1995,5(2)：198-200.
[24]邓常青,贺石林,彭延占,等.以双氧水损伤血管内膜所致的家兔颈总动脉血栓模型[J].中国病理生理杂志,1990,6(5)：318-391.
[25]彭延古,雷田香,李路丹,等.全蝎提取液对家兔实验性动脉血栓的影响[J].微循环学杂志,2006,16(4)：59-60.
[26]王莉莉,李志超.梅其柄,等.MN9202保护血栓大鼠尾动脉血管作用[J].第四军医大学学报,2000,21(2)：214-216.
[27]胡三觉,田巧莲,顾建文,等.一种新的体内血栓形成动物模型[J]中华血液学杂志,1993,14(10)：541-542.
[28]丁书文,魏陵阱,史红霞,等.白酒对角义菜胶所致大鼠尾部血栓形成的影响[J].中国预防医学杂志,2004,5(3)：161-163.
[29]张维,赵秀梅,刘育英,等.大鼠提睾肌动脉溶栓模型的建立与应用[J].中国微循环,2004,8(6)：395-398.
[30]赵钢,冯幼启.光化学脑梗塞动物的模型的研究进展[J].国际脑血管病杂志,1992,12：58.
[31]李树清,叶翼明.光化学诱导的血栓形成性脑缺血及其代谢改变[J].中国病理生理杂志,1994,10(001)：95-99.
[32]姚勇,蔡季平,周韵秋.光化学法建立视网膜中央动脉阻塞模型[J].中国微循环.2000,4(4)：232-234.
[33]耿昱,陈怀红,郑筱祥,等.东菱克栓酶对大鼠急性肠系膜微动脉血栓溶栓作用的实验研究[J].中国微循环.1999,3(2)：99-101.
[34]Nanda SK, Hatchell DL, Tiedeman JS. A new method for vascular occlusion Photochemical initiation of thrombosis[J]. Archives of ophthalmology,1987,105(8):1121-1124.
[35]常丽英,张新江,张苏明.用于溶栓研究的血栓栓塞性脑梗死动物模型[J].国外医学：脑血管疾病分册,2001,9(4)：229-231.
[36]Thomas GR, Thibodeaux H, Errett CJ, et al. A long-half-life and fibrin-specific form of tissue plasminogen activator in rabbit models of embolic stroke and peripheral bleeding[J]. Stroke,1994,25(10): 2072-2078.
[37]Zhang Z, Zhang RL, Jiang Q, et al. A new rat model of thrombolytic focal cerebral ischemia[J]. Journal of Cerebral Blood Flow & Metabolism,1997,17(2):123-135.
[38]Sakurama T, Kitamura R, Kaneko M. Tissue-type plasminogen activator improves neurological functions in a rat model of thromboembolic stroke[J]. Stroke,1994,25(2):451-456.