低氧环境下黄芪甲苷促进人骨髓间充质干细胞分泌血管内皮细胞相关因子的实验研究
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摘要
目的观察在低氧环境下,黄芪甲苷能否诱导人骨髓间充质干细胞(BMSCs)向血管内皮样细胞分化,提高BMSCs经血管内皮生长因子(VEGF)诱导分化后的血管内皮样细胞的功能。方法将人BMSCs分为4组:对照组(单纯人BMSCs培养)、VEGF组(含VEGF载体的腺病毒的完全培养基加入人BMSCs)、黄芪甲苷组(含黄芪甲苷粉剂加入人BMSCs)和联合诱导组(含黄芪甲苷粉剂和VEGF载体的腺病毒的完全培养基加入人BMSCs),5%O2干预2周,对分化的细胞进行形态学观察,流式细胞仪检测细胞表型CD31和CD105的表达,ELISA检测细胞培养上清液中VEGF及内皮型一氧化氮合酶(eNOS)因子的含量,Real-time PCR和Western blot检测内皮素(ET)和前列环素(PGI)的表达。结果 (1)2周后,镜下观察对照组与黄芪甲苷组细胞形态无明显变化,VEGF组和联合诱导组细胞似铺路石子样生长。(2)流式结果显示,对照组与黄芪甲苷组细胞不表达CD31,高表达CD105;VEGF组和联合诱导组细胞高表达CD31,低表达CD105,且VEGF组CD31表达高于联合诱导组(P<0.05),而CD105低于联合诱导组(P<0.05)。(3)ELISA检测发现,与对照组比较,各干预组的VEGF及eNOS因子表达均增高(均P<0.05),且大小顺序为VEGF组>联合诱导组>黄芪甲苷组。(4)RT-PCR及Western blot检测发现,与对照组比较,各干预组ET水平均升高(P<0.05),PGI表达量均降低(P<0.05);且联合诱导组ET蛋白表达水平最高,VEGF组PGI表达量最低。结论在低氧环境下,黄芪甲苷促进人BMSCs分泌VEGF相关因子;黄芪甲苷联合VEGF可以促进VEGF诱导分化后的血管内皮样细胞分泌ET。
Objective To observe whether astragaloside could induce human bone marrow mesenchymal stem cells( BMSCs) to differentiate into vascular endothelial cells( VECs),or improve the function of VECs under hypoxia condition.Methods The third passage BMSCs were divided into four groups,i. e.,the blank control group( cultured with conventional culture medium),the VEGF induction group [cultured with adenovirus vector containing vascular endothelial growth factor( VEGF) ],the Astragaloside group( cultured with astragaloside),and the combined group( cultured with adenovirus vector containing VEGF and astragaloside). After intervened by 5% O2 for 2 weeks,morphologies of differentiated cells were observed. The expressions of cell phenotype CD31 and CD105 were detected by flow cytometry. The levels of VEGF and endothelial nitric oxide synthase( eNOS) in supernate were detected by ELISA. The expressions of endothelin( ET) and prostacy-clin( PGI) were detected by Real-time PCR( RT-PCR) and Western blot. Results( 1) No obvious changes occurred in the cell morphology in the blank control group and the Astragaloside group after two weeks. Cells in the VEGF group and the combined group exhibited a colony-like growth.( 2) Results of flow cytometry showed that CD31 was negatively expressed in the blank control group and the Astragaloside group,but the expression of CD105 was high. However,the expression of CD31 increased,and the expression of CD105 decreased in the VEGF group and the combined group. The positive rate of CD31 was significantly higher in the VEGF group than in the combined group(P < 0. 05),and the positive rate of CD105 expression was lower in the VEGF group than in the combined group(P < 0. 05).( 3) ELISA showed,as compared with the blank control group,the expressions of VEGF and eNOS increased significantly in all the intervention groups(P < 0. 05),ranked as the VEGF group > the combined group > the Astragaloside group.( 4) RT-PCR and Western blot showed,as compared with the blank control group,ET level increased(P < 0. 05) and PGI decreased(P < 0. 05) in all the intervention groups. Further more,the expression of ET was the highest in the combined group,and the expression of PGI was the lowest in the VEGF group. Conclusions Astragaloside promoted BMSCs to secrete VEGF related factors under hypoxia condition.Astragaloside combined VEGF could promote the secretion of ET by VECs after VEGF induced differentiation.
Objective To observe whether astragaloside could induce human bone marrow mesenchymal stem cells( BMSCs) to differentiate into vascular endothelial cells( VECs),or improve the function of VECs under hypoxia condition.Methods The third passage BMSCs were divided into four groups,i. e.,the blank control group( cultured with conventional culture medium),the VEGF induction group [cultured with adenovirus vector containing vascular endothelial growth factor( VEGF) ],the Astragaloside group( cultured with astragaloside),and the combined group( cultured with adenovirus vector containing VEGF and astragaloside). After intervened by 5% O2 for 2 weeks,morphologies of differentiated cells were observed. The expressions of cell phenotype CD31 and CD105 were detected by flow cytometry. The levels of VEGF and endothelial nitric oxide synthase( eNOS) in supernate were detected by ELISA. The expressions of endothelin( ET) and prostacy-clin( PGI) were detected by Real-time PCR( RT-PCR) and Western blot. Results( 1) No obvious changes occurred in the cell morphology in the blank control group and the Astragaloside group after two weeks. Cells in the VEGF group and the combined group exhibited a colony-like growth.( 2) Results of flow cytometry showed that CD31 was negatively expressed in the blank control group and the Astragaloside group,but the expression of CD105 was high. However,the expression of CD31 increased,and the expression of CD105 decreased in the VEGF group and the combined group. The positive rate of CD31 was significantly higher in the VEGF group than in the combined group(P < 0. 05),and the positive rate of CD105 expression was lower in the VEGF group than in the combined group(P < 0. 05).( 3) ELISA showed,as compared with the blank control group,the expressions of VEGF and eNOS increased significantly in all the intervention groups(P < 0. 05),ranked as the VEGF group > the combined group > the Astragaloside group.( 4) RT-PCR and Western blot showed,as compared with the blank control group,ET level increased(P < 0. 05) and PGI decreased(P < 0. 05) in all the intervention groups. Further more,the expression of ET was the highest in the combined group,and the expression of PGI was the lowest in the VEGF group. Conclusions Astragaloside promoted BMSCs to secrete VEGF related factors under hypoxia condition.Astragaloside combined VEGF could promote the secretion of ET by VECs after VEGF induced differentiation.
引文
[1]贾立山,周云,张亚,等.体外诱导大鼠骨髓间充质干细胞向血管内皮细胞的分化[J].中国组织工程研究与临床康复,2009,13(19):3698-3702.
[2]陈毅,刘丹平.三种细胞因子体外联合诱导兔骨髓间充质干细胞向血管内皮细胞的分化[J].中国组织工程研究与临床康复,2008,12(21):4093-4096.
[3]胡继宏,贾佳,路娟,等.低氧下血管内皮细胞生长因子转染人骨髓间充质干细胞向血管内皮样细胞分化[J].中国组织工程研究,2017,21(9):1352-1356.
[4]刘永琦,李静雅,蔡玲,等.黄芪多糖诱导大鼠骨髓间充质干细胞分化的特性[J].中国中医药信息杂志,2014,21(6):60-64.
[5]冼邵祥,杨忠奇,汪朝晖,等.黄芪甲苷体外诱导骨髓间充质干细胞分化为心肌样细胞的实验研究[J].广州中医药大学学报,2007,24(1):37-40.
[6]刘琳,白海,王存邦,等.黄芪多糖对多发性骨髓瘤患者骨髓间充质干细胞增殖与细胞因子表达的影响[J].现代生物医学进展,2013,13(10):1873-1877.
[7]胡继宏,路娟,赵翊,等.黄芪主要活性成分及其提取物对鼠骨髓间充质干细胞增殖作用的影响[J].时珍国医国药,2016,27(5):1070-1072.
[8]彭小娟,白海,王存邦,等.黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响[J].现代生物学进展,2014,14(8):1452-1455.
[9]刘丹丹.人骨髓间充质干细胞向内皮细胞体外诱导分化的实验研究[D].长春:吉林大学,2004.
[10]Arutyunyan I,Fatkhudinov T,Kananykhina E,et al.Role of VEGF-A in angiogenesis promoted by umbilical cord-de-rived mesenchymal stromal/stem cells:in vitro study[J].Stem Cell Res Ther,2016,28(7):46-59.
[11]杜晓,陈小平,张开明,等.人骨髓间充质干细胞体外向血管内皮细胞诱导分化的实验研究[J].中西医结合心脑血管病杂志,2009,7(8):940-941.
[12]孔根现.VEGF分别与b FGF、a FGF联合诱导骨髓间充质干细胞分化为血管内皮细胞[D].广州:南方医科大学,2013.
[13]马俊伟.骨髓间充质干细胞向血管内皮细胞诱导分化的实验研究[D].沈阳:中国医科大学,2009.
[14]Yang LX,Xu ZY,Zhang BR,et al.In vitro induction of bone marrow stromal stem cells to differentiate into endothelial cells[J].Acad J Sec Milit Med Univ,2003,24(12):1300-1304.
[15]沈凌.黄芪对低氧环境下血管内皮细胞生长因子/基质细胞衍生因子-1诱导人骨髓间充质干细胞分化、迁移的干预作用的研究[D].北京:北京中医药大学,2010.
[16]Bandara N,Gurusinghe S,Chen H,et al.Minicircle DNAmediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells[J].Stem Cell Res Ther,2016,29(7):48-62.
[17]赵保明,李凤良,宋喜明,等.谷胱甘肽对糖基化终末产物环境中人脐静脉内皮细胞前列环素和内皮素-1分泌的影响[J].郑州大学学报(医学版),2011,46(4):599-601.
[18]Ikhapoh IA,Pelham CJ,Agrawal DK,et al.Synergistic effect of angiotensinⅡon vascular endothelial growth factor-A-mediated differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells[J].Stem Cell Res Ther,2015,26(6):4-16.
[19]Groop PH,Forsblom C,Thomas MC.Mechanisms of disease:Pathway-selective insulin resistance and microvascular complications of diabetes[J].Nat Clin Pract Endocrinol Metab,2005,1(2):100-110.
[2]陈毅,刘丹平.三种细胞因子体外联合诱导兔骨髓间充质干细胞向血管内皮细胞的分化[J].中国组织工程研究与临床康复,2008,12(21):4093-4096.
[3]胡继宏,贾佳,路娟,等.低氧下血管内皮细胞生长因子转染人骨髓间充质干细胞向血管内皮样细胞分化[J].中国组织工程研究,2017,21(9):1352-1356.
[4]刘永琦,李静雅,蔡玲,等.黄芪多糖诱导大鼠骨髓间充质干细胞分化的特性[J].中国中医药信息杂志,2014,21(6):60-64.
[5]冼邵祥,杨忠奇,汪朝晖,等.黄芪甲苷体外诱导骨髓间充质干细胞分化为心肌样细胞的实验研究[J].广州中医药大学学报,2007,24(1):37-40.
[6]刘琳,白海,王存邦,等.黄芪多糖对多发性骨髓瘤患者骨髓间充质干细胞增殖与细胞因子表达的影响[J].现代生物医学进展,2013,13(10):1873-1877.
[7]胡继宏,路娟,赵翊,等.黄芪主要活性成分及其提取物对鼠骨髓间充质干细胞增殖作用的影响[J].时珍国医国药,2016,27(5):1070-1072.
[8]彭小娟,白海,王存邦,等.黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响[J].现代生物学进展,2014,14(8):1452-1455.
[9]刘丹丹.人骨髓间充质干细胞向内皮细胞体外诱导分化的实验研究[D].长春:吉林大学,2004.
[10]Arutyunyan I,Fatkhudinov T,Kananykhina E,et al.Role of VEGF-A in angiogenesis promoted by umbilical cord-de-rived mesenchymal stromal/stem cells:in vitro study[J].Stem Cell Res Ther,2016,28(7):46-59.
[11]杜晓,陈小平,张开明,等.人骨髓间充质干细胞体外向血管内皮细胞诱导分化的实验研究[J].中西医结合心脑血管病杂志,2009,7(8):940-941.
[12]孔根现.VEGF分别与b FGF、a FGF联合诱导骨髓间充质干细胞分化为血管内皮细胞[D].广州:南方医科大学,2013.
[13]马俊伟.骨髓间充质干细胞向血管内皮细胞诱导分化的实验研究[D].沈阳:中国医科大学,2009.
[14]Yang LX,Xu ZY,Zhang BR,et al.In vitro induction of bone marrow stromal stem cells to differentiate into endothelial cells[J].Acad J Sec Milit Med Univ,2003,24(12):1300-1304.
[15]沈凌.黄芪对低氧环境下血管内皮细胞生长因子/基质细胞衍生因子-1诱导人骨髓间充质干细胞分化、迁移的干预作用的研究[D].北京:北京中医药大学,2010.
[16]Bandara N,Gurusinghe S,Chen H,et al.Minicircle DNAmediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells[J].Stem Cell Res Ther,2016,29(7):48-62.
[17]赵保明,李凤良,宋喜明,等.谷胱甘肽对糖基化终末产物环境中人脐静脉内皮细胞前列环素和内皮素-1分泌的影响[J].郑州大学学报(医学版),2011,46(4):599-601.
[18]Ikhapoh IA,Pelham CJ,Agrawal DK,et al.Synergistic effect of angiotensinⅡon vascular endothelial growth factor-A-mediated differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells[J].Stem Cell Res Ther,2015,26(6):4-16.
[19]Groop PH,Forsblom C,Thomas MC.Mechanisms of disease:Pathway-selective insulin resistance and microvascular complications of diabetes[J].Nat Clin Pract Endocrinol Metab,2005,1(2):100-110.