丹参凝集素促进血管平滑肌细胞的增殖机制研究
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摘要
目的探究丹参凝集素蛋白(SMLP)对大鼠血管平滑肌细胞(VSMCs)增殖的影响机制。方法通过蛋白分离纯化技术从中药丹参中分离得到具有活性的凝集素蛋白,噻唑蓝(MTT)法检测SMLP对大鼠VSMCs的增殖作用,蛋白质印迹法(Western blot)检测相关增殖基因的表达水平。构建pet-28a-sml原核表达载体,利用蛋白质亲和层析等技术纯化体外表达的SMLP,并通过红细胞凝集实验确认纯化的SMLP的活性。结果丹参中分离的SMLP能够促进大鼠VSMCs增殖,增殖相关基因蛋白激酶B(AKT)基因与哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化水平明显增加,而凋亡相关基因JNK1和Bax表达水平明显降低。成功构建了pet-28a-smlp原核表达载体,并在体外表达纯化了SMLP;红细胞凝集实验表明,体外表达纯化的SMLP具有活性。结论 SMLP能够促进大鼠VSMCs的增殖;成功建立SMLP的体外表达纯化方法,为进一步研究其功能奠定了基础。
Objective To explore the mechanism of the effect of Salvia miltiorrhiza lectin(SMLP)on the proliferation of vascular smooth muscle cells(VSMCs)in rats.Methods The SMLP was isolated from Salvia miltiorrhiza by protein separation and purification technology.The MTT assay was performed to detect the proliferation of VSMCs in rats after SMLP treatment.The expression levels of proliferation-related genes were detected by Western blot.The prokaryotic expression vector of pet-28 a-sml was constructed,and SMLP was purified by affinity chromatography.The activity of purified SMLP was tested by RBC agglutination experiment.Results The SMLP isolated from Salvia miltiorrhiza could promote the proliferation of VSMCs in rats,and the expression levels of proliferation-related genes(p-AKT and p-mTOR)were significantly increased,while the expression levels of apoptosis-related genes(JNK1 and Bax)were decreased obviously.The prokaryotic expression vector pet-28 a-sml was successfully constructed,and the SMLP was purified in vitro.The RBC agglutination experiment showed that the purified SMLP protein was functional in vitro.Conclusion SMLP can promote the proliferation of VSMCs in rats.The expression and purification method of SMLP in vitro was established,which lays a foundation for further study on the function of the protein.
Objective To explore the mechanism of the effect of Salvia miltiorrhiza lectin(SMLP)on the proliferation of vascular smooth muscle cells(VSMCs)in rats.Methods The SMLP was isolated from Salvia miltiorrhiza by protein separation and purification technology.The MTT assay was performed to detect the proliferation of VSMCs in rats after SMLP treatment.The expression levels of proliferation-related genes were detected by Western blot.The prokaryotic expression vector of pet-28 a-sml was constructed,and SMLP was purified by affinity chromatography.The activity of purified SMLP was tested by RBC agglutination experiment.Results The SMLP isolated from Salvia miltiorrhiza could promote the proliferation of VSMCs in rats,and the expression levels of proliferation-related genes(p-AKT and p-mTOR)were significantly increased,while the expression levels of apoptosis-related genes(JNK1 and Bax)were decreased obviously.The prokaryotic expression vector pet-28 a-sml was successfully constructed,and the SMLP was purified in vitro.The RBC agglutination experiment showed that the purified SMLP protein was functional in vitro.Conclusion SMLP can promote the proliferation of VSMCs in rats.The expression and purification method of SMLP in vitro was established,which lays a foundation for further study on the function of the protein.
引文
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[14]YU S,CHEN Y,CHEN S,et al.Klotho inhibits proliferation and migration of angiotensinⅡ-induced vascular smooth musclecells(VSMCs)by modulating NF-κB p65,Akt,and extracellular signal regulated kinase(ERK)signaling activities[J].Med Sci Monit,2018,24:4851-4860.
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[16]阎卉芳,徐昊,彭熙炜,等.黄芪和当归配伍对大鼠血管内膜增生血管平滑肌细胞增殖的影响[J].中国临床药理学与治疗学,2018,23(4):361-369.
[17]张东雪,张胜雷,张璐,等.SET8-shRNA抑制血管平滑肌细胞增殖和迁移[J].中国动脉硬化杂志,2018,26(2):152-156.
[18]常遵辉,朱芩,陈璟,等.高糖促进大鼠血管平滑肌细胞A7r5增殖及相关基因表达[J].宁夏医科大学学报,2016,38(2):154-158.
[19]王鼎,宋兵,钟旋,等.植物凝集素刺激外周血单个核细胞增殖及分泌因子表达的变化[J].中国组织工程研究,2014,18(23):3707-3714.
[20]谈浩琪,张楚楚,屈雪,等.植物凝集素在生物医用领域中的应用[J].高分子通报,2016,29(5):91-102.
[2]温进坤,韩梅.血管平滑肌细胞[M].北京:科学出版社,2005:6-8.
[3]罗寅,马占龙.血管平滑肌细胞在动脉粥样硬化病变中作用的研究进展[J].陕西医学杂志,2012,41(3):360-361.
[4]张文通,李俊,吴玉婷,等.山柰酚-3-O-芸香糖苷对血管平滑肌细胞增殖,迁移及TGFBR1信号通路活化的影响[J].中国病理生理杂志,2018,34(5):832-838.
[5]王冰瑶,吴晓燕,樊官伟.丹参素保护心血管系统的药理作用机制研究进展[J].中草药,2014,45(17):2571-2575.
[6]李向军,范文成,李奉勤,等.丹参有效成分提取的研究[J].中国现代中药,2011,13(5):33-35.
[7]LI X,DU J R,YU Y,et al.TanshinoneⅡA inhibits smooth muscle proliferation and intimal hyperplasia in the rat carotid balloon-injured model through inhibition of MAPK signaling pathway[J].J Ethnopharmacol,2010,129(2):273-279.
[8]HO J H,HONG C Y.Salvianolic acids:small compounds with multiple mechanisms for cardiovascular protection[J].J Biomed Sci,2011,18:30.
[9]李春红,陈岑,夏之宁,等.植物源活性蛋白的药理作用及分析方法研究进展[J].中国中药杂志,2015,40(13):2508-2517.
[10]杨瑾,杨照华.中草药治疗心血管疾病进展研究[J].内蒙古中医药,2013,34(51):120-121.
[11]赵志芳.丹参对心血管疾病治疗的研究进展[J].海峡药学,2016,28(5):41-44.
[12]STABILE E,ZHOU Y F,SAJI M,et al.Akt controls vascular smooth muscle cell proliferation in vitro and in vivo by delaying G1/S exit[J].Circ Res,2003,93(11):1059-1065.
[13]WANG A B,LI H L,ZHANG R,et al.A20attenuates vascular smooth muscle cell proliferation and migration through blocking PI3k/Akt singling in vitro and in vivo[J].J Biomed Sci,2007,14:357-371.
[14]YU S,CHEN Y,CHEN S,et al.Klotho inhibits proliferation and migration of angiotensinⅡ-induced vascular smooth musclecells(VSMCs)by modulating NF-κB p65,Akt,and extracellular signal regulated kinase(ERK)signaling activities[J].Med Sci Monit,2018,24:4851-4860.
[15]FANG H,YANG S,LUO Y,et al.Notoginsenoside R1inhibits vascular smooth muscle cell proliferation,migration and neointimal hyperplasia through PI3K/Akt signaling[J].Sci Rep,2018,8(1):7595.
[16]阎卉芳,徐昊,彭熙炜,等.黄芪和当归配伍对大鼠血管内膜增生血管平滑肌细胞增殖的影响[J].中国临床药理学与治疗学,2018,23(4):361-369.
[17]张东雪,张胜雷,张璐,等.SET8-shRNA抑制血管平滑肌细胞增殖和迁移[J].中国动脉硬化杂志,2018,26(2):152-156.
[18]常遵辉,朱芩,陈璟,等.高糖促进大鼠血管平滑肌细胞A7r5增殖及相关基因表达[J].宁夏医科大学学报,2016,38(2):154-158.
[19]王鼎,宋兵,钟旋,等.植物凝集素刺激外周血单个核细胞增殖及分泌因子表达的变化[J].中国组织工程研究,2014,18(23):3707-3714.
[20]谈浩琪,张楚楚,屈雪,等.植物凝集素在生物医用领域中的应用[J].高分子通报,2016,29(5):91-102.