Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes： 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester（CFSE）, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein（Di I-Ac LDL）, and green fluorescent protein（GFP）. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo.RESULTS： EPCs labeled with CFSE and Di I-Ac LDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28 d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and Di I-Ac LDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina.CONCLUSION： The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and Di I-Ac LDL are suitable for short-term EPClabeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

C-reactive protein decreases interleukin-8 production in human endothelial progenitor cells by inhibition of p38 MAPK pathway

Background C-reactive protein （CRP） has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation, and it is also speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells （EPCs）. Interleukin-8 （IL-8） is an important mediator of the paracrine mitogenic effect of EPCs which has direct angiogenic effects on mature endothelial cells. We, herein, investigated the direct effect of CRP on IL-8 production and gene expression in cultured human EPCs. Methods EPCs were isolated from the peripheral venous blood of healthy male volunteers. Cells were cultured in EndoCultTM liquid medium in the absence and presence of CRP at clinically relevant concentrations （5 to 25 μg/ml） for different durations （3 to 48 hours）. IL-8 protein and mRNA of cultured EPCs were evaluated using ELISA and real-time PCR. Results The results showed that CRP at a concentration of 10 μg/ml significantly reduced IL-8 secretion of cultured EPCs with a peak at 25 μg/ml, and also decreased mRNA expression in EPCs with a peak at 12 hours. In addition, preincubation of EPCs with SB203580, an inhibitor of p38 mitogen-activated protein kinase （MAPK） decreased CRP inhibition of IL-8 mRNA expression at 12 hours in EPCs. Conclusions Our study, for the first time, demonstrates that CRP directly inhibits EPCs IL-8 secretion, a key cytokine player of angiogenesis induced by EPCs. Inhibition occurred in part via an effect of CRP to active the p38 MAPK signal transduction pathway in EPC. The ability of CRP to inhibit EPCs IL-8 secretion may represent an important mechanism that further links inflammation to cardiovascular disease.

Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

Objective:To study the role of bone marrow mesenchymal stem cells(BMSCs)in construction of vascularized engineered tissue.Methods:hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein(GFP)-CMV.Then the recombinant shuttle plasmid was transfected into BMSCs with Lipofectamine^(TM)2000 for packaging and amplifying.hVTGF165 mRNA expression in BMSCs cells was tested.Results:The sequence of hVEGFI65 in pShutlle-GFP-hVFGF165 plasmid was confirimed by double-enzyme cleavage method and sequencing.hVECF165 was highly expressed in BMSCs.Conclusions:The GFP/hVECF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells,which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

A vascular endothelial growth factor activating transcription factor increases the endothelial progenitor cells population and induces therapeutic angiogenesis in a type 1 diabetic mouse with hindlimb ischemia

Background Therapeutic angiogenesis has been shown to promote blood vessel growth and improve tissue perfusion. Vascular endothelial growth factor （VEGF） plays an important role in angiogenesis. However, it has side effects that limit its therapeutic utility in vivo, especially at high concentrations. This study aimed to investigate whether an intramuscular injection of a genetically engineered zinc finger VEGF-activating transcription factor modulates the endothelial progenitor cells （EPC） and promotes therapeutic angiogenesis in a hindlimb ischemia model with type 1 diabetes. Methods AIIoxan （intravenous injection） was used to induce type I diabetes in C57BL/6 mice （n=58）. The ischemic limb received ZFP-VEGF （125 pg ZFP-VEGF plasmid in 1% poloxamer） or placebo （1% poloxamer） intramuscularly. Mice were sacrificed 3, 5, 10, or 20 days post-injection. Limb blood flow was monitored using laser Doppler perfusion imaging. VEGF mRNA and protein expression were examined using real-time PCR and ELISA, respectively. Capillary density, proliferation, and apoptosis were examined using immunohistochemistry techniques. Flow cytometry was used to detect the EPC population in bone marrow. Two-tailed Student＇s paired t test and repeated-measures analysis of variance were used for statistical analysis. Results ZFP-VEGF increased VEGF mRNA and protein expression at 3 and 10 days post-injection, and increased EPC in bone marrow at day 5 and 20 post-injection compared with controls （P〈0.05）. ZFP-VEGF treatment resulted in better perfusion recovery, a higher capillary density and proliferation, and less apoptosis compared with controls （P〈0.05）. Conclusions Intramuscular ZFP-VEGF injection promotes therapeutic angiogenesis in an ischemic hindlimb model with type 1 diabetes. This might be due to the effects of VEGF on cell survival and EPC recruitment.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Bone marrow progenitor cells do not contribute to liver fibrogenic cells

AIM:To investigate the contribution of bone marrow(BM) cells to hepatic fibrosis.METHODS:To establish a model of chimerism,C57Bl/6 female mice were subjected to full-body irradiation(7 Gy) resulting in BM myeloablation.BM mononuclear cells obtained from male transgenic mice expressing enhanced green fluorescent protein(GFP) were used for reconstitution.Engraftment was confirmed by flow cytometry.To induce liver injury,chimeric animals received carbon tetrachloride(CCl4) 0.5 mL/kg intraperitoneally twice a week for 30 d(CCl4 30 d) and age-matched controls received saline(Saline 30 d).At the end of this period,animals were sacrificed for post mortem analysis.Liver samples were stained with hematoxylin and eosin to observe liver architectural changes and with Sirius red for collagen quantification by morphometric analysis.α-smooth muscle actin(α-SMA) was analyzed by confocal microscopy to identify GFP+ cells with myofibroblast(MF) characteristics.Liver tissue,BM and peripheral blood were collected and prepared for flow cytometric analysis using specific markers for detection of hepatic stellate cells(HSCs) and precursors from the BM.RESULTS:Injury to the liver induced changes in the hepatic parenchymal architecture,as reflected by the presence of inflammatory infiltrate and an increase in collagen deposition(Saline 30 d = 11.10% ± 1.12% vs CCl4 30 d = 12.60% ± 0.73%,P = 0.0329).Confocal microscopy revealed increased reactivity against α-SMA in CCl4 30 d compared to Saline 30 d,but there was no co-localization with GFP+ cells,suggesting that cells from BM do not differentiate to MFs.Liver flow cytometric analysis showed a significant increase of CD45+/GFP+ cells in liver tissue(Saline 30 d = 3.2% ± 2.2% vs CCl4 30 d = 5.8% ± 1.3%,P = 0.0458),suggesting that this increase was due to inflammatory cell infiltration(neutrophils and monocytes).There was also a significant increase of common myeloid progenitor cells(CD117+/CD45+) in the livers of CCl4-treated animals(Saline 30 d = 2.16% ± 1.80% vs CCl4 30 d = 5.60% ± 1.30%,P = 0.0142).In addition the GFP-/CD38+/CD45-subpopulation was significantly increased in the CCl4 30 d group compared to the Saline 30 d group(17.5% ± 3.9% vs 9.3% ± 2.4%,P = 0.004),indicating that the increase in the activated HSC subpopulation was not of BM origin.CONCLUSION:BM progenitor cells do not contribute to fibrosis,but there is a high recruitment of inflammatory cells that stimulates HSCs and MFs of liver origin.? 2012 Baishideng.All rights reserved.

Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP- C1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluoresce- nce microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection. Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

ABI家族成员3结合蛋白在血管紧张素Ⅱ诱导内皮祖细胞功能障碍中的作用及机制研究

目的探讨ABI家族成员3结合蛋白(ABI family member 3-binding protein,ABI3BP)在血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导内皮祖细胞功能障碍中的作用及机制。方法为探讨ABI3BP在AngⅡ诱导内皮祖细胞功能障碍中的作用,将细胞分为4组,sh-NC组[转染阴性对照短发夹RNA(LV-scramble-shRNA)+磷酸盐缓冲液(phosphate buffered saline,PBS)]、sh-ABI3BP组[转染ABI3BP shRNA(LV-ABI3BP-shRNA)+PBS]、sh-NC+AngⅡ组(LV-scramble-shRNA+AngⅡ)和sh-ABI3BP+AngⅡ组(LV-ABI3BP-shRNA+AngⅡ)。采用Transwell实验检测细胞迁移能力,黏附实验检测细胞黏附能力,Matrigel检测细胞成管能力,原位末端标记法检测细胞凋亡。Western blot检测整合素β1-黏着斑激酶(focal adhesion kinase,FAK)-P53信号通路变化情况。结果与sh-NC组比较,sh-NC+AngⅡ组迁移细胞数量、黏附细胞数量、小管形成数量显著降低,细胞凋亡率、整合素β1、磷酸化FAK(p-FAK)/FAK及P53蛋白表达显著增高,差异有统计学意义(P<0.05)。与sh-NC+AngⅡ组比较,sh-ABI3BP+AngⅡ组迁移细胞数量[(88.67±8.33)个vs(62.33±7.37)个]、黏附细胞数量[(104.33±6.03)个vs(68.33±10.05)个]、小管形成数量[(36.33±3.21)个vs(19.33±3.06)个]显著增高,细胞凋亡率、整合素β1、p-FAK/FAK及P53蛋白表达水平显著降低,差异有统计学意义(P<0.05)。结论AngⅡ可上调ABI3BP表达,敲低ABI3BP基因表达可改善AngⅡ诱导的内皮祖细胞功能障碍,其机制可能与抑制整合素β1-FAK-P53信号通路有关。

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

Background Macrophage stimulating protein （MSP） is produced by human bone marrow endothelial cells. In this study we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor （SCF）, interleukin 3 （IL-3）, interleukin 6 （IL-6）, granulocyte macrophage-colony stimulating factor （GM-CSF）, erythropoietin （EPO） （Cys） and MSP or of Cys and bone marrow endothelial cell conditioned medium （EC-CM）. Methods Human bone marrow CD34^＋ cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit （CFU-GM） and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte （CFU-GEMM） were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium （NBT） reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. Results MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow （BM） CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys＋EC-CM, Cys＋MSP or Cys compared with 0 hour control in liquid culture system after 6 days. Conclusion MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

蛋白激酶D1在大鼠骨髓源性内皮祖细胞中的促血管新生作用

目的探讨蛋白激酶D1(PKD1)对大鼠骨髓源性内皮祖细胞(EPCs)黏附、迁移、增殖、血管形成能力及内皮型一氧化氮合成酶(e NOS)表达的影响。方法体外培养、分离和鉴定大鼠骨髓源性EPCs,观察PKD1及其特异性阻断剂CID755673对EPCs的黏附、迁移、增殖、血管形成能力的影响,以及对EPCs中e NOS的mRNA表达和蛋白表达的影响。结果 EPCs体外细胞培养实验表明,PKD1可明显促进EPCs的黏附、迁移和增殖,提升EPCs的血管形成能力,上调EPCs中e NOS的mRNA表达和蛋白表达水平。结论 PKD1具有调控EPCs促血管新生的作用,其促血管新生的作用可能以一种依赖e NOS的方式进行。

瑞舒伐他汀抑制C-反应蛋白诱导的晚期内皮祖细胞炎症因子的表达

目的:观察瑞舒伐他汀对C-反应蛋白(CRP)诱导晚期内皮祖细胞分泌单核细胞趋化蛋白1(MCP-1)、白细胞介素8(IL-8)、血管细胞黏附分子1(VCAM-1)及细胞间粘附分子-1(ICAM-1)的影响。方法:密度离心法获取人脐血单个核细胞并培养获得晚期内皮祖细胞。以不同浓度和不同时间CRP刺激晚期内皮祖细胞,以及将晚期内皮祖细胞用不同浓度(10-8mol/L、10-7mol/L和10-6mol/L)瑞舒伐他汀预孵育12 h后再用CRP 50 mg/L刺激,观察细胞的炎症变化。采用荧光定量PCR方法检测IL-8、MCP-1、VCAM-1和ICAM-1 mRNA表达情况,用ELISA检测细胞上清液MCP-1和VCAM-1蛋白表达情况。结果:IL-8、MCP-1、VCAM-1和ICAM-1 mRNA表达量及MCP-1和VCAM-1蛋白表达量均随CRP刺激浓度的增高逐渐增加;50 mg/L CRP不同时间刺激下IL-8、VCAM-1和ICAM-1 mRNA表达量在6 h达高峰,MCP-1则在3 h即达高峰,而MCP-1和VCAM-1蛋白水平均随CRP刺激时间增加逐渐增高。而以不同浓度瑞舒伐他汀预孵育,可以浓度依赖性地抑制CRP诱导的晚期内皮祖细胞各炎症因子的表达。结论:不同浓度、不同时间的CRP刺激可增加晚期内皮祖细胞炎症因子表达,进一步证明CRP并非仅仅为炎症标志物;瑞舒伐他汀可显著抑制CRP诱导晚期内皮祖细胞炎症因子的表达,从一个新的角度阐明了瑞舒伐他汀的作用机制。

血府逐瘀汤对血管紧张素Ⅱ诱导大鼠骨髓源内皮祖细胞衰老及p53、SIRT1蛋白表达的影响

目的观察血府逐瘀汤对血管紧张素Ⅱ(AngⅡ)诱导大鼠骨髓源内皮祖细胞(EPCs)衰老及对p53、SIRT1蛋白表达的影响,探讨其抗衰老的作用机制。方法体外培养EPCs,加入100 nmol/L AngⅡ诱导细胞衰老,建立EPCs衰老模型,随机分为正常组、模型组、miR-34a抑制剂组、血府逐瘀汤5%药物血清组、血府逐瘀汤10%药物血清组、血府逐瘀汤15%药物血清组。β-半乳糖苷酶染色法检测内皮祖细胞衰老,Western blot检测EPCs中p53蛋白、SIRT1蛋白的表达。结果与正常组比较,模型组EPCs衰老数量明显增多(P<0.01),p53蛋白表达量明显升高(P<0.01),SIRT1蛋白表达量明显降低(P<0.01);与模型组比较,miR-34a抑制剂组和5%、10%、15%药物血清组EPCs衰老数量显著减少(P<0.01),miR-34a抑制剂组和10%、15%药物血清组p53蛋白表达显著降低(P<0.05,P<0.01),miR-34a抑制剂组和10%、15%药物血清组SIRT1蛋白表达明显升高(P<0.05,P<0.01);与miR-34a抑制剂组比较,5%、10%、15%药物血清组EPCs衰老数量显著增多(P<0.05,P<0.01),p53蛋白表达明显升高(P<0.05,P<0.01),SIRT1蛋白表达明显下降(P<0.01);与5%药物血清组比较,10%、15%药物血清组EPCs衰老数量明显减少(P<0.01),p53蛋白表达显著降低(P<0.01),SIRT1蛋白表达显著升高(P<0.01);与10%药物血清组比较,15%药物血清组EPCs衰老数量明显减少(P<0.01),p53蛋白表达差异不明显,SIRT1蛋白表达明显升高(P<0.01)。结论血府逐瘀汤能延缓EPCs的衰老,可明显降低衰老EPCs中p53蛋白的表达,显著增加衰老EPCs中SIRT1蛋白的表达,其中以15%药物血清组效果最佳。

蛋白激酶D1促血管新生的体内外实验分析

目的:探讨蛋白激酶D1(PKD1)促血管新生的作用,为心肌梗死等缺血性疾病以PKD1为治疗靶点提供新的思路。方法:体外培养、分离和鉴定内皮祖细胞(EPCs),观察PKD1及其特异性阻断剂CID755673对EPCs中血管内皮生长因子(VEGF)及其受体KDR表达的影响。复制大鼠心肌梗死模型,分析PKD1及CID755673干预对大鼠心肌梗死后受损心肌组织病理形态学、微血管和内皮细胞变化以及VEGF、KDR表达的影响。结果:EPCs体外细胞培养实验表明,PKD1可明显上调EPCs中VEGF和KDR的表达水平。大鼠心肌梗死动物实验结果表明,PKD1干预后的大鼠心肌组织排列较为有序,结构较为清晰,内皮细胞胞膜光滑、完整,周细胞可见,心肌组织中的VEGF和KDR表达水平显著上调。结论:PKD1有明显的促血管新生作用,该作用可能是通过VEGF介导而实现的。

p53-miR-34a-SIRT1反馈环在血管内皮祖细胞复制性衰老过程中的调控作用及机制

目的:研究p53-miR-34a-SIRT1反馈环在血管内皮祖细胞(EPC)复制性衰老过程中的调控作用及机制。方法:培养并鉴定由脐带血来源的EPC;观察第三代和第六代EPC在衰老、凋亡、周期和血管形成等方面的差异;检测第三代和第六代EPC中p53、乙酰化的p53(Ac-p53)、沉默信息调节因子2相关酶1(SIRT1)的表达情况;构建携带微小核糖核酸(miR)-34a抑制因子的慢病毒载体,明确外源性miR-34a抑制因子能否延缓第六代EPC的凋亡。结果:成功培养了脐带血来源的EPC。第六代EPC的衰老率和凋亡率明显高于第三代EPC,细胞周期主要停留在G0/G1期;第六代EPC的p53表达量高于第三代EPC,Ac-p53和SIRT1表达则相对较低,组间差异均有统计学意义(P均<0.05)。转染携带miR-34a抑制因子的慢病毒载体后,第六代EPC衰老情况有所改善,血管形成能力也增强,晚期凋亡率明显减少。结论:p53-miR-34a-SIRT1是EPC复制性衰老过程中的重要反馈调节机制,miR-34a抑制因子可能是延缓EPC衰老的靶点。

C反应蛋白对外周血内皮祖细胞数量及功能的影响

目的研究C反应蛋白对人外周血内皮祖细胞数量及功能的影响。方法从外周血中分离出单个核细胞,体外培养7天,在贴壁细胞中加入不同浓度的C反应蛋白(1 mg/L、2.5 mg/L和5.0 mg/L)作用不同时间(24 h、48 h和72 h),用四唑盐比色试验(MTT)和细胞集落形成单位计数的方法评价C反应蛋白对内皮祖细胞增殖的影响;采用趋化试验评价不同浓度的C反应蛋白对血管内皮生长因子诱导的内皮祖细胞趋化能力的影响;检测细胞上清中一氧化氮的浓度变化;逆转录—聚合酶链式反应检测细胞内皮源性一氧化氮合酶表达强度变化。结果C反应蛋白减少内皮祖细胞的集落形成单位数量及抑制内皮祖细胞的增殖能力;随着C反应蛋白浓度的增加,内皮祖细胞的趋化能力受到抑制;同样细胞分泌的一氧化氮减少,内皮源性一氧化氮合酶表达减弱。结论C反应蛋白可能通过抑制内皮祖细胞的增殖和趋化能力促进内皮功能不全的发展。

冠心病患者高敏C反应蛋白与循环内皮祖细胞的关系及临床意义

目的研究冠心病患者血清高敏C反应蛋白(hs-CRP)与循环内皮祖细胞(EPCs)水平之间的关系及临床意义。方法将46例研究对象随机分为稳定型心绞痛(SAP)组(n=18)、急性冠脉综合征(ACS)组(n=24)及对照组(n=36),并根据冠状动脉造影结果分为单支病变组(n=18)、双支病变组(n=14)及三支病变组(n=10)。胶乳凝集反应法测定冠状动脉造影患者高敏CRP浓度,同时采集研究对象外周血进行EPCs的分离培养,倒置相差显微镜下计数细胞克隆形成单位评估循环EPCs水平。结果ACS组hs-CRP浓度明显高于对照组和SAP组,而SAP组及ACS组循环EPCs水平明显低于对照组(P<0.05);双支病变组与三支病变组hs-CRP水平较对照组显著升高(P<0.05),冠状动脉病变(单支、双支、三支)组EPCs水平均显著低于对照组(P<0.01)。高敏C反应蛋白与循环内皮祖细胞水平呈负相关(r=-0.429,P<0.05)。结论CRP和EPCs与冠心病及冠状动脉病变程度具有相关性,且CRP和EPCs两者之间呈负相关。提示CRP可能通过抑制EPCs数量从而减弱EPCs参与损伤内皮修复的能力,与冠心病发生及临床表现相关。

通心络对晚期糖基化终产物诱导的内皮祖细胞存活的影响及其机制

目的探讨通心络对晚期糖基化终产物(AGEs)诱导的内皮祖细胞(EPCs)存活的影响及相关的分子机制。方法分离出人外周血单个核细胞后经体外诱导分化为EPCs,采用Western印迹法检测通心络对糖基化人血清白蛋白(AGE-HSA)刺激的EPCs的丝裂原活化蛋白激酶(MAPK)通路以及AGEs受体(RAGE)表达的影响,同时观察不同浓度通心络对AGE-HSA刺激的EPCs增殖及凋亡的影响。结果通心络超微粉溶液可抑制由AGE-HSA诱导的EPCs MAPK信号通路的激活,其中以100mg/L的通心络对p38和细胞外信号调节酶(ERK)MAPK信号通路的抑制作用最为显著(P值均<0.01)。结论通心络主要通过抑制AGE-HSA诱导EPCs MAPK(主要是p38和ERK)信号通路的激活,下调EPCs RAGE的表达,进而抑制EPCs的凋亡并促进其增殖。

绿色荧光蛋白和红色荧光蛋白共转染骨髓间充质干细胞、乳鼠心肌细胞、Eahy926细胞表达特征的共聚焦分析

目的利用携带绿色荧光蛋白(GFP)、红色荧光蛋白(RFP)报告基因的慢病毒载体共转染,观察2种报告基因在人骨髓间充质干细胞(hMSC)、乳鼠心肌细胞(Myo)和内皮细胞株(Eahy926)中的整合表达特征。方法应用人类1型免疫缺陷病毒改造而构建慢病毒载体,将GFP、RFP以同时或先后方式共转染hMSC、乳鼠Myo和Eahy926细胞,激光共聚焦显微镜观察2种报告基因在不同细胞中的共表达特点。结果 3种细胞均能被GFP、RFP共转染,其中hMSC和乳鼠Myo均有竞争加随机转染方式,Eahy926只有随机转染方式;3种细胞转染GFP、RFP的效率有所不同,共表达2种报告基因的细胞表现为以核为中心的表达区域重叠。结论 hMSC、乳鼠Myo和Eahy926细胞均可共转染2种报告基因,但整合方式有所不同,这将对利用多个报告基因同时示踪细胞多种生命特征提供有力帮助。

表达HBx的肝前体细胞的小鼠肝内移植模型的构建

目的构建表达乙肝病毒X基因(HBx)的肝前体细胞,并经小鼠门静脉注射建立肝内移植模型,为进一步研究HBx对肝前体细胞的作用和影响及其在原发性肝癌发病机制中的作用奠定了基础。方法分别将表达HBx基因的重组腺病毒Ad-HBx-GFP和空载体Ad-GFP转染小鼠肝前体细胞,再经小鼠门静脉注射后3、7、14 d取血清和肝组织,检测血清丙氨酸氨基转移酶(ALT)变化情况,观察绿色荧光蛋白表达情况,免疫组织化学法观察HBx阳性染色,并取表达高峰在第7天的肝组织,进行Western blot法和实时荧光定量PCR(Real-time PCR)法分别检测HBx的蛋白及mRNA表达。结果成功构建了表达HBx并靶向针对肝前体细胞的小鼠模型,绿色荧光蛋白表达量、免疫组化阳性细胞数量及血清ALT均在注射后第7天达到高峰,RT-PCR及Western blot均检测到第7天14-19/Ad-HBx-GFP组小鼠肝组织目的基因及蛋白的特异性表达,与14-19/Ad-GFP组及生理盐水注射组相比结果具有统计学差异。结论成功构建了表达HBx并靶向针对肝前体细胞的小鼠模型,操作可靠,重复性好,效果稳定,不仅对探讨原发性肝癌的发生机制奠定了基础,也为肿瘤动物模型的构建提供了新的思路。

高敏C反应蛋白对骨髓内皮祖细胞的黏附和增殖抑制作用

目的观察体外培养的骨髓内皮祖细胞(EPCs)在C反应蛋白作用后功能的改变,探讨C反应蛋白影响EPCs修复损伤血管内膜的可能机制。方法使用密度梯度离心法从骨髓获取单个核细胞并进行体外培养。对贴壁细胞进行细胞化学分析,以激光共聚焦显微镜鉴定FITC标记的荆豆凝血素I(FITC-UEA-I)和DiI一标记的乙酰化低密度脂蛋白(DiI-acLDL)双染色阳性细胞为正在分化的EPCs。加入不同浓度C反应蛋白,测定C反应蛋白作用后不同时间EPCs迁移、黏附、增殖能力。结果C反应蛋白作用后骨髓EPCs迁移、黏附和增殖能力明显减退。结论C反应蛋白可使骨髓EPCs迁移、黏附、增殖功能受损,且这种变化与C反应蛋白浓度与作用时间呈正相关。