重组人组织激肽释放酶结合蛋白抗氧化活性及抗凝活性分析
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摘要
探究Kallistatin蛋白在体外3种细胞模型中的抗氧化活性及其体外抗凝、溶栓活性.首先,在体外构建HBZY-1,HaCaT,HTR8-s/Vneo细胞的H2O2,Fe-HQ氧化应激模型,并将Kallistatin蛋白应用于构建的氧化应激模型中.然后,采用多浓度梯度法筛选最佳氧化剂浓度,利用细胞活性实验检测以上3种细胞的活性,并进行体外抗凝、溶栓实验考察.结果表明:在H2O2模型中,当Kallistatin蛋白浓度为0.6μmol·L-1时,3种细胞活力与对照组(LD-Hanks缓冲液)相近,低剂量蛋白组(0.06μmol·L-1)的细胞活力明显低于高剂量蛋白组细胞;在Fe-HQ模型中,不同细胞中Kallistatin蛋白剂量的作用不同,Kallistatin蛋白具有体外抗氧化活性,不同应激条件下不同细胞对蛋白剂量的依赖性不同;体外抗凝、溶栓实验表明此蛋白具有相关活性.
To investigate the anti-oxidative activity and anticoagulant and thrombolytic activity of Kallistatin in three kinds of cell models in vitro,H2 O2 and Fe-HQ oxidative stress models of HBZY-1,HaCaT and HTR8-s/Vneo cells were constructed in vitro,and the Kallistatin protein was used in these oxidative stress models.Multi-concentration gradient method was adopt to select the optimal oxidant concentration.CCK experiment was used to detect the activity of three kinds of cells,and the anticoagulant and thrombolytic experiments were carried out in vitro.The results shows that in the H2 O2 model,the viability of three kinds of cell was similar to the control group(LD-hanks buffer)when the concentration of Kallistatin protein was 0.6μmol·L-1.The cell viability of low-dose protein group(0.06μmol·L-1)was significantly lower than that of the high dose.In the Fe-HQ model,the effect of Kallistatin protein doses differed in different cells.Thus,Kallistatin protein has antioxidant activity in vitro,and it is dose-dependent in different cells under different stress conditions.Furthermore,in vitro anticoagulation and thrombolysis experiments showed that this protein has related activity.
To investigate the anti-oxidative activity and anticoagulant and thrombolytic activity of Kallistatin in three kinds of cell models in vitro,H2 O2 and Fe-HQ oxidative stress models of HBZY-1,HaCaT and HTR8-s/Vneo cells were constructed in vitro,and the Kallistatin protein was used in these oxidative stress models.Multi-concentration gradient method was adopt to select the optimal oxidant concentration.CCK experiment was used to detect the activity of three kinds of cells,and the anticoagulant and thrombolytic experiments were carried out in vitro.The results shows that in the H2 O2 model,the viability of three kinds of cell was similar to the control group(LD-hanks buffer)when the concentration of Kallistatin protein was 0.6μmol·L-1.The cell viability of low-dose protein group(0.06μmol·L-1)was significantly lower than that of the high dose.In the Fe-HQ model,the effect of Kallistatin protein doses differed in different cells.Thus,Kallistatin protein has antioxidant activity in vitro,and it is dose-dependent in different cells under different stress conditions.Furthermore,in vitro anticoagulation and thrombolysis experiments showed that this protein has related activity.
引文
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[16]MIAO R Q,AGATA J,CHAO L,et al.Kallistatin is a new inhibitor of angiogenesis and tumor growth[J].Blood,2002,100(9):3245-3252.DOI:10.1182/blood-2002-01-0185.
[2]徐炉李.重组人Kallistatin蛋白的发酵罐制备技术及抗肝纤维化相关生物活性研究[D].厦门:华侨大学,2012.
[3]刘晔,齐荔红,王硕丰,等.肝星状细胞HSC-T6体外肝纤维化模型的建立[J].药学实践杂志,2005,23(6):339-342.DOI:10.3969/j.issn.1006-0111.2005.06.006.
[4]DIAO Yong,ZHAO Xiaofeng,LIN Junsheng,et al.Protection of the liver against CCl-4-induced injury by intramuscular electrotransfer of a kallistatin-encoding plasmid[J].World Journal of Gastroenterology,2011,17(1):111-117.DOI:10.3748/wjg.v17.i1.111.
[5]郑晨娜,段训威,贾东方,等.重组人组织激肽释放酶结合蛋白对小鼠溃疡性结肠炎的抗炎作用[J].药学学报,2016,51(7):1077-1082.DOI:10.16438/j.0513-4870.2015-1167.
[6]MOUAWAD J,SAADEH F,TABOSH H,et al.The photoprotective effects of 2-benzoyl-3-phenylquinoxaline 1,4-dioxide against UVB-induced damage in HaCaT cells[J].Medical Oncology,2016,33(8):21-23.DOI:10.1007/s12032-016-0802-4.
[7]ZHANG Ting,ZHAO Chun,LUO Liang,et al.High concentraction of taurocholic acid induced apoptosis in HTR-8/SVneo cells via overexpression of ERp29and activation of p38[J].Placenta,2014,35(7):496-500.DOI:10.1016/j.placenta.2014.03.023.
[8]CHAO Julie,TILLMAN D M,WANG Maoyin,et al.Identification of a new tissue-kallikrein-binding protein[J].Biochem J,1986,239(2):325-331.DOI:10.1042/bj2390325.
[9]CAO Sheng,YAQOOB U,DAS A,et al.Neuropilin-1promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-beta signaling in hepatic stellate cells[J].Journal of Clinical Investigation,2010,120(7):2379.DOI:10.1172/JCI41203.
[10]SHIAU A L,TEO M L,CHEN S Y,et al.Inhibition of experimental lung metastasis by systemic lentiviral delivery of Lallistatin[J].BMC Cancer,2010,10:245-253.DOI:10.1186/1471-2407-10-245.
[11]BERRES M L,KOENEN R R,RUELAND A,et al.Antagonism of the chemokine Ccl5ameliorates experimental liver fibrosis in mice[J].Journal of Clinical Investigation,2010,120(11):4129-4140.DOI:10.1172/JCI41732.
[12]TSE D,SUN Xueying,JIANG Hongchi,et al.Adeno-associated virus-mediated expression of kallistatin suppresses local and remote hepatocellular carcinomas[J].Journal of Gene Medicine,2008,10(5):508-517.DOI:10.1002/jgm.1180.
[13]王希春.固态发酵高溶栓活性豆豉及其抗氧化特性的研究[D].无锡:江南大学,2008.
[14]初金鑫,蔡文娣,韩宝芹,等.单环刺螠纤溶酶UFE-1的性质和溶栓活性[J].天然产物研究与开发,2010,4(22):661-664.DOI:10.3969/j.issn.1001-6880.2010.04.028.
[15]SHEN Bo,GAO Lin,HSU Y,et al.Kallistatin attenuates endothelial apotosis through inhibition of oxidative stress and activation of Akt-eNOS signaling[J].Am J Physiol Heart Circ physiol,2010,299(5):1419-1427.DOI:10.1152/ajpheart.00591.2010.
[16]MIAO R Q,AGATA J,CHAO L,et al.Kallistatin is a new inhibitor of angiogenesis and tumor growth[J].Blood,2002,100(9):3245-3252.DOI:10.1182/blood-2002-01-0185.