美洲大蠊提取物诱导血管内皮细胞迁移的初步研究
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摘要
血管内皮细胞迁移在多种生理、病理现象中扮演着至关重要的角色。血管内皮细胞迁移有助于维持血管壁内皮的完整性和新血管的生长:在伤口愈合过程中,增殖的血管内皮细胞首先迁移到受损血管处,再以出芽或非出芽的方式形成新的血管;在组织修复的初始阶段,血管内皮细胞迁移到新生的肉芽组织中形成初始状态的毛细血管为新生的肉芽组织提供营养;另外,血管内皮细胞的迁移也是肿瘤的必要条件。研究能够诱导血管内皮迁移的中药对科研和临床有重要的意义,美洲大蠊提取液在临床中被广泛运用于皮肤创伤和胃溃疡治疗,且疗效明确。有大量的研究分别从促进胞基质表达,调节细胞因子分泌量,提高中性粒细胞数量,提高巨噬细胞吞噬能力及促使体内SOD值回升等角度解释美洲大蠊提取物促进伤口愈合的原因。但少有美洲大蠊提取物对血管内皮细胞作用的研究报道。因此,本研究通过传统的细胞迁移实验方法结合微流控芯片技术,对美洲大蠊提取液诱导血管内皮细胞迁移进行初步研究。
对美洲大蠊虫体进行有效成分的提取;使用传统的细胞划痕法、Transwell小室法和琼脂糖平板法对不同浓度条件下的美洲大蠊提取液诱导血管内皮细胞迁移进行研究;运用插管抽丝法制作一款能够在较短时间内产生稳定浓度梯度的琼脂糖微流控装置,并借助该装置检测美洲大蠊提取液诱导血管内皮细胞迁移。
运用划痕法和Transwell小室法初步证实美洲大蠊提取物能诱导血管内皮细胞迁移:其中5%(体积比)的美洲大蠊提取物对血管内皮的迁移没有发生明显的迁移作用,在浓度为5-30%范围内,血管内皮细胞的迁移呈在线性关系。利用琼脂糖凝胶为基质的微流控装置中以20%浓度的美洲大蠊提取物为诱导剂诱导6h后,将细胞培养去等分为4个区,其中1区和2区分别有大约7%和3%的细胞减少,而3区和4区分别有大约9%和2%的细胞数目增加。
初步证明美洲大蠊提取物能够诱导血管内皮细胞迁移;同时插管抽丝法制作的琼脂糖微流控装置能够产生稳定的浓度梯度,可以开发成用于检测细胞迁移的装置。
Endothelial cell migration play improtant role in many physiological and pathologic process.The integrity of vessel endothelium and angiogenesis depend on endothelial cell migration:in the process of wound healing,neovascularization by budding or other way is the consquence of the proliferous EC migrate to the breakage of vessel;in the elementary step of tissue repair,the capillary network in original state supplying granulation tissue with nutrientsubstance is the consquence of the proliferous EC migrate to anagenetic granulation tissue;additionally,the migration of EC is prerequisite for tumorigeness. Research on what chinese medicine induce EC migrate will be make contribution to clinic. The extraction from periplaneta americana were ofen used for treatment of wound healling and gastric ulcer effectively.There are a great deal of research on mechanism of periplaneta americana for wound healling,such as it adjust the secretory volume of EC,incerase the quantity of neutrophilic granulocyte, advance the endocytosis-ability of macrophage or promote the SOD level rally. But rerely research on what will happen to extraction of periplaneta americana act on EC. Therefore, in our reserch get help from tradional laboratory facilities and micro-fluidic chip to prove the chemotaxis of endothelial cells in response to the extraction from periplaneta americana.
The solution is extract from periplaneta americana then using the traditional methods of scratch assay , transwell chamber and agarose plate test the extraction of periplaneta americana in various consistency induce EC migrate. A microfluidic device which using withdrawing thread form agarose hydrogel was designed and fabricated to generate stable concentration gradients immediately . Using this device monitored the chemotactic response of endothelial cell to the extraction of periplaneta americana.
The methods of scratch assay and transwell chamber approved conformably that extraction of periplaneta americana can induce EC migrate: the extraction of medium and periplaneta americana volume ratio at 5% were unable to induce human endothelial cells migration. In contrast, to the extent that volume ratio is 10% to 30%, the relation of extraction concentration and the quantity of EC migration is linear dependence. There are respectively 7% and 3% of human endothelial cells were observed to migrate towards higher concentrations when the Medium and Periplaneta americana volume ratio was 20%.
对美洲大蠊虫体进行有效成分的提取;使用传统的细胞划痕法、Transwell小室法和琼脂糖平板法对不同浓度条件下的美洲大蠊提取液诱导血管内皮细胞迁移进行研究;运用插管抽丝法制作一款能够在较短时间内产生稳定浓度梯度的琼脂糖微流控装置,并借助该装置检测美洲大蠊提取液诱导血管内皮细胞迁移。
运用划痕法和Transwell小室法初步证实美洲大蠊提取物能诱导血管内皮细胞迁移:其中5%(体积比)的美洲大蠊提取物对血管内皮的迁移没有发生明显的迁移作用,在浓度为5-30%范围内,血管内皮细胞的迁移呈在线性关系。利用琼脂糖凝胶为基质的微流控装置中以20%浓度的美洲大蠊提取物为诱导剂诱导6h后,将细胞培养去等分为4个区,其中1区和2区分别有大约7%和3%的细胞减少,而3区和4区分别有大约9%和2%的细胞数目增加。
初步证明美洲大蠊提取物能够诱导血管内皮细胞迁移;同时插管抽丝法制作的琼脂糖微流控装置能够产生稳定的浓度梯度,可以开发成用于检测细胞迁移的装置。
Endothelial cell migration play improtant role in many physiological and pathologic process.The integrity of vessel endothelium and angiogenesis depend on endothelial cell migration:in the process of wound healing,neovascularization by budding or other way is the consquence of the proliferous EC migrate to the breakage of vessel;in the elementary step of tissue repair,the capillary network in original state supplying granulation tissue with nutrientsubstance is the consquence of the proliferous EC migrate to anagenetic granulation tissue;additionally,the migration of EC is prerequisite for tumorigeness. Research on what chinese medicine induce EC migrate will be make contribution to clinic. The extraction from periplaneta americana were ofen used for treatment of wound healling and gastric ulcer effectively.There are a great deal of research on mechanism of periplaneta americana for wound healling,such as it adjust the secretory volume of EC,incerase the quantity of neutrophilic granulocyte, advance the endocytosis-ability of macrophage or promote the SOD level rally. But rerely research on what will happen to extraction of periplaneta americana act on EC. Therefore, in our reserch get help from tradional laboratory facilities and micro-fluidic chip to prove the chemotaxis of endothelial cells in response to the extraction from periplaneta americana.
The solution is extract from periplaneta americana then using the traditional methods of scratch assay , transwell chamber and agarose plate test the extraction of periplaneta americana in various consistency induce EC migrate. A microfluidic device which using withdrawing thread form agarose hydrogel was designed and fabricated to generate stable concentration gradients immediately . Using this device monitored the chemotactic response of endothelial cell to the extraction of periplaneta americana.
The methods of scratch assay and transwell chamber approved conformably that extraction of periplaneta americana can induce EC migrate: the extraction of medium and periplaneta americana volume ratio at 5% were unable to induce human endothelial cells migration. In contrast, to the extent that volume ratio is 10% to 30%, the relation of extraction concentration and the quantity of EC migration is linear dependence. There are respectively 7% and 3% of human endothelial cells were observed to migrate towards higher concentrations when the Medium and Periplaneta americana volume ratio was 20%.
引文
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[2] Pepper MS.Manipulating angiogenesis from basic science to the bed side[J]. Arterioscler Thromb Vascbiol. 1997,17(4):605-619.
[3]付小兵,王德文.创伤修复基础[M].北京:人民军医出版社, 1997:129 - 204.
[4]王正国.创伤愈合与组织修复[M].济南:山东科学技术出版社,1998: 3 - 15.
[5] Petite H,Viateau V,Bensaid W,et al.Tissue-engineered bone regeneration.Nat Biotech 2000;18(9):959-63.
[6]任晋生,罗兴洪.浅谈中药的开发研究[J].山西中医学院学报,2006 Vol.7 No.2:40-41.
[7]盘莉,潜力巨大的动物类中药开发,中国处方药[J] 2010.01 No.94:24-26
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[10]高英兰乙醇对大鼠血管内皮细胞的促凋亡作用岭南心血管病杂志2006年12月第12卷第6期431-434.
[11] Thomas M. Biomolecular gradients in cell culture systems. Lab Chip, 2008, 8, 34–57
[12] Campell SC. Advances in angiogenesis research:relevance to urological Oncology[J]. J Urol,1997,158(5):1663-1674.
[13] R. A. Firtel and C. Y. Chung, The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients,Bioessays, 2000, 22(7), 603–615.
[14] Jonathan W. Song Microfluidic Endothelium for Studying the Intravascular Adhesion of Metastatic Breast Cancer Cells, PLoS ONE | www.plosone.org, 2009 ,6, Volume 4,Issue 6,e5756.
[15] Shing-Yi Cheng, A hydrogel-based microfluidic device for the studies of directed cell migration(J)lab on chip, 2007, 7:763–769.
[16] Mohan K. S. Verma. Embedded Template-Assisted Fabrication of Complex Microchannels in PDMS and Design of a Microfluidic Adhesive. Langmuir 2006, 22, 10291-10295.
[17]贾月飞蒋稼欢基于微丝的PDMS微流动通道制作技术科学通报[J]2008年第53卷第17期: 2116-2124.
[18] Bouis D ,Hospers GA ,Meijer C , et al1 Endothelium in vitro :a review of human vascular endothelial cell lines for blood vessele related research[J].1 A ngiogenesis , 2001 ,4(2) :9121021.
[19]屈纪富,程天民.参与皮肤伤口愈合的细胞及其作用的研究进展.现代康复2001年12月第5卷第12期[J] :74-75.
[20] Wang Di,Gotlieb Ai. Fibroblast growth factor 2 enhances early stages of in vitro endothelial repair by microfilament bundle reorganization and cell elongation[J].Exp MolPathol, 1999,66(3):179-190.
[21]黄华王贵学血管内皮细胞迁移及相关影响因素研究进展国际生物医学工程杂志2006年12月第29卷第6期357-361.
[22]段小军,杨柳促进组织工程化骨血管化的研究进展与策略.中国临床康复第7卷第26期.3628-3629.
[23] Elcin YM,Dixit V,Gitnick G.Extensive in vivo angiogenesis following controlled release of human vascular endothelical cell growth factor:implications for tissue engineering and wound healing.Artif Organs 2001;25(7):558-65.
[24] Yancopoulos GD.Davis S.Gale NW.et al.Vascular specific growth factors and blood vessel formation[J]. Nature.2000,407(6801):242-248
[25] N ish io E, Fukush ima K, Sh iozok iM , et al. N it ric ox2 idedono r SNA P induces apop to sis in smoo th muscle cells th rough cGM P2independent mechanism. B iochem B iophys Res Commun, 1996; 221 (1)∶163
[26] Yano Y,Seishima M,Tokoro Y。et a1.Stimulatory efects oflipo protein(a)and low·density lipoprotein on human umbilical vein en·dothelial cellmi grationan dproliferation arepartiallymediatedby6·broblast growth factor-2[J].Biochim Biophys Acta(BBA)/Lipids Lipid Metabol,1998,1393(1):26-34.
[27] Okamoto H,Yatomi Y,Ohmofi T,et a1.Sphingosine 1-pho6phate stimulates Gi·an d Bho-mediated vaacul~ endothelial cell spreading and migration[J].Thromtxrsis Res,2000~99(3):259-265.
[28] Dimmeler S。Dembach E,Zeiher AM. Phosphorylation of the endothelial nitric oxide synthase at Ser-l177 is required for VEGF-induced endothelial cell migration[J].FEBS Letters,2000,477(3):258-262.
[29] Morbideli L’Brogeli L,GrangerHJ,eta1.Endothelial cellmigrationis induced by soluble P--selecdn[J].Life Sci Inehd Phannacol Letters,1997,62(1):PL7-PL1 l_
[30] Lau YT.Ma WC.Nitric oxide inhibits mi gration ofcultured endothelial[J].Cells Biochem Biophys Res Commun,1996,221(3):670-674.
[31] Yamaguebi N。Anan d-ALpte B, Lee M, et a1. Endcetatin inhibits VEGF-induced endothelial cell migration an d tumor growth indepondenfly offine binding[J].EMBOJ。1999。18(16):4414—23.
[32] Lo CM et al. Biophys J, 2000, 79 : 144
[33] Schaper W,Adili F,Balzer J,et al. Venous shunting of collateral flow after femoral artery occlusion markedly increases fluid shear stress and collateral vessel growth in a chronic pig model. J Vasc Res,2002,39:62.34 Hu YL’Li S,Miao H,et a1.Roles of mieretubule dynamics and small gtpase rac in endothelial cel migration and lamelipodium formation underflow[J].J Vase Res,2002,39:465-476.
[35]李勇康复新液治疗胃溃疡的作用机制中国药物与临床2008年6月第8卷第6495-496.
[36] Ulrike Haessler, An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies, (J)Biomed Microdevices 2009, 11:827–835
[37] Amir Shamloo ,Endothelial cell polarization and chemotaxis in a micro?uidic device, (J)lab on chip 2008, 8:1292–1299。
[38] A. Hatch, et al., A rapid diffusion immunoassay in a T-sensor,Nat. Biotechnol., 2001, 19(5), 461–465.
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