中国组织工程研究与临床康复 第 15 卷 第 15 期
2011
–04–09 出版
Journal of Clinical Rehabilitative Tissue Engineering Research April 9, 2011 Vol.15, No.15
Effect of intermittent high glucose on proliferation and apoptosis of endothelial progenitor cells from human peripheral blood as well as the production of malondialdehyde and antioxidant**☆ Xu Han-song1, Kong De-ming1, Xiang Hui2, Xie Xiao-yun3, Lin An-hua4 Abstract BACKGROUND: Studies have demonstrated that intermittent high glucose can have a more severe impact on vascular endothelial function in comparison with persistent hyperglycemia. OBJECTIVE: To investigate the effect of intermittent high glucose on the proliferation and apoptosis of endothelial progenitor cells (EPCs) from human peripheral blood in vitro as well as the production of malondialdehyde (MDA) and antioxidant. METHODS: Total mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation and then the cells were placed on fibronectin-coated culture dishes. After 7 days of culture, the adherent cells were identified as EPCs by laser scanning confocal microscope. The cells were synchronized and then stimulated with glucose 5.5 mmol/L (normal control group), 20 mmol/L (constant high glucose group), and 5.5/20 mmol/L (intermittent high glucose group, 5.5 and 20 mmol/L glucose culture solution was changed every 8 hours) for 72 hours. EPCs proliferation and apoptosis was measured by MTT assay and flow cytometry, respectively. The content of MDA and the activity of superoxide dismutase (SOD) in culture solution were detected with colorimetry. RESULTS AND CONCLUSION: After EPCs were exposed to constant high glucose (20 mmol/L) and intermittent high glucose (5.5/20 mmol/L) for 72 hours, proliferated cells were significantly reduced and the apoptosis rate was significantly increased compared with those exposed to normal glucose (P < 0.01). Furthermore, there was a significant increase in MDA contents as well as a significant reduce in SOD activities in the constant high glucose and intermittent high glucose group (P < 0.01), especially in the latter group. These findings indicated that both intermittent high glucose and constant glucose could inhibit the proliferation and promote the apoptosis of EPCs; however, intermittent high glucose appears to worsen the effects on EPCs. This is maybe due to the increased oxidative stress.
Materials Main reagents and instruments are listed as follows:
INTRODUCTION Studies have shown that intermittent high glucose has a more severe impact than persistent hyperglycemia on vascular endothelial function[1-2]. Endothelial progenitor cells (EPCs) are a kind of precursor cells which can be differentiated into vascular endothelial cells. EPCs are not only involved in human embryonic angiogenesis, but also in postnatal angiogenesis and endothelial repair, which is major factor to maintain the dynamic equilibrium between endothelial injury and repair. An important risk for occurrence and development of vascular complications due to diabetes mellitus is the number of EPCs and vascular endothelial dysfunction[3-4]. Oxidative stress is the cause of vascular endothelial dysfunction in diabetic patients. In the present study, we observed the effects of intermittent high glucose on EPCs proliferation and apoptosis, malondialdehyde (MDA), as well as antioxidant factor synthesis.
MATERIALS AND MEHTODS Design A comparative cytological observation. Time and setting The study was performed at the Central Laboratory, Guiyang College of TCM in December, 2009.
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Reagent and instrument
Source
Human lymphocyte separation medium
Tianjin Haoyang Biological Manufacture Co., Ltd.China Chemicon, USA Hyclone, USA Pepro Tech, USA
Human fibronectin M199 medium Recombinant human vascular endothelial growth factor (rhVEGF), recombinant human basic fibroblast growth factor (rhbFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1) FITC-Ulex europaeus agglutinin , Dil labeled acetylated low-density lipoprotein (Dil-Ac-LDL), D-glucose powder, mannitol powder MTT dimethyl sulfoxide (DMSO), trypsin PE labeled anti-CD133 antibody, FITC labeled anti-CD34 antibody, endothelial growth factor receptor 2 (EGFR-2) antibody Annexin V-FITC Apoptosis Detection Kit MDA detection kit, superoxide dismutase assay kit
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BEKMAN Coulter flow cytometry Multi-wavelength laser scanning confocal microscope
Sigma, USA
1 Department of Endocrinology, the Second Affiliated Hospital of Guiyang College of TCM, Guiyang 550003, Guizhou Province, 2 China; Department of Psychology, Guizhou Provincial People’s Hospital, Guiyang 550003, Guizhou Province, 3 China; Department of Endocrinology, Third Xiangya Hospital of Central South University, Changsha 410008, Hunan Province, 4 China; Department of Endocrinology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
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Xu Han-song , Doctor, Associate professor, Department of Endocrinology, the Second Affiliated Hospital of Guiyang College of TCM, Guiyang 550003, Guizhou Province, China xuhansong911@163. com Supported by: the National Natural Science Foundation of China, No. 30960491*; Guangzhou Provincial Governor Found for Outstanding Education Professionals*. Received: 2010-12-15 Accepted: 2011-02-26 (20101210009/WLM)
Amresco, USA Biolegend, USA
Jingmei Biotech Co., Ltd., Shanghai, China Nanjing Jiancheng Bioengineering Institute, China BECKMAN Leica, Germeny
Xu HS, Kong DM, Xiang H, Xie XY, Lin AH. Effect of intermittent high glucose on proliferation and apoptosis of endothelial progenitor cells from human peripheral blood as well as the production of malondialdehyde and antioxidant. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(15): 2755-2759. [http://www.crter.cn http://en.zglckf.com]
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Peripheral venous blood samples were collected from health adults at the Center of Physical Examination, the Second Affiliated Hospital of Guiyang College of TCM, aged (38±4) years. Written informed content was obtained from all the subjects. Methods Isolation, culture and identification of human peripheral blood EPCs 10 mL fasting peripheral venous blood sample was isolated from a healthy adult to obtain mononuclear cells by using density gradient centrifugation following treated with sodium heparin anticoagulant[5-6]. The obtained mononuclear cells were cultured in fibronectin-coated 6-well culture plates. 0.5 mL M199 medium containing 20% fetal bovine serum FBS, 10 μg/L rhVEGF, 2 μg/L rhbFGF, 10 μg/L EGF, 12 μg/L IGF-1, 1×105 U/L penicillin and 1×105 U/L streptomycin was added. The cell solution was cultured in a 5% CO2 incubator with saturated humidity at 37 for 4 days. After non-adherent cells were rinsed with phosphate buffer solution (PBS), the medium was changed for further 7-day culture. Then, non-adherent cells were removed by using PBS, and adherent cells were identified[2]. The cultured cells were cocultured with 2.4 mg/L Dil-Ac-LDL at 37 for 1 hour followed by 10-minute 20 g/L paraformaldehyde fixation. After rinsing with PBS, 10 mg/L FITC labeled Ulex europaeus agglutinin was added to the cell samples and cultured at 37 for 1 hour. Under the laser scanning confocal microscope, Ulex lectin FITC- and Dil-Ac-LDL double stained positive cells were differentiating EPCs[5-6]. The adherent cell suspension at a proper concentration was incubated with PE-labeled anti-CD133 monoclonal antibody, FITC labeled anti-CD34 monoclonal antibody and anti-endothelial growth factor receptor 2 antibody at 4 for 30 minutes, and then rinsed with PBS twice. 300 μL PBS was added to the solution to suspend cells. The flow cytometry was used for surface markers[5-6].
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Experimental grouping Following cultured in M199 medium without FBS, the adherent cells were divided into four groups: normal control group (5.5 mmol/L glucose), constant high glucose group (20 mmol/L glucose), and intermittent high glucose group (5.5 and 20 mmol/L glucose culture solution was changed every 8 hours), hypertonic control group (20 mmol/L mannitol). The medium in each group was changed every 8 hours, and the culture process lasted for 72 hours. EPCs proliferation After digestion with 0.25% trypsin and culture in M199 medium, the mononuclear cell suspension at a density of 1 × 104 per well was placed into fibronectin-coated 96 well plates with each well volume of 200 μL. The culture plates were placed into the 5% CO2 incubator, at 37 and saturated humidity for 24 hours. Then, 5 g/L MTT solution of 20 μL was added to each well for continuous culture of 4 hours. After that, the supernatant was removed, and 150 μL DMSO was added to each well. The micro oscillator was used for 10 minutes to fully dissolve crystals. Absorbance at 490 nm was measured by
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ELISA analyzer. EPCs apoptosis The cell concentration was adjusted to 109/L following digestion and rinsing with PBS twice. 100 μL of cell suspension was placed into a flow tube and mixed with 5 μL AnnexinV/FITC and 10 μL 20 mg/L propidium iodide (PI). The mixture was incubated at room temperature in the dark for 15 minutes, and then was diluted by 400 μL PBS. The flow cytometry showed that Annexin-V +PI- cells were considered to be early apoptotic cells, Annexin-V +PI+ were apoptotic and necrotic cells, Annexin-V-PI+ were damaged cells, and Annexin-V-PI- were living cells. Early apoptotic rate = the number of apoptotic cells/total number of cells measured × 100%. Measurement of MDA content and SOD activity The conditioned cell medium was centrifuged at 1 000 r/min for 10 minutes, and the supernatant was preserved at -70 . Detection was carried out according to kit instructions, and the sample absorbance value was measured by using the spectrophotometer.
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Main outcome measures Proliferation condition and apoptotic rate of EPCs as well as MDA content and SOD activity. Statistical analysis All the data were expressed as Mean±SD, and analyzed by using SPSS 16.0 software. Analysis of variance and t test were used for intergroup comparison, and a value of P < 0.05 was considered as significant.
RESULTS Identification of EPCs The mononuclear cells isolated formed several cell masses at 3-4 days, and some spindle cells sprouted from the edge of cell mass at 5-6 days. On the 7th day, the adherent cells increased in number, some aggregated to form “Blood Island”( Figure 1).
a: Mononuclear cells just after isolated from peripheral blood showed as round-shape
b: After 7 days in culture, non-adherent cells were removed and adherent cells exhibited a spindle-shaped
Figure 1 Morphology of cultured mononuclear cells (×100)
Under the laser confocal microscope, Dil-Ac-LDL (red, excitation wavelength 543 nm) and Ulex lectin FITC- (green, excitation wavelength 477 nm) double stained positive cells were differentiating EPCs (Figure 2).
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Effect of intermittent high glucose on EPCs proliferation and apoptosis After EPCs were exposed to constant high glucose (20 mmol/L) and intermittent high glucose (5.5/20 mmol/L) for 72 hours, proliferated cells were significantly reduced and the apoptotic rate was increased dramatically (P < 0.01), especially those exposed to intermittent high glucose (P < 0.01; Table 1). a: Dil labeled acetylated low density lipoprotein (Dil-ac-LDL) staining
b: FITC-UEA-I staining
Effect of intermittent high glucose on MDA content and SOD activity There was a significant increase in MDA contents as well as a significant reduce in SOD activities in the constant high glucose and intermittent high glucose group (P < 0.01), especially in the latter group (P < 0.01; Table 1).
c: Dil-ac-LDL and FITC-UEA-I double staining
Table 1 Effect of intermittent high glucose on the proliferation and apoptosis of endothelial progenitor cells (EPCs) from human peripheral blood as well as malondialdehyde (MDA) content _ and superoxide dismutase (SOD) activity (x±s)
Figure 2 Identification of endothelial progenitor cells after 7 d
in culture (Laser confocal microscope, bar: 20 µm)
The results of flow cytrometry examination showed that CD34 positive cells accounted for (32.15±8.68)%, CD133 positive cells accounted for (18.73±7.12)%, and cells positive for EGFR-2 were (69.45±8.21)% (Figure 3).
Group Normal control Hypertonic control Constant high glucose Intermittent high glucose
Proliferation of EPCs (A)
Apoptosis rate of EPCs MDA content (%) (μmol/L)
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646.12±31.90
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Figure 3 The phenotype of endothelial progenitor cells
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DISCUSSION Studies have shown that intermittent high glucose causes apoptosis of cultured human umbilical vein endothelial cells more easily than constant high glucose[7-9], and it can cause a series of changes in vascular structure and function, leading to vascular lesions[10-13]. EPCs, precursor cells of endothelial cells, are involved in postnatal angiogenesis and endothelial repair, which are a key factor to maintain the homeostasis between endothelial injury and repair. Whether there is a reduce in the number of human peripheral blood EPCs and function impairment is the key for the occurrence and development of vascular diseases. It is proved that EPCs in the peripheral blood of diabetic patients have a reduction in number companied with functional impairment[14]. In vitro experiments show that high glucose plays a important role in EPCs injury[15]. However, the difference and related mechanism of intermittent high glucose and constant high glucose effects on EPCs are unclear. In the present study, Annexin V-FITC/PI double staining flow cytometry method was used to observe the changes in EPCs proliferation and apoptosis, MDA content and SOD activity following intermittent high glucose intervention to investigate the effect of intermittent high glucose on EPCs as well as oxidative stress mechanism. Some cells (such as neurons) are vulnerable to damage
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because of digestion and percussion during the experiments, which lead to a high proportion of late apoptotic and necrotic cells. It is difficult to distinguish apoptotic cells and necrotic cells. In the early stage of cell apoptosis, phospholipids phosphatidylserine migrates from inside to outside of the cell membrane. Annexin V-FITC/PI double staining can distinguish normal cells, early apoptotic cells and dead cells, based on which the apoptotic rate is more reliable. The results showed that constant high glucose (20 mmol/L) and intermittent high glucose (5.5/20 mmol/L) could both lead to a reduce of EPCs proliferation and an increase of apoptotic rate after action on EPCs for 72 hours. However, the intermittent high glucose had a better effect, indicating that the intermittent high glucose has a greater impact on EPCS as compared to the constant high glucose. This is a good explain for why diabetic patients with instable blood glucose are more likely to have macrovascular complications. Mannitol could not cause significant increase in apoptotic EPCs, which is consistent with overseas research[8], indicating that high glucose and intermittent high glucose rather than osmotic pressure changes result in increased EPCs apoptosis. It has been shown that intermittent high glucose can accelerate the apoptosis of vascular endothelial cells, which may be related to oxidative stress[1]. Oxidative stress is a common physiopathological mechanism for diabetic vascular complications and the important factor for endothelial dysfunction due to high glucose[16-22]. MDA is a lipid peroxide, and can reflect the degree of lipid peroxidation and oxidative damage[23]. SOD is an indicator that indirectly reflects the body’s ability to eliminate oxygen free radicals. In this study, MDA and SOD were selected as observation indicators to reflect the effect of high glucose and intermittent high glucose on EPCs under oxidative stress conditions. The results shown that the activates of SOD decreased and MDA contents increased in both high glucose and intermittent high glucose groups, especially in the latter group. It is indicated that intermittent high glucose has a greater effect on oxidation and antioxidant ability of EPCs than high glucose. It suggests that a long-term intermittent high glucose in diabetic patients can aggravate endothelial oxidative stress and finally lead to the occurrence and development of vascular complications.
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Quagliaro L, Piconi L, Assaloni R, et al. Intermittent high glucose enhances apoptosis related to oxidative stress in human umbilical vein endothelial cells: the role of protein kinase C and NAD(P)H-oxidase activation. Diabetes. 2003;52(11):2795-2804. Azuma K, Kawamori R, Toyofuku Y, et al. Repetitive fluctuations in blood glucose enhance monocyte adhesion to the endothelium of rat thoracic aorta. Arterioscler Thromb Vasc Biol. 2006;26(10):2275-2280. Xu HS, Lei MX, Kong DM, et al. Changes in the proliferation,differentiation and cell cycle of endothelial progenitor cells from peripheral blood in patients with type 2 Diabetes. Zhongguo Tangniaobing Zazhi. 2008;16(12):738-740.
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Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002;106(22):2781-2786. Wang H, Wang L, Li KZ, et al. Culture and identification of endothelial progenitor cells from human peripheral blood. Zhongguo Linchuang Kangfu. 2006;10(1):47-49. Liu ZH, Lei MX, Wang AM, et al. Isolation, induction and differentiation of human blood-derived endothelia progenitor cells. Zhongnan Daxue Xuebao: Yixue Ban. 2005;30(5): 566-569. Piconi L, Quagliaro L, Da Ros R, et al. Intermittent high glucose enhances ICAM-1, VCAM-1, E-selectin and interleukin-6 expression in human umbilical endothelial cells in culture: the role of poly(ADP-ribose) polymerase. J Thromb Haemost. 2004; 2(8):1453-1459. Risso A, Mercuri F, Quagliaro L, et al. Intermittent high glucose enhances apoptosis in human umbilical vein endothelial cells in culture. Am J Physiol Endocrinol Metab. 2001;281(5):E924-930. Guevara NV, Chen KH, Chan L. Apoptosis in atherosclerosis: pathological and pharmacological implications. Pharmacol Res. 2001;44(2):59-71. Pliszka B, Redowicz MJ, Stepkowski D. Interaction of the N-terminal part of the A1 essential light chain with the myosin heavy chain. Biochem Biophys Res Commun. 2001;281(4): 924-928. Booth G, Stalker TJ, Lefer AM, et al. Mechanisms of amelioration of glucose-induced endothelial dysfunction following inhibition of protein kinase C in vivo. Diabetes. 2002; 51(5):1556-1564. Nakamura N, Ueno Y, Tsuchiyama Y, et al. Isolated post-challenge hyperglycemia in patients with normal fasting glucose concentration exaggerates neointimal hyperplasia after coronary stent implantation. Circ J. 2003;67(1):61-67. Cryer PE, Davis SN, Shamoon H. Hypoglycemia in diabetes. Diabetes Care. 2003;26(6):1902-1912. Loomans CJ, de Koning EJ, Staal FJ, et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes. 2004; 53(1):195-199. Wang AM, Lei MX, Liu ZH, et al. The induction and differentiation of endothelial progenitor cell from human peripheral blood of type 2 diabetics and its influential factors. Zhongguo Tangniaobing Zazhi. 2006;14(3):3188-3191. Piconi L, Quagliaro L, Assaloni R, et al. Constant and intermittent high glucose enhances endothelial cell apoptosis through mitochondrial superoxide overproduction. Diabetes Metab Res Rev. 2006;22(3):198-203. Monnier L, Mas E, Ginet C, et al. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA. 2006;295(14):1681-1687. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005;54(6):1615-1625. Ceriello A. New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy. Diabetes Care. 2003;26(5):1589-1596. El-Mesallamy H, Hamdy N, Suwailem S, et al. Oxidative stress and platelet activation: markers of myocardial infarction in type 2 diabetes mellitus. Angiology. 2010;61(1):14-18. Dominguez LJ, Galioto A, Pineo A, et al. Age, homocysteine, and oxidative stress: relation to hypertension and type 2 diabetes mellitus. J Am Coll Nutr. 2010;29(1):1-6. Singhania N, Puri D, Madhu SV, et al. Assessment of oxidative stress and endothelial dysfunction in Asian Indians with type 2 diabetes mellitus with and without macroangiopathy. QJM. 2008;101(6):449-455. Ozdemir G, Ozden M, Maral H, et al. Malondialdehyde, glutathione, glutathione peroxidase and homocysteine levels in type 2 diabetic patients with and without microalbuminuria. Ann Clin Biochem. 2005;42(Pt 2):99-104.
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人外周血内皮祖细胞增殖、凋亡及丙二醛、抗氧化因子合成与波动性高糖的影响**☆ 徐寒松 ，孔德明 ，向 慧 ，谢晓云 ，林安华 ( 贵阳中医学院第二附属医院内分泌科，贵州省贵阳市 550003； 贵州省人民医院心理科， 贵州省贵阳市 550003； 中南大学湘雅三医院内分泌科，湖南省长沙市 410008； 江西省人民医院内分泌科，江西省南昌市 330006) 1
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徐寒松☆，男，1973 年生，贵州省安顺市人， 汉族，2007 年中南大学湘雅医院毕业，博士， 副教授，主要从事糖尿病慢性并发症方面的 研究。 摘要 背景：研究表明波动性高血糖较持续性高血 糖对血管内皮功能的损害可能更严重。 目的：观察波动性高糖对人外周血内皮祖细 胞增殖、凋亡及丙二醛、抗氧化因子合成的 影响。 方法：密度梯度离心法获取人外周血单个核 细胞。取经培养鉴定后的内皮祖细胞，细胞 同化后分别给予 5.5 mmol/L，20 mmol/L， 5.5/20 mmol/L 葡萄糖(5.5，20 mmol/L 的葡 萄糖培养液每 8 h 更换 1 次)及 20 mmol/L 甘露醇。干预 72 h 后，MTT 法检测内皮祖 细胞的增殖情况；流式细胞仪检测细胞凋亡 率；比色法测定培养液中丙二醛水平及超氧 化物歧化酶活力。 结果与结论：20 mmol/L 和 5.5/20 mmol/L 葡萄糖作用 72 h，内皮祖细胞增殖减少、凋 亡率增高(P < 0.01），培养液中丙二醛水平 增高、超氧化物歧化酶活力降低(P < 0.01)， 均以 5.5/20 mmol/L 葡萄糖作用最明显(P < 0.01)。说明波动性高糖较恒定性高糖更易抑
制内皮祖细胞增殖并促进其凋亡，其机制可 能与波动性高糖环境下氧化应激水平增高有 关。 关键词：内皮祖细胞；增殖；凋亡；丙二醛； 波动性高糖 doi:10.3969/j.issn.1673-8225.2011.15.023
中图分类号: R318 文献标识码: B 文章编号: 1673-8225(2011)15-02755-05
徐寒松，孔德明，向慧，谢晓云，林安华. 人外周血内皮祖细胞增殖、凋亡及丙二醛、 抗氧化因子合成与波动性高糖的影响[J].中 国组织工程研究与临床康复, 2011,15 (15):2755-2759. [http://www.crter.org http://cn.zglckf.com] (Edited by Li YK,/Wang L)
来自本文课题的更多信息--
基金资助：国家自然科学基金 ；贵州省优秀科技教育人才 省长基金。课题名称：黄芪组分对 2 型 糖尿病外周血内皮祖细胞体内再内皮 化能力的影响及分子机制研究。 (30960491)
ISSN 1673-8225 CN 21-1539/R
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年版权归《中国组织工程研究与临床康复》杂志社所有
杂志调查分析类文章体例：本刊中文部
文题： 作者： 单位： 摘要： 背景： 目的： 方法： 结果与结论： 关键词： 0 引言 1 对象和方法 设计： 时间及地点： ISSN 1673-8225 CN 21-1539/R
作者贡献：徐寒松进行实验设计， 实验实施为徐寒松、向慧、谢晓云、林 安华，实验评估为徐寒松、孔德明，资 料收集为向慧，徐寒松成文，徐寒松审 校，徐寒松对文章负责。 利益冲突：课题未涉及任何厂家及 相关雇主或其他经济组织直接或间接 的经济或利益的赞助。 伦理批准：实验征得了受试者的知 情同意。 本文创新性： 提供证据：检索 CNKI 数据库，检 索时间：建库至 2010-03，关键词：“内 皮祖细胞、增殖、凋亡、波动性高糖”， 未见与文章密切相关文献。 创新点说明：采用内皮细胞的前体 细胞——内皮祖细胞研究波动性高糖 对其增殖、凋亡的影响及氧化应激机 制，探讨波动性高糖在糖尿病血管并发 症发生发展中的重要地位，提示糖尿病 临床治疗应重视平稳控制血糖。
对象： 受试者的入选标准： 分组方法： 分组隐藏： 执行： 盲法(遮蔽)： 样本量 主要观察指标： 统计学分析： 2 结果 2.1 参与者数量分析 2.2 各阶段受试者流程 2.3 随访情况 CODEN: ZLKHAH
基线资料 2.5 主要结果 2.6 材料宿主反应/不良反应 2.7 病例综合评估 3 讨论 4 参考文献 5 辅文 基金资助： 作者贡献： 致谢： 利益冲突： 伦理批准： 本文创新性： 2.4
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