消渴丸对人血管内皮细胞生长和功能的影响
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摘要
背景
消渴丸成品药包括中药成分：黄芪、生地黄、天花粉、葛根、五味子、山药、玉米须；以及西药成分：格列本脲。二者之间有很好的定量关系。从西医的角度来看,消渴丸中定量的格列本脲有良好的降低血糖的作用,从而减轻了糖毒性作用,保护了胰岛功能；从中医角度上看,消渴丸具有滋阴补肾、益气生津等功效。既往研究表明,消渴丸对糖尿病治疗有较好的作用,对糖尿病各种并发症的预防及治疗也有一定意义。但到目前为止,有关消渴丸防治糖尿病及其并发症作用机制的研究还不够充分。为此,进一步对消渴丸的作用机制加以研究实属必要。血管内皮紊乱是糖尿病相关并发症发生和发展的关键一步,因此,选取血管内皮细胞作为研究对象来研究消渴丸对内皮细胞生长和功能的影响,可以为探讨消渴丸可能的保护机制提供方向。
目的
实验第一部分,摸索并总结合适的人脐静脉内皮细胞提取和培养方法,为接下来的药物实验提供研究对象。实验第二部分,通过体外细胞培养,采取直接添加药物的方法,观测消渴丸对人血管内皮细胞功能的影响,以及消渴丸中药成分对内皮细胞增殖的作用,以探讨消渴丸对人血管内皮细胞的保护作用及其可能的机制,为进一步研究消渴丸的药理作用作镝垫。
方法
原代培养获取人脐静脉内皮细胞,选取第3代细胞,接种于24孔板中,待细胞贴壁后,进行药物干预试验：分成3个浓度组,根据格列本脲浓度依次为2ng/ml、20ng/ml和200ng/ml,每个浓度组内再分为格列本脲组、消渴丸成品组、消渴丸纯中药成分组和空白对照组,每个组样本量为8。继续培养24h,显微镜下观察细胞生长状态,并吸取细胞培养液分别测定NO含量。消渴丸对内皮细胞增殖的影响试验：制备第3代人脐静脉内皮细胞悬液,分成四个组,每组8孔,1×105个细胞/孔。a空白对照组,b消渴丸中药成分粗提液低浓度组(2×10-5g/ml),c消渴丸中药成分粗提液中浓度组(4×10-5g/ml),d消渴丸中药成分粗提液高浓度组(2×10-4g/ml),培养1,3,5天后分别进行细胞计数。所得数据采用软件SPSS17.0进行分析,组与组之间均选择t检验。
结果
初步研究显示：三个浓度下,消渴丸成品组和消渴丸纯中药成分组NO含量相对于格列本脲组和空白对照组均明显升高(P<0.001),消渴丸成品组与消渴丸纯中药成分组相比,NO含量无明显差别(P>0.05),并且随着药物浓度的递增,NO含量也明显上升(P<0.05)。消渴丸对内皮细胞增殖的影响试验发现：与空白对照组相比较,高剂量的消渴丸中药成分粗提液对血管内皮细胞有抑制其生长的作用,且差异具有统计学意义(P<0.05)。
结论
消渴丸在体外细胞培养环境中,可以提高NO的含量,而且随着药物浓度的递增,NO浓度也随之增加,并且发现,这主要是来自消渴丸的中药成分的作用。这可能是消渴丸保护血管内皮的一种机制。另外,高剂量的消渴丸中药成分粗提液对血管内皮细胞有抑制其生长的作用,且差异具有统计学意义(P<0.05)。
Objective
To investigate the effect of a chinese triditional medicine, which is called "Xiaokewan", on the release of nitric oxide (NO) inculture endothelial cell from human umbilical vein。 And the effect of this medicine on the proliferation of human umbilical vein endothelial cells (HUVECs) was also studied。
Methods
HUVECs, were isolated by collagenase digestion。 The3th generation of cultured HUVECs were used with the cell count of1×105/ml in each core The experiment consisted of4groups:(A) glibenclamide group;(B) full medicine of "Xiaokewan" group;(C) medicine of "xiaokewan" without group;(D) control group; and three different concentrations for each group were compared。For the study of effect of "Xiaokewan" on the proliferation of HUVECs,4groups were assigned randomly:(a) control group;(b) low concentration group;(c) moderate concentration group;(d) high concentration group。At day1、day3and day5count the cells in each core。
Results
"Xiaokewan" promoted the secretion of NO, and this effect was mainly due to the medicine without glibenclamide。High concentration of "Xiaokewan" inhibited the proliferation of HUVECs。
Conclusions
For each concentration," Xiaokewan" can promote the secretion of NO of HUVECs in vitro and high concentration of "Xiaokewan" inhibit the proliferation of HUVECs。
消渴丸成品药包括中药成分：黄芪、生地黄、天花粉、葛根、五味子、山药、玉米须；以及西药成分：格列本脲。二者之间有很好的定量关系。从西医的角度来看,消渴丸中定量的格列本脲有良好的降低血糖的作用,从而减轻了糖毒性作用,保护了胰岛功能；从中医角度上看,消渴丸具有滋阴补肾、益气生津等功效。既往研究表明,消渴丸对糖尿病治疗有较好的作用,对糖尿病各种并发症的预防及治疗也有一定意义。但到目前为止,有关消渴丸防治糖尿病及其并发症作用机制的研究还不够充分。为此,进一步对消渴丸的作用机制加以研究实属必要。血管内皮紊乱是糖尿病相关并发症发生和发展的关键一步,因此,选取血管内皮细胞作为研究对象来研究消渴丸对内皮细胞生长和功能的影响,可以为探讨消渴丸可能的保护机制提供方向。
目的
实验第一部分,摸索并总结合适的人脐静脉内皮细胞提取和培养方法,为接下来的药物实验提供研究对象。实验第二部分,通过体外细胞培养,采取直接添加药物的方法,观测消渴丸对人血管内皮细胞功能的影响,以及消渴丸中药成分对内皮细胞增殖的作用,以探讨消渴丸对人血管内皮细胞的保护作用及其可能的机制,为进一步研究消渴丸的药理作用作镝垫。
方法
原代培养获取人脐静脉内皮细胞,选取第3代细胞,接种于24孔板中,待细胞贴壁后,进行药物干预试验：分成3个浓度组,根据格列本脲浓度依次为2ng/ml、20ng/ml和200ng/ml,每个浓度组内再分为格列本脲组、消渴丸成品组、消渴丸纯中药成分组和空白对照组,每个组样本量为8。继续培养24h,显微镜下观察细胞生长状态,并吸取细胞培养液分别测定NO含量。消渴丸对内皮细胞增殖的影响试验：制备第3代人脐静脉内皮细胞悬液,分成四个组,每组8孔,1×105个细胞/孔。a空白对照组,b消渴丸中药成分粗提液低浓度组(2×10-5g/ml),c消渴丸中药成分粗提液中浓度组(4×10-5g/ml),d消渴丸中药成分粗提液高浓度组(2×10-4g/ml),培养1,3,5天后分别进行细胞计数。所得数据采用软件SPSS17.0进行分析,组与组之间均选择t检验。
结果
初步研究显示：三个浓度下,消渴丸成品组和消渴丸纯中药成分组NO含量相对于格列本脲组和空白对照组均明显升高(P<0.001),消渴丸成品组与消渴丸纯中药成分组相比,NO含量无明显差别(P>0.05),并且随着药物浓度的递增,NO含量也明显上升(P<0.05)。消渴丸对内皮细胞增殖的影响试验发现：与空白对照组相比较,高剂量的消渴丸中药成分粗提液对血管内皮细胞有抑制其生长的作用,且差异具有统计学意义(P<0.05)。
结论
消渴丸在体外细胞培养环境中,可以提高NO的含量,而且随着药物浓度的递增,NO浓度也随之增加,并且发现,这主要是来自消渴丸的中药成分的作用。这可能是消渴丸保护血管内皮的一种机制。另外,高剂量的消渴丸中药成分粗提液对血管内皮细胞有抑制其生长的作用,且差异具有统计学意义(P<0.05)。
Objective
To investigate the effect of a chinese triditional medicine, which is called "Xiaokewan", on the release of nitric oxide (NO) inculture endothelial cell from human umbilical vein。 And the effect of this medicine on the proliferation of human umbilical vein endothelial cells (HUVECs) was also studied。
Methods
HUVECs, were isolated by collagenase digestion。 The3th generation of cultured HUVECs were used with the cell count of1×105/ml in each core The experiment consisted of4groups:(A) glibenclamide group;(B) full medicine of "Xiaokewan" group;(C) medicine of "xiaokewan" without group;(D) control group; and three different concentrations for each group were compared。For the study of effect of "Xiaokewan" on the proliferation of HUVECs,4groups were assigned randomly:(a) control group;(b) low concentration group;(c) moderate concentration group;(d) high concentration group。At day1、day3and day5count the cells in each core。
Results
"Xiaokewan" promoted the secretion of NO, and this effect was mainly due to the medicine without glibenclamide。High concentration of "Xiaokewan" inhibited the proliferation of HUVECs。
Conclusions
For each concentration," Xiaokewan" can promote the secretion of NO of HUVECs in vitro and high concentration of "Xiaokewan" inhibit the proliferation of HUVECs。
引文
1)张宝庚,陈铁镇,张晶范等。人脐带静脉及大鼠主动脉内皮细胞的培养[J]。中华心血管病杂志,1985；13(1)：52。
2) Jaffe E. A., Nachman R. L., Becker C. G., et al. Culture of human endothelial cells derived from umbilical veins:identification by morphologic and immunologic criteria[J]. Clin Invest,1973; 52 (6):2745.
3) Springer, T. A.,1994。 Traffic signals for leukocyte recirculation and leukocyte emigration:the multistep paradigm. Cell 76:301。
4) Bahra, P., Rainger, G. E, Wautier, J. L., Luu, N. T., Nash, G. B.,1998. Each step during transendothelial migration of flowing neutrophils is regulated by the stimulatory concentration of tumour necrosis factor-alpha.Cell Adhes. Commun. 6:491-501.
5) Kaplanski, G.., Fabrigoule, M., Boulay, V., Dinarello, C. A., Bongrand, P., Kaplanski, S., Famarier, C.,1997.Thrombin induces endothelial type Ⅱ activation in vitro.IL-land TNF-a-independent IL-8 secretion and E selectin expression. J. Immunol.158:5435.
6) Kaplanski, G., Marin, V., Fabrigoule, M., Boulay, V., Benoliel, A.-M., Bongrand, P., Kaplanski, S., Farnarier, C.,1998. Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1(ICAM-1, CD54) and vascular cell adhesion molecule-1(VCAM-1, CD106). Blood 92:1259.
7) Ross, R.,1999. Atherosclerosis-an inflammatory disease. N. Engl. J. Med. 340:115.
8) Pauli, B.U., Augustin-Voss, H.G, El-Sabban, M.E., Johnson, B.C., Hammer, D.A., 1990. Organ preference of metastasis. Cancer Metastasis Rev.9:175.
9) Maciag, T., Hoover, G.A., Stemerman, M.B., Weinstein, R.,1981. Serial propagation of human endothelial cells in vitro. J. Cell Biol.91:420.
10)孙惠文,张梅。人脐静脉内皮细胞的培养观察与鉴定[J]。实用医药杂志,2006,23(9)：1072。
11)Mutin, G., George, F., Leslaule, G., Sampol, J.,1996. Reevaluation of trypsin-EDTA for endothelial cell detachment before flow cytometry analysis. Endothelium 4:289.
12)Gillis, C., Jonson, B., Haegerstrand, A.,1995. Effect of sera, basic fibrobalst growth factor, heparin and cyclic AMP-stimulation on proliferation of human vascular endothelial cells. Cell. Mol. Biol.41:1131.
13)Thorton, S., Mueller, S.N., Levine, E.M.,1983. Human endothelial cells:use of heparin in cloning and long-term serial cultivation. Science 222:623.
14) Weinstein, R., Wenc, K.,1986. Growth factor responses of human arterial endothelial cells in vitro. In Vitro Cell. Dev. Biol.22:549.
15)Fajardo, L.P.,1989. Special report, the complexity of endothelial cells. Am. J. Clin. Pathol.92:241.
16)Hickey, M.J., Kanwar, S., McCafferty, D.-M., Granger, D.N., Eppihimer, M.J., Kubes, P.,1999. Varying of E-selectin and P-selectin in different microvascular beds in response to antigen. J. Immunol.162:1137.
17)刘成海,中药复方体外药理学研究思考,中药新药与临床药理,2000,11(1)：53—56。
18)Gimbrone, M.A., Cotran, R.S., Folkman J. Human Vascular endothelial cells in culture:growth and DNA synthesis[J]. Cell Biol,1974; 60 (3):673.
19) Weibel, E.R., Palade, G.E. New cytoplasmic components in arterial endothelia[J]. Cell Biol,19645 23:101.
20)张林生,王逸兰,陆道培。脐血血浆支持造血祖细胞体外集落培养的研究。实验血液血杂志,1996,4：87-91。
21) Wolff B, Grabe H.J., Schluter C., et al. Endothelial nitric oxide synthase Glu 298 Asp gene polymorphism, blood pressure and hypertension in a general population sample[J]. J Hypertens,2005,23 (7):1361-1366.
22)Yoon S., Shin C., Park H.Y., et al. Endothelial nitric oxide synthase gene is associated with vessel stenosis in Korean population[J]. Clin Chim Acta,2005, 353 (2-2):177-185.
23)Kishinoto T., Misawa Y., Kaetu A., et al. eNOS Glu 298 Asp polymorphism and hypertension in a cohort study in Japanese[J]. Prev Med, 2004,39 (5):927-931.
24)Zhu H., Wang X., Dong Y., et al. Influence of the eNOS gene on development of blood pressure and left ventricular mass longitudinal findings in multiethnic youth[J]. Phamacogenet Genomics,2005,15 (9):669-675.
25)卞杰勇,周岱。人脐静脉内皮细胞体外培养的影响因素。细胞生物学杂志,1997,19(2)：66-68。
26)Furchgott R.F. Endothelium-derived relaxing factor:discovery, early studies, and identification as nitric oxide. Biosci Rep,1999,19:235-251.
27)Furchgott R.F., Zawadzki J.V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature,1980,288:373-376.
28) Murad F. Shattuck Lecture. Nitric oxide and cyclic GMP in cell signaling and drug development. N Engl J Med,2006,355:2003-2011.
29)Ignarro L.J. Nitric oxide:a unique endogenous signaling molecule in vascular biology. Biosci Rep,1999,19:51-71.
30)Raij L. Nitric oxide in the pathogenesis of cardiac disease. J Clin Hypertens (Greenwich),2006,8:30-39.
31)Levy R.M., Prince J.M., Billiar T.R. Nitric oxide:a clinical primer. Crit Care Med,2005,33:S492-S495.
32)Cengel A., Sahinarslan A. Nitric oxide and cardiovascular system. Anadolu Kardiyol Derg,2006,6:364-368.
33) Murad F. Discovery of some of the biological effects of nitric oxide and its role in cell signaling. Biosci Rep,1999,19:133-154.
34) Kim J.A., Montagnani M., Koh K.K., et al. Reciprocal relationships between insulin resistance and endothelial dysfunction:molecular and pathophysiological mechanisms. Circulation,2006,113:1888-1904.
35)Nagy G., Koncz A., Femandez D., et al. Nitric oxide, mitochondrial hyperpolarization, and T cell activation. Free Radic Biol Med,2007,42: 1625-1631.
36)Ignarro L.J., Napoli C., Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide:an overview. Circ Res,2002,90:21-28.
37)阮秋蓉,宋建新,邓仲端等。山莨菪碱下行调节培养的内皮细胞表达纤溶酶原激活物抑制剂1[J]。中国动脉硬化杂志,2001,9(3)：194-197。
38)邓春雷,陈熙。四物汤对血管内皮细胞分泌组织因子及其抑制物的影响[J]。南华大学学报,2001,29(2)：120-122。
39)聂克。黄芪当归药对心血管系统作用的研究概况。中国中西医结合杂志,1996,16(6)：379-381。
40)李春梅。红参对人体血管内皮细胞的作用。国外医学.中医中药分册,1999,21(4)：15-16。
41) Li XD, Lu KH, Guo SZ, et al. Culture of vascular endothelial cell[J]. Chin J Aesthetic Med,2001,10 (3):18-20.
42)王建民,刘荫秋,赖西南。正常脐静脉离体后不同时间内皮某些物质含量的变化[J]。第三军医大学学报,1995,17(1)：86。
43) Jonathan B., Rosenberg, Judith S., et al. Genetic induction of a releasable pool of factor VIII in human endothelial cells[J]. Arterioscler Thromb Vasc Biol,2000, 20 (12):2689-2695.
44)Mario P., Afazal J., Daniel P., et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors[J]. Blood,2000,95 (3):952-958.
45)Relou A.M., Damen C.A., Van der Schaft. Effect of culture conditions on endothelial cell growth and responsiveness[J]. Tissue Cell,1998,30(5):525-530.
46) Evans G.R., Brandt K., Klatz S., et al. Bioactive poly conduits seeded with Schwann cells for peripheral nerve regeneration[J]. Biomaterials,2002,23 (1): 841.
47) Liang PF, Zhang PH, Yang XH, et al. Experimental study on induction of endothelial apoptosis by bum serum and subeschar tissue fluid[J].中华烧伤杂志,2004,20(5)：275-277.
48)Savore C, Zhang C, Muir C, et al. Perlecan knockdown inmetastatic prostate cancer cells reduces heparin-binding growth factor responses in vitro and tumor growth in vivo[J]. Clin Exp Metastasis,2005,22 (5):377-390.
49) Kuvin J.T., Kmmelstiel C.D. Infection causes of atherosclerosis[J]. Am Heart J, 1999,137:216-226.
50)Harb T.S., Zareba W., Moss A.J., et al. Association of C-reactive protein and serum amyloid A with recurrent coronary events in stable patients after healing of acute myocardial infarction[J]. AMJ Cardiol,2002,89:216-221.
51) Allen P.B., Russell P.T., Frank K., et al. Elevated C-reactive protein values and atherosclerosis in sudden coronary death:Association with different pathologies[J]. Circulation,2002,17:2019-2029.
52) Tomoda H., Aoki N. Prognostic value of C-reactive protein levels within six hours after onset of acute myocardial infarction[J]. Am Heart J,2000,140:324-328.
53) Gupta S. Chronic infection in aetiology of atherosclerosis-focus on Chlamydia pneumonias[J]. Atherosclerosis,1999,143:1-6.
54)Buffon A., Liuzzo G., Biasucci L.M., et al. Preprocedural serum levels of C-reactive protein predict early complications and late restenosis after coronary angioplasty[J].J Am Coll Cardiol,1999,34:1512-1521.
55) Aso Y., Okumura K., Yoshida N., et al. Plasma interleukin-6 is associated with coagulation in poorly controlled patients with type 2 diabetes[J]. Diabet Med, 2005,20 (11):930.
56)Barber P.A., Auer R.N., Buchan Alu, et al. Understanding and managing ischemic stroke[J]. Can J Physiol Pharmacol,2001,79 (3):283.
57) Pradhan A.D., Ridker P.M. Do atherosclerosis and type 2 diabetes share a common inflammatory basis[J].Eur Heart J,2002,23 (11):831.
58)Temel kova K.T., Siegert G, Bergmann S, et al. Subclinical inflammation is trongly related to insulin resistance but not to impaired insulin secretion in a high risk population for diabetes[J]. Metabolism,2004,51:743-749.
59)Byberg L, Siegbahn A, Berglund L, et al. Plasminogen activator unhibitor 1 activity is independently relared to both insulin sensitivity and serum triglycerdes in 70-year old men[J].Arterioselerosis,2002,18 (2):258-264.
60) Pederson od, Gram J, Jespersen J, et al. Plasminogen activitor inhibitor type-1 determines plasmin formation in patients with ischemic heart disease[J]. Thromb Haemost,1997,78:1656.
61) Abbott R.D., Brand F.N., Kannel W.B., et al. Cout and coronary heart disease. The Framingham study J Clin Epidemiol,1988,41 (3):237.
62) Lehto S, Nislcanen L, ronneman T, et al. Serum uric acid is a strong predictor of stroke in patients with non-insulin-dependent diabetes mellitus. Stroke,1998, 29 (3):635-639.
63)Freedman D.S., Willismson D.F., Gunter E.W., et al. relation of serumuric acidto mortality and ischemic heart disease. The nhganes 1 Epidemiologic follow-up study. A M J Epidemiol,1995,141 (7):637.
64) Brand K, Page S, Rogler G, et al. Activated teanscription factor nuclear factor kappa B is present in the atherosclerotic lesion. J Clin Invest,1996,97(7):1715, 1722.
65) Munro J.M., Cotran R.S. The pathogenesis of atherosclerosis:atherogenesis and inflammation. Laboratory Investigation,1988,58:249-261.
66) E Fougerolles A.R., Qin X, Springer T.A. Characterization of the function of intercellular adhesion molecule (ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses. J Exp Med,1994,179 (2):619-629.
67) NI Qing, SHI Zhen-sheng. Treatment of Couty Nephropathy with TCM. Xinjiang Journal of TCM,1997, (1):11.
68)CUI Shu-zhen, WANG Yue-zhong, LI Xiu-ying, et al. Clinical Observation on Treatment of Acute Renal Failure with Complexity of Huangdanye. Journal of Traditional Chinese Medicine and Chinese materia medica of Jilin,1997, (2): 22-23.
69)Schultz G.S., Grant M.B., Neovascular growth factor[J]. Eye,1991,5:170-180.
70)Umeda M. Effector of certain herbai medicine on the biotransfomation of arachidonio acid:a new pharmacological testing method using serum[J]. J Ethnothnopharmacol,1988,23:91-98.
71) Wamw H. Effect of shaosaikoto a Japanese and Chinese herbal medicine mixture on mitogenix activity of lipopolysaccharide, a new pharmacological testing method[J]. J Ethnothnopharmacol,1987,211:45-53.
72)李仪奎。中药血清药理学实验方法的若干问题[J]。中药新药与临床药理,1999,2：95-98。
73)杨奎,周明媚,姜远平等。中药血清药理血的方法学研究一给药方案的研究[J]。中药药理与临床,1999,15：43-46。
74)Amal J.F., Michek J.B., Harrisom D.G Nitric oxide in the pahtogenesis of hypertension. Curr Opin Nephrol Hypertension.1995,4:182-188.
75) Harrisom D.G Endothelial dysfunction in atherosclerosis. Basic Res Cardiol, 1994,1:87-102.
76) Amal J.F., Dinh-xuan A.T., Pueyo M., et al. Endothelium-derived nitric oxide and vascular physiology and pathyology. Cell Mol Life Sci,1999,55:1078-1087.
77)Takemoto M, Liao J.K. Pletiotropic effects of 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arterioscler Thromb Vasc Biol,2001,21: 1712-1719.
78)Burridge K, Wennerberg K. Rho and Rac take center stage. Cell,2004,116: 167-179.
79)Laufs U, Liao J.K. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem,1998,273:24226-24271.
80) Wagner A.H., Kohler T,Ruckschloss U, et al. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors throgh attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol,2000,20:61-69.
81) Ming X.F., Viswambharan H, Barandier C, et al. Rho GTPase/Rho Kinase negative regulate endothelial nitric oxide synthase phosphorylation through the inhibition of protein KinaseB/AKT in human endothelial cells. Molecular and Cellular Biolgoy,2002,22:8467-8477.
2) Jaffe E. A., Nachman R. L., Becker C. G., et al. Culture of human endothelial cells derived from umbilical veins:identification by morphologic and immunologic criteria[J]. Clin Invest,1973; 52 (6):2745.
3) Springer, T. A.,1994。 Traffic signals for leukocyte recirculation and leukocyte emigration:the multistep paradigm. Cell 76:301。
4) Bahra, P., Rainger, G. E, Wautier, J. L., Luu, N. T., Nash, G. B.,1998. Each step during transendothelial migration of flowing neutrophils is regulated by the stimulatory concentration of tumour necrosis factor-alpha.Cell Adhes. Commun. 6:491-501.
5) Kaplanski, G.., Fabrigoule, M., Boulay, V., Dinarello, C. A., Bongrand, P., Kaplanski, S., Famarier, C.,1997.Thrombin induces endothelial type Ⅱ activation in vitro.IL-land TNF-a-independent IL-8 secretion and E selectin expression. J. Immunol.158:5435.
6) Kaplanski, G., Marin, V., Fabrigoule, M., Boulay, V., Benoliel, A.-M., Bongrand, P., Kaplanski, S., Farnarier, C.,1998. Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1(ICAM-1, CD54) and vascular cell adhesion molecule-1(VCAM-1, CD106). Blood 92:1259.
7) Ross, R.,1999. Atherosclerosis-an inflammatory disease. N. Engl. J. Med. 340:115.
8) Pauli, B.U., Augustin-Voss, H.G, El-Sabban, M.E., Johnson, B.C., Hammer, D.A., 1990. Organ preference of metastasis. Cancer Metastasis Rev.9:175.
9) Maciag, T., Hoover, G.A., Stemerman, M.B., Weinstein, R.,1981. Serial propagation of human endothelial cells in vitro. J. Cell Biol.91:420.
10)孙惠文,张梅。人脐静脉内皮细胞的培养观察与鉴定[J]。实用医药杂志,2006,23(9)：1072。
11)Mutin, G., George, F., Leslaule, G., Sampol, J.,1996. Reevaluation of trypsin-EDTA for endothelial cell detachment before flow cytometry analysis. Endothelium 4:289.
12)Gillis, C., Jonson, B., Haegerstrand, A.,1995. Effect of sera, basic fibrobalst growth factor, heparin and cyclic AMP-stimulation on proliferation of human vascular endothelial cells. Cell. Mol. Biol.41:1131.
13)Thorton, S., Mueller, S.N., Levine, E.M.,1983. Human endothelial cells:use of heparin in cloning and long-term serial cultivation. Science 222:623.
14) Weinstein, R., Wenc, K.,1986. Growth factor responses of human arterial endothelial cells in vitro. In Vitro Cell. Dev. Biol.22:549.
15)Fajardo, L.P.,1989. Special report, the complexity of endothelial cells. Am. J. Clin. Pathol.92:241.
16)Hickey, M.J., Kanwar, S., McCafferty, D.-M., Granger, D.N., Eppihimer, M.J., Kubes, P.,1999. Varying of E-selectin and P-selectin in different microvascular beds in response to antigen. J. Immunol.162:1137.
17)刘成海,中药复方体外药理学研究思考,中药新药与临床药理,2000,11(1)：53—56。
18)Gimbrone, M.A., Cotran, R.S., Folkman J. Human Vascular endothelial cells in culture:growth and DNA synthesis[J]. Cell Biol,1974; 60 (3):673.
19) Weibel, E.R., Palade, G.E. New cytoplasmic components in arterial endothelia[J]. Cell Biol,19645 23:101.
20)张林生,王逸兰,陆道培。脐血血浆支持造血祖细胞体外集落培养的研究。实验血液血杂志,1996,4：87-91。
21) Wolff B, Grabe H.J., Schluter C., et al. Endothelial nitric oxide synthase Glu 298 Asp gene polymorphism, blood pressure and hypertension in a general population sample[J]. J Hypertens,2005,23 (7):1361-1366.
22)Yoon S., Shin C., Park H.Y., et al. Endothelial nitric oxide synthase gene is associated with vessel stenosis in Korean population[J]. Clin Chim Acta,2005, 353 (2-2):177-185.
23)Kishinoto T., Misawa Y., Kaetu A., et al. eNOS Glu 298 Asp polymorphism and hypertension in a cohort study in Japanese[J]. Prev Med, 2004,39 (5):927-931.
24)Zhu H., Wang X., Dong Y., et al. Influence of the eNOS gene on development of blood pressure and left ventricular mass longitudinal findings in multiethnic youth[J]. Phamacogenet Genomics,2005,15 (9):669-675.
25)卞杰勇,周岱。人脐静脉内皮细胞体外培养的影响因素。细胞生物学杂志,1997,19(2)：66-68。
26)Furchgott R.F. Endothelium-derived relaxing factor:discovery, early studies, and identification as nitric oxide. Biosci Rep,1999,19:235-251.
27)Furchgott R.F., Zawadzki J.V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature,1980,288:373-376.
28) Murad F. Shattuck Lecture. Nitric oxide and cyclic GMP in cell signaling and drug development. N Engl J Med,2006,355:2003-2011.
29)Ignarro L.J. Nitric oxide:a unique endogenous signaling molecule in vascular biology. Biosci Rep,1999,19:51-71.
30)Raij L. Nitric oxide in the pathogenesis of cardiac disease. J Clin Hypertens (Greenwich),2006,8:30-39.
31)Levy R.M., Prince J.M., Billiar T.R. Nitric oxide:a clinical primer. Crit Care Med,2005,33:S492-S495.
32)Cengel A., Sahinarslan A. Nitric oxide and cardiovascular system. Anadolu Kardiyol Derg,2006,6:364-368.
33) Murad F. Discovery of some of the biological effects of nitric oxide and its role in cell signaling. Biosci Rep,1999,19:133-154.
34) Kim J.A., Montagnani M., Koh K.K., et al. Reciprocal relationships between insulin resistance and endothelial dysfunction:molecular and pathophysiological mechanisms. Circulation,2006,113:1888-1904.
35)Nagy G., Koncz A., Femandez D., et al. Nitric oxide, mitochondrial hyperpolarization, and T cell activation. Free Radic Biol Med,2007,42: 1625-1631.
36)Ignarro L.J., Napoli C., Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide:an overview. Circ Res,2002,90:21-28.
37)阮秋蓉,宋建新,邓仲端等。山莨菪碱下行调节培养的内皮细胞表达纤溶酶原激活物抑制剂1[J]。中国动脉硬化杂志,2001,9(3)：194-197。
38)邓春雷,陈熙。四物汤对血管内皮细胞分泌组织因子及其抑制物的影响[J]。南华大学学报,2001,29(2)：120-122。
39)聂克。黄芪当归药对心血管系统作用的研究概况。中国中西医结合杂志,1996,16(6)：379-381。
40)李春梅。红参对人体血管内皮细胞的作用。国外医学.中医中药分册,1999,21(4)：15-16。
41) Li XD, Lu KH, Guo SZ, et al. Culture of vascular endothelial cell[J]. Chin J Aesthetic Med,2001,10 (3):18-20.
42)王建民,刘荫秋,赖西南。正常脐静脉离体后不同时间内皮某些物质含量的变化[J]。第三军医大学学报,1995,17(1)：86。
43) Jonathan B., Rosenberg, Judith S., et al. Genetic induction of a releasable pool of factor VIII in human endothelial cells[J]. Arterioscler Thromb Vasc Biol,2000, 20 (12):2689-2695.
44)Mario P., Afazal J., Daniel P., et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors[J]. Blood,2000,95 (3):952-958.
45)Relou A.M., Damen C.A., Van der Schaft. Effect of culture conditions on endothelial cell growth and responsiveness[J]. Tissue Cell,1998,30(5):525-530.
46) Evans G.R., Brandt K., Klatz S., et al. Bioactive poly conduits seeded with Schwann cells for peripheral nerve regeneration[J]. Biomaterials,2002,23 (1): 841.
47) Liang PF, Zhang PH, Yang XH, et al. Experimental study on induction of endothelial apoptosis by bum serum and subeschar tissue fluid[J].中华烧伤杂志,2004,20(5)：275-277.
48)Savore C, Zhang C, Muir C, et al. Perlecan knockdown inmetastatic prostate cancer cells reduces heparin-binding growth factor responses in vitro and tumor growth in vivo[J]. Clin Exp Metastasis,2005,22 (5):377-390.
49) Kuvin J.T., Kmmelstiel C.D. Infection causes of atherosclerosis[J]. Am Heart J, 1999,137:216-226.
50)Harb T.S., Zareba W., Moss A.J., et al. Association of C-reactive protein and serum amyloid A with recurrent coronary events in stable patients after healing of acute myocardial infarction[J]. AMJ Cardiol,2002,89:216-221.
51) Allen P.B., Russell P.T., Frank K., et al. Elevated C-reactive protein values and atherosclerosis in sudden coronary death:Association with different pathologies[J]. Circulation,2002,17:2019-2029.
52) Tomoda H., Aoki N. Prognostic value of C-reactive protein levels within six hours after onset of acute myocardial infarction[J]. Am Heart J,2000,140:324-328.
53) Gupta S. Chronic infection in aetiology of atherosclerosis-focus on Chlamydia pneumonias[J]. Atherosclerosis,1999,143:1-6.
54)Buffon A., Liuzzo G., Biasucci L.M., et al. Preprocedural serum levels of C-reactive protein predict early complications and late restenosis after coronary angioplasty[J].J Am Coll Cardiol,1999,34:1512-1521.
55) Aso Y., Okumura K., Yoshida N., et al. Plasma interleukin-6 is associated with coagulation in poorly controlled patients with type 2 diabetes[J]. Diabet Med, 2005,20 (11):930.
56)Barber P.A., Auer R.N., Buchan Alu, et al. Understanding and managing ischemic stroke[J]. Can J Physiol Pharmacol,2001,79 (3):283.
57) Pradhan A.D., Ridker P.M. Do atherosclerosis and type 2 diabetes share a common inflammatory basis[J].Eur Heart J,2002,23 (11):831.
58)Temel kova K.T., Siegert G, Bergmann S, et al. Subclinical inflammation is trongly related to insulin resistance but not to impaired insulin secretion in a high risk population for diabetes[J]. Metabolism,2004,51:743-749.
59)Byberg L, Siegbahn A, Berglund L, et al. Plasminogen activator unhibitor 1 activity is independently relared to both insulin sensitivity and serum triglycerdes in 70-year old men[J].Arterioselerosis,2002,18 (2):258-264.
60) Pederson od, Gram J, Jespersen J, et al. Plasminogen activitor inhibitor type-1 determines plasmin formation in patients with ischemic heart disease[J]. Thromb Haemost,1997,78:1656.
61) Abbott R.D., Brand F.N., Kannel W.B., et al. Cout and coronary heart disease. The Framingham study J Clin Epidemiol,1988,41 (3):237.
62) Lehto S, Nislcanen L, ronneman T, et al. Serum uric acid is a strong predictor of stroke in patients with non-insulin-dependent diabetes mellitus. Stroke,1998, 29 (3):635-639.
63)Freedman D.S., Willismson D.F., Gunter E.W., et al. relation of serumuric acidto mortality and ischemic heart disease. The nhganes 1 Epidemiologic follow-up study. A M J Epidemiol,1995,141 (7):637.
64) Brand K, Page S, Rogler G, et al. Activated teanscription factor nuclear factor kappa B is present in the atherosclerotic lesion. J Clin Invest,1996,97(7):1715, 1722.
65) Munro J.M., Cotran R.S. The pathogenesis of atherosclerosis:atherogenesis and inflammation. Laboratory Investigation,1988,58:249-261.
66) E Fougerolles A.R., Qin X, Springer T.A. Characterization of the function of intercellular adhesion molecule (ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses. J Exp Med,1994,179 (2):619-629.
67) NI Qing, SHI Zhen-sheng. Treatment of Couty Nephropathy with TCM. Xinjiang Journal of TCM,1997, (1):11.
68)CUI Shu-zhen, WANG Yue-zhong, LI Xiu-ying, et al. Clinical Observation on Treatment of Acute Renal Failure with Complexity of Huangdanye. Journal of Traditional Chinese Medicine and Chinese materia medica of Jilin,1997, (2): 22-23.
69)Schultz G.S., Grant M.B., Neovascular growth factor[J]. Eye,1991,5:170-180.
70)Umeda M. Effector of certain herbai medicine on the biotransfomation of arachidonio acid:a new pharmacological testing method using serum[J]. J Ethnothnopharmacol,1988,23:91-98.
71) Wamw H. Effect of shaosaikoto a Japanese and Chinese herbal medicine mixture on mitogenix activity of lipopolysaccharide, a new pharmacological testing method[J]. J Ethnothnopharmacol,1987,211:45-53.
72)李仪奎。中药血清药理学实验方法的若干问题[J]。中药新药与临床药理,1999,2：95-98。
73)杨奎,周明媚,姜远平等。中药血清药理血的方法学研究一给药方案的研究[J]。中药药理与临床,1999,15：43-46。
74)Amal J.F., Michek J.B., Harrisom D.G Nitric oxide in the pahtogenesis of hypertension. Curr Opin Nephrol Hypertension.1995,4:182-188.
75) Harrisom D.G Endothelial dysfunction in atherosclerosis. Basic Res Cardiol, 1994,1:87-102.
76) Amal J.F., Dinh-xuan A.T., Pueyo M., et al. Endothelium-derived nitric oxide and vascular physiology and pathyology. Cell Mol Life Sci,1999,55:1078-1087.
77)Takemoto M, Liao J.K. Pletiotropic effects of 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arterioscler Thromb Vasc Biol,2001,21: 1712-1719.
78)Burridge K, Wennerberg K. Rho and Rac take center stage. Cell,2004,116: 167-179.
79)Laufs U, Liao J.K. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem,1998,273:24226-24271.
80) Wagner A.H., Kohler T,Ruckschloss U, et al. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors throgh attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol,2000,20:61-69.
81) Ming X.F., Viswambharan H, Barandier C, et al. Rho GTPase/Rho Kinase negative regulate endothelial nitric oxide synthase phosphorylation through the inhibition of protein KinaseB/AKT in human endothelial cells. Molecular and Cellular Biolgoy,2002,22:8467-8477.
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