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1.
AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.  相似文献   

2.
AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34+ and CD34- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34+ and CD34-,total MNC led to a significant increase in the expansion of CD34+ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34+, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34+ and AC133+cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34+ and CD34- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.  相似文献   

3.
AIM: To determine the combined effect of transmuscle laser revascularization (TMR) and endothelial progenitor cells(EPCs) treatment on ischemic hindlimb of nude rats.METHODS: Mononuclear cells (MNCs) isolated from human umbilical cord-blood (HUCB) by density gradient centrifugation were expanded in vitro. Immunocytochemistry and flow cytometry studies were performed. EPCs were labeled with 1, 1- dioctadecyl-1 to 3, 3, 3, 3- tetramethyl-indocarbocyanine perchlorate (DiI) before injected into the laser induced channels or ischemic region. Acute ischemic limb was created in 4 groups of nude rats by ligating right external iliac artery. All animals were divided randomly into the following four groups: TMR+EPCs group: local transplantation of EPCs into laser channels; TMR group: transmuscular channels were created without EPCs; EPCs group: EPCs were injected into ischemic hindlimb; control group: ischemic model without TMR or EPCs. All rats underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtained a flow ratio ［femoral artery flow index (FAFI): right femoral artery flow /left femoral artery flow］ at baseline (after ligating artery immediately) and 28 days postoperation, and then the samples of ischemic limb muscle underwent histochemical and immunohistologic analysis. RESULTS: The attached cells expressed endothelial cell (ECs) markers (KDR, CD34, CD31, AC133 and von Willebrand factor) and exhibited function similar to that of ECs judged by Ac-LDL incorporation. Flow cytometric analysis disclosed that AT cells were positive for CD34 (62%±7%) and AC133 (57.2%±9.8%) at day 7 of culture. 28 days after therapy, FAFI was significantly higher in the TMR +EPCs (0.66±0.09, P<0.01) and EPCs group (0.59±0.09, P<0.05) compared to control group (0.47±0.05). It was significantly higher in TMR +EPCs-，EPCs- and TMR group compared to baseline (TMR+EPCs group: 0.66±0.09 vs 0.39±0.07, P<0.01; TMR group: 0.54±0.12 vs 0.40±0.09, P<0.05; EPCs group: 0.59±0.09 vs 0.38±0.08, P<0.01; control group: 0.47 ±0.05 vs 0.39±0.08, P>0.05). FAFI in the control group was unchanged and no difference was found between TMR group and control group. TMR+EPCs (5.66±0.77), TMR (4.96±0.31) as well as EPCs (4.68±0.44) treatment resulted in an increased number of capillaries in the treated regional area compared with control group (2.60±0.31, P<0.01).CONCLUSION: Nd: YAG-laser revascularization combined with the application of EPCs transplantation significantly ameliorates perfusion and augments neovascularization in this ischemic hindlimb model.  相似文献   

4.
AIM: To investigate the effect of hyperoxia exposure on the paracrine function of endothelial progenitor cells (EPCs), and to explore the effects of paracrine factors of EPCs on the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ) exposed to hyperoxia. METHODS: Bone marrow-derived mononuclear cells were isolated and cultured in EGM-2MV medium for 7~10 d to obtain and identify EPCs. EPCs were cultured in room air (RA) or 60% O2. The normoxia EPC-conditioned medium (E-CM-RA) and hyperoxia EPC-conditioned medium (E-CM-O2) were collected. The levels of VEGF, FGF10, PDGF-BB and EGF in E-CM-RA and E-CM-O2 were detected by ELISA. AECⅡ from adult rats were isolated, purified and cultured for 2 d, then divided into RA group, O2 group, O2+E-CM-RA group and O2+E-CM-O2 group. The proliferation of AECⅡ was detected by MTT assay and cell counting. The mRNA expression of SP-C and AQP5 was quantified by real-time PCR. RESULTS: The expression of VEGF and FGF10 in E-CM-O2 group decreased significantly compared with E-CM-RA group (P<0.01). There were significant differences in AECⅡ viability and number among the 4 groups at 12 h, 24 h, 2 d and 3 d (P<0.01). Compared with RA group, AECⅡ viability and number in O2 group decreased significantly at 12 h, 24 h, 2 d and 3 d (P<0.05). The AECⅡ viability and number in O2+E-CM-RA group were significantly higher than those in O2 group at 12 h, 24 h, 2 d and 3 d (P<0.05). However, no significant difference in AECⅡ viability and number between O2+E-CM-O2 group and O2 group at 12 h, 2 d and 3 d was observed. There were significant differences in the mRNA expression of SP-C and AQP5 in the 4 groups at 24 h, 2 d and 3 d (P<0.01). Compared with RA group, the mRNA expression of SP-C in O2 group was significantly inhibited (P<0.01), but the mRNA expression of AQP5 was promoted (P<0.01) at 24 h, 2 d and 3 d. Compared with O2 group, the mRNA level of SP-C in O2+E-CM-RA group and O2+E-CM-O2 group (P<0.05) at 24 h, 2 d and 3 d was increased, and the mRNA expression of AQP5 (P<0.01) at 2 d and 3 d was inhibited.CONCLUSION: EPCs secrete VEGF and FGF10, and hyperoxia impairs this paracrine function. Hyperoxia exposure inhibits AECⅡ proliferation and the mRNA expression of SP-C, but promotes the mRNA expression of AQP5. EPC-conditioned medium improves the proliferation of hyperoxia-exposed AECⅡ, and inhibits the transformation of AECⅡ. Hyperoxia exposure impairs the paracrine function of EPCs, and weakened the effects of E-CM-O2 on AECⅡ.  相似文献   

5.
AIM: To investigate the gender differences and influence of menstrual cycle on the number and activity of adult circulating endothelial progenitor cells (EPCs), and the effect of estradiol on EPCs. METHODS: Ten men and 10 women were enrolled in the study. Peripheral blood samples of the men were collected only once and peripheral blood samples of the women were collected at each menstrual cycle phase (menstrual, pre-ovulatory and mid-luteal phases). The number of CD34+/CD133+/kinase insert domain-containing receptor (KDR)+ EPCs was determined by flow cytometry analysis and the level of circulating estradiol was measured by radioimmunoassay. Mononuclear cells were isolated from the blood and cultured in vitro. After cultured for 7 days, the number and the adhesive capacity of EPCs were observed. The effect of estradiol on the EPCs were detected by transmembrane migration assay and proliferation assay. RESULTS: The number of circulating EPCs was significantly higher in women than that in men (P<0.01), and it was higher at the pre-ovulatory phase and the mid-luteal phase than that at the menstrual phase (P<0.05). After cultured in vitro, the activity of EPCs did not reveal gender difference. In the cells treated with estradiol at concentration of ≥1×10-9 mol/L, the capacities of transmembrane migration and proliferation were significantly increased (P<0.05). CONCLUSION: There are the gender differences of adult circulating EPCs between men and women. The number and activity of adult circulating EPCs may be regulated by menstrual cycle. In addition, estrogen plays an important role in the arousing of EPCs.  相似文献   

6.
AIM: To explore the effects of transforming growth factor-α (TGF-α) in the monoclonal formation, proliferation, migration and adhesiveness of human endothelial progenitor cells (EPCs). METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α (final concentrations of 1, 5, 10 μg/L, respectively). At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10 μg/L TGF-α plus 1: 1 000 EGFR-TKI) were set. The effects of TGF-α on monoclonal formation, proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment, thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays, respectively. The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor (VEGF) were measured by Western blotting. RESULTS: Different concentrations of TGF-α all significantly induced the monoclonal formation, proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI. The results of Western blotting showed that TGF-α also induced the expression of EGFR and VEGF with a certain concentration effect (P<0.01). CONCLUSION: By combining with EGFR induced the expression of VEGF, TGF-α significantly promotes the related cell function of monoclonal formation, proliferation, migration, adhesiveness in EPCs.  相似文献   

7.
AIM: To investigate whether Chinese yellow wine has influences on homocysteine (Hcy)-induced dysfunction in rat endothelial progenitor cells (EPCs). METHODS: Rat bone marrow was extracted to harvest mononuclear cells (MNCs) by density gradient centrifugation. The MNCs were plated on fibronectin-coated culture dishes, and were induced into EPCs by EGM-2 complete medium supplemented with cell growth factor. The adherent cells were collected 7 d later for all studies. EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptaking and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The viability, migration, apoptosis and in vitro vasculogenic activity of the EPCs were determined by MTT assay, Transwell chamber assay, apoptosis kit and in vitro vasculogenesis kit, respectively. RESULTS: Compared with control group, the viability, migration and in vitro vasculogenic capacity of the EPCs in Hcy group were significantly decreased (P<0.01). Compared with Hcy group, yellow wine group and red wine group both significantly improved the viability, migration and in vitro vasculogenic capacity of Hcy-induced EPCs (P<0.01). Compared with control group, yellow wine group and red wine group both significantly improved the above-mentioned functions of EPCs (P<0.05). However, no significant difference of apoptosis in all groups was observed. CONCLUSION: Hcy may result in dysfuction of EPCs. Treatment with yellow wine improves Hcy-induced EPC functions.  相似文献   

8.
AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.  相似文献   

9.
AIM: To investigate the role of nitric oxide in proliferation and secretion of vascular endothelial cells induced by vascular endothelial growth factorr (VEGF). METHODS: The in vitro cultured vascular endothelial cells of rabbit aorta were divided into control group, VEGF-treated group and VEGF+L-NAME treated group, the absorbance (A) value of vascular endothelial cells, endothelin-1(ET-1) and von Willebrand factor (vWF) in the supernatant were examined by WST-1 assay, radioimmunoassay and ELISA. RESULTS: The A value in VEGF and VEGF+L-NAME treated group were higher than that in control group (P＜0.01). A value in VEGF group was higher than that in VEGF+L-NAME group the ET-1 and vWF were markedly decreased in VEGF group compared with the control and VEGF+L-NAME treated group (P＜0.05, P＜0.01). These results indicated that VEGF promoted the proliferation and inhibited the secretion of ET-1 and vWF in vascular endothelial cells, and L-NAME inhibited the effect of VEGF. CONCLUSION:Nitric oxide is an important mediator in the process of stimulating proliferation and regulating secretion of vascular endothelial cells by VEGF.  相似文献   

10.
AIM:To investigate the effects of atorvastatin reloading in pre-percutaneous coronary intervention (PCI) period on endothelial progenitor cell (EPC) count and inflammatory cytokine expression in the stable angina pectoris patients who had previously received long-term statin treatment. METHODS:The patients with stable angina pectoris that had received long-term statin therapy and planned to accept PCI were randomized into 3 groups: 80 mg atorvastatin 12 h and 40 mg 2 h before coronary angioplasty (80 mg reloading), pre-operatively with 40 mg/d atorvastatin for 7 d (40 mg reloading), and without atorvastatin reloading (no reloading). CD45-/CD133+/CD34+, CD45-/CD34+/KDR+ and CD45-/CD144+/KDR+ EPCs in 100 μL peripheral blood were determined by flow cytometry 1 h prior to PCI and 1 h, 6 h and 24 h after PCI. The serum concentrations of soluble intercellular adhesion molecule 1 (sICAM-1), C-reactive protein (CRP) and troponin I (TnI) were analyzed immediately prior to and 24 h after PCI. RESULTS: (1) In 80 mg reloading group, the numbers of circulating CD45-/CD133+/CD34+ and CD45-/CD34+/ KDR+ early differentiation stage EPCs 1 h and 6 h after coronary angioplasty was significantly elevated compared with those before PCI (P<0.05). (2) In control group, the serum concentrations of sICAM-1 and CRP 24 h after PCI were significantly elevated (P<0.05) compared with preoperative values. (3) The rise in serum TnI concentration from pre- to post-operation in 80 mg reloading group was lower than that in control group. CONCLUSION: The method of atorvastatin reload before PCI affects the number of EPCs in peri-operative period. High dose of atorvastatin application before PCI triggers early EPC circulation. The serum levels of post-operative inflammatory cytokine sICAM-1 as well as CRP are reduced by atorvastatin reloading before PCI.  相似文献   

11.
LI Yan  TANG Ke-xin  LI Hong  ZHANG Jie  CHENG Min 《园艺学报》2011,27(12):2296-2301
AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.  相似文献   

12.
AIM: To investigate the effects of serum from rats at different ages on the functional activities of rat bone marrow-derived endothelial progenitor cells (EPCs). METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 month-old) and aged (19 to 26 month-old) Sprague-Dawley rats by Ficoll density gradient centrifugation and cultured with medium DMEM/F12 (containing 10% fetal bovine serum, endothelial cell growth supplement ECGs 100 mg/L, 1×105 U/L of penicillin and streptomycin, respectively), 48 h later, the suspending cells were translocated to be cultured in new flasks coated with fibronectin, the secondary attached cells were used to perform the further experiments. EPCs were characterized as double positive for Dil-ac-LDL uptake and lectin binding. The cells were further identified by CD31 and vWF expression. Serum from young (1 to 2 month-old) and aged (19 to 26 month-old) rats was collected and used to culture EPCs. The experiments were divided into four groups: A= aged rat EPCs + aged rat serum; B= aged rat EPCs + young rat serum; C= young rat EPCs +aged rat serum and D= young rat EPCs + young rat serum. After cultured in DMEM/F12 supplemented with 10% serum from rats at different age (no fetal bovine serum addition in this medium), the average fluorescence intensities of EPCs stained with Dil-ac-LDL were tested by laser scanning co-focal microscopy. Migration and proliferation were assayed by modified Boyden chamber and MTT, respectively. Cell adhesion was performed by replacing EPCs onto cultured DAPI-labeled confluent smooth muscle cell monolayer and the adherent cells were counted. RESULTS: Young rat serum significantly improved the ability of aged rat EPCs for uptake of Dil-ac-LDL (P<0.01), increased the migration (P<0.01), adhesion (P<0.05) and proliferation activity (P<0.01) of aged rat EPCs, whereas aged rat serum obviously decreased the migration (P<0.05) and adhesion (P<0.05) activity of the young rat EPCs. CONCLUSION: Young rat serum significantly improves the activity of aged rat EPCs. On the contrary, aged rat serum partially inhibits the activity of young rat EPCs.  相似文献   

13.
AIM: To investigate the impact of various levels of glucose on endothelial progenitor cells (EPCs) proliferation, senescence, and nitric oxide (NO) secretion,and the effect of insulin under high glucose conditions.METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation, cultured with medium 199, and identified to be EPCs at 7th day by flk-1 and AC133 double staining. EPCss were harvested and incubated with glucose (5, 10, 20, 40 mmol/L) or insulin (0.1, 1, 10, 100 nmol/L) under high glucose conditions for 24 h or 7 days. Proliferative capacity, senescence level and NO secretion (after 24 h of incubation) were subsequently determined.RESULTS: High glucose (40 mmol/L) markedly inhibited EPCs proliferation, accelerated EPCs senescence, and decreased NO production (all P<0.05). Compared with high glucose (40 mmol/L) group, insulin intervention promoted EPCs proliferation, inhibited EPCs senescence (prominent at 1 nmol/L, P<0.05), and enhanced NO secretion (prominent at 10 nmol/L, P<0.05).CONCLUSION: High glucose harms EPCs proliferation and function, while insulin alleviates this jeopardy, indicating the protective role of insulin for the cardiovascular system.  相似文献   

14.
AIM: To observe the effects of vascular endothelial growth factor (VEGF) on the proliferation and differentiation of neural stem cells (NSCs) of rats in vitro.METHODS: NSCs isolated from the hippocampal gyrus of SD rats were primary cultured and subcultured,and then divided into two groups: (1) the cells in VEGF group were treated with 150 μg/L VEGF in the culture system,and VEGF was removed at the 7 th day;(2) control group (without VEGF treatment).The cellular morphology of two groups was observed by contrast phase microscope.Nestin and NF-200 expressing cells were detected via immunofluorescence method.The percentages of the immunostaining positive cells in each group at the 7 th day and at the 11 th day were determined.RESULTS: At the 7 th day,the percentage of nestin positive cells in VEGF group was 52.19%±7.95%,vs 29.26%±4.12% in control group (P<0.01).The percentage of NF positive cells in VEGF group was 22.33%±4.13%,vs 38.62%±5.31% in control group (P<0.01).At the 3 th day after VEGF was removed,the percentage of NF positive cells in VEGF group was 43.10%±3.70%,vs 30.56%±4.16% in control group (P<0.01).CONCLUSION: VEGF stimulates the proliferation of neural stem cells and inhibits their differentiation.  相似文献   

15.
AIM: Endothelial progenitor cells (EPCs) are a group of stem cells/progenitor cells, which exist in postnatal body and can be of specially homing to the foci of angiogenesis and then differentiate into endothelial cells.This investigation was to study the method for culturing endothelia progenitor cells (EPCs) in vitro, and to observe its feasibility and condition formed vessel-like structure.METHODS: The cells were isolated from born marrow, peripheral blood, umbilical cord blood or spleen in different laboratories.The EPCs derived from human umbilical cord blood and rabbit peripheral blood were cultured in vitro through adhesion selection and were differentiated into endothelial cells under the induction of special cytokines.The expression of CD34, VEGFR-2, AC133 and VE-cadherin were detected by fluorescence-activated cell sorting.The endothelial cell lineage was confirmed by DiI-ac-LDL up-taking and Ⅷ factor immunocy tochemistry.RESULTS: The EPCs derived from human umbilical cord blood and rabbit peripheral blood were cultured in vitro successfully, forming cord-like and tube-like structure.The EPCs derived from rabbit peripheral blood differentiated more mature and formed vessel-like structure.CONCLUSION: The EPCs derived from human umbilical cord blood and rabbit peripheral blood formed vessel-like structure in vitro.EPCs may be a potential resource of vessel tissue engineering.  相似文献   

16.
AIM: To investigate the effects of triptolide on endothelial progenitor cells from peripheral blood.METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation.The cells were plated on fibronectin-coated culture dishes.After 7 d of culture,adherent cells were characterized by demonstrating the expression of CD34,CD31 and vWF with immunohistochemistry.Adherent cells were stimulated with triptolide (2.5,5.0,10.0,20.0 μg/L) or vehicle control for 24 h.Activities of EPCs in terms of proliferation and migration were determined by MTT assay and modified Boyden chamber assay,respectively.EPCs adhesion assay was performed by replating MNCs on fibronectin-coated dishes,and then the adherent cells were counted.RESULTS: The proliferative,migratory and adhesive capacities of EPCs decreased significantly after 24 h incubation with triptolide,maximum at 20 μg/L (compared to that in control group).In patients with coronary heart disease,the biological function of EPCs was lower than that in patients without coronary heart disease in low dosage of triptolide but almost the same in high dosage group.CONCLUSION: Triptolide may inhibit functional activities of EPCs,the reendothelization and neovascularization of vessel.  相似文献   

17.
AIM: To investigate the effect of vascular endothelial growth factor(VEGF) on the biological function of peripheral endothelial progenitor cells (EPCs) and to find the suitable concentration to promote the growth of EPCs.METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After culture for 4 days,attached cells were incubated with VEGF in a series of concentrations (0,10,20 and 50 μg/L) for 72 h,then attached cells were characterized with immunohistochemistry.EPC proliferation and migration activity were assayed with MTT assay and modified Boyden chamber assay,respectively.EPC adhesion assay was performed by replating MNCs on fibronectin-coated dishes,and then the adherent cells were counted.RESULTS: The EPCs from MNCs were successfully isolated and were differentiated to endothelial cells (ECs).Incubation of isolated human MNCs with VEGF increased the proliferative,migratory and adhesive capacities of EPCs,and this effect was most prominent when the concentration of VEGF was 20 μg/L after 72 hours.At the same concentration of VEGF,the functions of EPCs from patients with masculine coronary arteriography were lower than those of EPCs from patients with negative coronary arteriography.CONCLUSION: Functional activities of EPCs are decreased in patients with masculine coronary arteriography.The results suggest that the low concentration of VEGF may improve functional activities of EPCs.  相似文献   

18.
AIM: To evaluate the immunogenicity of a novel orthopedics materials (graded zirconia-hydroxyapatite composite) in vitro by using peripheral blood mononuclear cells (PBMCs) from healthy young people, and simple zirconia-hydroxyapatite composited material was used as control materials. METHODS: Proliferation of PBMCs cultured in different liquid after 5 days was measured by MTT methods. ELISA was used to detect TNF-α and IL-6 concentration in the supernatant of PBMCs cultured in the extracts after 24 hours. Flow cytometery was used to measure CD69 and CD25 in activated PBMCs cultured in the extracts of the two kinds of materials after 24 hours. RESULTS: The proliferation rate of the simple composite group was significantly lower than that in negative group (P<0.05), but there was no difference between the graded composited group and negative group. After 24 hours culture with LPS, the concentrations of TNF-α and IL-6 in the simple composited group were significantly higher than that in graded composited group, respectively (P<0.05). Cultured with PHA for 24 hours, the ratio of CD69 and CD25 positive PBMCs in the simple composited group was all significantly higher than that in graded composited material group (P<0.01). CONCLUSION: The number of PBMCs activated by the graded composited material is less than the simple composited material and the immunogenicity of the graded composited material is lower than the simple composited material.  相似文献   

19.
WANG Li-ping  LI Li  YAO Ji-wen  LI Bo 《园艺学报》2016,32(7):1180-1188
AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.  相似文献   

20.
AIM: To study the effect of Xiaozhong (detumescence)-Zhitong (analgesia) mixture on the function of vascular endothelial cells of rat skin flaps and the expression of VEGF-Dll4/Notch signaling pathway-related proteins. METHODS: Vascular endothelial cells of rat skin flaps were isolated and cultured. The cells were divided into control group, hypoxia group, hypoxia+detumescence analgesia group, hypoxia+detumescence analgesia+axitinib (VEGF receptor inhibitor) group, and hypoxia+detumescence analgesia+MK-0752 (Notch signaling pathway blocker) group. The serum levels of VEGF were measured by ELISA. The number of dead and living cells at 1 d, 2 d and 3 d after hypoxia was determined by cell calcein-AM and PI double staining. The protein expression levels of VEGF-A, Notch and Dll4 in the cells at 24 h and 48 h were detected by Western blot. RESULTS: Compared with control group, the content of VEGF was increased significantly after 24 h and 48 h, and the protein expression of VEGF-A, Notch and Dll4 was increased significantly (P<0.05). Compared with hypoxia group, the content of VEGF was increased significantly after the intervention of Xiaozhong-Zhitong mixture, the death rate was decreased significantly, and the protein expression of VEGF-A, Notch and Dll4 was increased significantly (P<0.05). Compared with Xiaozhong-Zhitong mixture group, the protective effect of Xiaozhong-Zhitong mixture on hypoxia-induced vascular endothelial cell injury was weakened by VEGF receptor inhibitor, the cell mortality was significantly increased, the content of VEGF was decreased, and the protein expression of VEGF-A, Notch and Dll4 was decreased (P<0.05). After intervention with Notch signaling pathway blocker, the cell viability remained unchanged, the expression level of VEGF-A was increased, and the increased Notch and Dll4 protein expression was effectively resisted (P<0.01). CONCLUSION: Xiaozhong-Zhitong mixture improves the function of vascular endothelial cells of rat skin flaps, and its mechanism may be related to the influence of the signal transduction pathway of VEGF-Dll4/Notch.  相似文献   

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