Circulating endothelial progenitor cells are reduced in hemodialysis patients

OBJECTIVE: The risk of cardiovascular disease in hemodialysis patients is far greater than in the general population. Endothelial progenitor cells (EPCs) circulating in the peripheral blood contribute to neovascularization in the ischemic tissue. EPCs are considered to be included in CD34 positive (CD34+) or AC133 positive (AC133+) mononuclear cells (MNCs). This study's aim was to determine the number and functional activity of EPCs in hemodialysis patients and age-matched control subjects. METHODS: The numbers of CD34+ MNCs and AC133+ MNCs in the peripheral blood were quantified by flow cytometry. The peripheral blood EPCs were also examined by an in vitro culture assay. The levels of serum vascular endothelial growth factor (VEGF) were measured by sandwich enzyme immunoassay. RESULTS: The numbers of CD34+ MNCs and AC133+ MNCs were significantly reduced by 56% and 49%, respectively, in hemodialysis patients (n = 50) compared with control subjects (n = 36). The number of EPCs determined by the culture assay was also significantly reduced by 41% in hemodialysis patients compared with control subjects. Multivariate analysis revealed that none of the atherosclerotic risk factors were independent predictors of reduced CD34+ MNC counts. The serum VEGF levels in hemodialysis patients were not different from those in control subjects and did not correlate with CD34+ MNC counts. CONCLUSION: Circulating EPCs are significantly reduced in hemodialysis patients, which might be related to impaired neovascularization and cardiovascular disease in these patients.     

脂联素对氧化低密度脂蛋白损伤内皮祖细胞的影响及作用机制

目的观察脂联素对氧化低密度脂蛋白（ox-LDL）损伤人外周血内皮祖细胞（EPCs）的影响,并探讨其作用机制。方法密度梯度离心法获取外周血单个核细胞,通过流式细胞仪鉴定EPCs。培养7d后,收集贴壁细胞并随机分为正常对照组,ox-LDL组（10 mg·L~（-1））及脂联素干预组（ox-LDL 10mg·L~（-1）加脂联素,浓度分别为1.25、2.5和5mg·L~（-1））。干预24h后采用细胞活力噻唑蓝（MTT）法测定细胞活力,黏附能力测定实验测定其黏附能力,并测定各组细胞上清液中白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）含量。结果 ox-LDL与EPCs作用后,各组细胞的细胞活力、黏附能力均显著下降,IL-6、TNF-α含量均显著升高;脂联素干预24h后,明显提高了EPCs的黏附能力、细胞活力,降低了IL-6、TNF-a含量。结论脂联素对EPCs具有保护作用,其机制可能与抑制炎症反应有关。     

Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells

Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using CD34⁺ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using CD34⁺ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including CD34⁺, CD34⁺/CD133⁺, CD34⁺/CD31⁺ and CD34⁺/CXCR4⁺. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis.     

Mobilization of progenitor cells into peripheral blood by gamma-tocotrienol: A promising radiation countermeasure

Gamma-tocotrienol (GT3), a vitamin E isoform, is shown to induce high levels of granulocyte colony stimulating factor (G-CSF) in mice. G-CSF is a key cytokine used for stimulation of hematopoiesis, and mobilization of hematopoietic stem and progenitor cells into peripheral blood. GT3 is also shown to induce vascular endothelial growth factor (VEGF), another important cytokine necessary for vasculogenesis and endothelial progenitor mobilization. Since GT3 induces both these cytokines, we tested whether GT3 mobilizes hematopoietic and endothelial progenitors in mice. GT3 (200 mg/kg) was injected in 10-week-old CD2F1 mice and mobilization of progenitors in peripheral blood was analyzed at 24, 48, and 72 h post-administration. Circulating hematopoietic progenitor cells (HPCs, Lin^?, cKit⁺), endothelial progenitor cells (EPCs, Lin^?, CD34⁺, Flk⁺), and stromal progenitor cells (SPCs, Lin^?, CD29⁺, CD105⁺) in peripheral blood mononuclear cells (PBMCs) were analyzed simultaneously by flow cytometry. Mobilized HPCs, EPCs and SPCs in PBMC were also measured by colony-forming unit (CFU) assay in progenitor-specific media. Three groups of mice received vehicle, GT3 and GT3 plus AMD3100, a receptor antagonist used to enhance mobilization. GT3 induced significant mobilization of all three progenitor cell types compared to vehicle in peripheral blood; AMD3100 enhanced GT3-induced mobilization even further. Mobilization of progenitor cells in peripheral blood by GT3 indicates that GT3 can be used as an alternative to G-CSF and VGEF to mobilize HPCs and EPCs.     

脂联素对内皮祖细胞氧化损伤的保护作用及机制研究

目的观察脂联素对氧化低密度脂蛋白（oxidative low density lipoprotein,Ox-LDL）诱导的人外周血内皮祖细胞（endothelial progenitor cells,EPCs）氧化损伤的保护作用,并探讨其可能的机制。方法密度梯度离心法获取外周血单个核细胞,通过流式细胞仪鉴定内皮祖细胞。培养7d后,收集贴壁细胞并随机分为正常对照组、Ox-LDL组（10μg·mL-1）及脂联素干预组（Ox-LDL10μg·mL-1加脂联素,浓度分别为1.25,2.5,5μg·mL-1）,干预24h后采用细胞活力噻唑蓝（MTT）法测定细胞活力。并取各组细胞上清液行超氧化物歧化酶（SOD）、丙二醛（MDA）含量检测。结果脂联素可以改善Ox-LDL诱导氧化损伤的EPCs的活力,增加SOD活性,降低MDA含量。结论脂联素对氧化损伤的EPCs具有保护作用,其机制可能与提高抗氧化酶活性有关。     

Effects of encapsulated rabbit mesenchymal stem cells on ex vivo expansion of human umbilical cord blood hematopoietic stem/progenitor cells

The expansion of umbilical cord blood mononuclear cells (UCB MNCs) was investigated in a novel co-culture system by means of encapsulation of rabbit bone marrow (BM) mesenchymal stem cells (MSCs) in alginate beads (Alg beads). Three kinds of media were applied and the experiments lasted for 7 days. The total nucleated cell density was measured every 24 h. Flow cytometric assay for CD34⁺ cells and methylcellulose colony assays were carried out at 0, 72 and 168 h. It was found that the encapsulated MSCs illustrated remarkable effects on UCB MNCs expansion regardless of whether serum is present in culture media or not. At the end of 168 h co-culture, the total nucleated cell number was multiplied by 15 ± 2.9 times, and CD34⁺ cells 5.3 ± 0.3 times and colony-forming units in culture (CFU-Cs) 5.6 ± 1.2 times in the serum-free media supplemented with conventional dose of cytokines, which was very similar to the results in the containing 20% serum media. While in the control, i.e. MNC expansion without encapsulated MSCs, however, total nucleated cells density changed mildly, CD34⁺ cells and CFU-Cs showed little effective expansion. It is demonstrated that the encapsulated stromal cells can support the expansion of UCB MNCs effectively under the experimental condition.     

Stem cell mobilization and collection in patients with liver cirrhosis

Background Bone marrow‐derived stem cells (BMSC) and granulocyte colony‐stimulating factor (G‐CSF) have been proved to contribute to tissue regeneration after liver injury. Aims To test the safety of G‐CSF and define the exact dose capable of mobilizing BMSC in the majority of patients with liver cirrhosis; and to assess the feasibility of leukapheresis to collect BMSC from peripheral blood. Methods In this study, we treated 18 patients affected by liver cirrhosis with increasing doses of G‐CSF to mobilize CD34⁺ and CD133⁺ BMSC into the peripheral blood. Results The dose‐finding phase demonstrated that 15 μg/kg/day of G‐CSF is the optimal dose to mobilize both CD34⁺ and CD133⁺ stem cells. Circulating BMSC were collected by a single step leukapheresis in three patients and the mean number of CD34⁺ and CD133⁺ cells cryopreserved was 1.3 ± 0.7 and 1.2 ± 0.5 × 10⁶/kg, respectively. No severe adverse events were observed during the drug administration and stem cell collection. Noteworthy is, none of the patients showed a significant modification of liver function. Conclusions Our study demonstrates that G‐CSF administration and BMSC collection from the peripheral blood is possible and safe in patients with liver cirrhosis. The optimal dose to mobilize BMSC in cirrhotics is 15 μg/kg/day. At this dose, G‐CSF does not seem to modify the residual liver function in cirrhotic patients.     

SCID repopulating cells derived from unmobilised adult human peripheral blood

SUMMARY

Severe combined immunodeficient (SCID)-repopulating cells (termed SRC) with lymphohaematopoietic differentiation potential reside at an extremely low frequency in unmobilised adult human peripheral blood. Recently, an ex vivo method of increasing the relative numbers of at least four distinct human stem cell classes, that include CD34⁺ haematopoietic progenitor cells, in mononuclear cells (MNC) obtained from unmobilised adult human peripheral blood has been described. This process is triggered by a monoclonal antibody (mAb) against the human monomorphic region of the beta chain of HLA-DP, DQ and DR (clone CR3/43). Herein, we assess the ability of human male donor-derived MNC, following ex vivo culturing for 3 hr in haematopoietic-conducive conditions (HCC) (3-hr MNC/HCC), to form SRC in female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. All 3-hr MNC/HCC-recipient animals exhibited significant levels (>0.5%) of human cell engraftment in the

bone marrow, thymus and spleen when compared to animals receiving MNC cultured in the absence of CR3/43. Phenotypic characterisation of the bone marrow cell populations of engrafted mice demonstrated significant levels of human lymphohaematopoietic cell lineages, comprised of T lymphocytes, monocytes, erythrocytes and megakaryocytes, including platelets. In addition, significant levels of clonogenic human CD34⁺ cells were also detected by in vitro surrogate assay. The thymi of engrafted animals contained maturating human thymocytes, while the spleen consisted mainly of T lymphocytes. Fluorescence in situ hybridisation (FISH) further identified the presence of human male X and Y chromosomes at engrafted sites, whilst the human origin of the cells was confirmed by a specific PCR assay for the human Cart-1 gene. In conclusion, the conversion of MNC to SRC in response to treatment with CR3/43 for 3 hr could have far-reaching clinical implications especially where time and donor-histocompatibility are limiting factors.     

Therapeutic angiogenesis by bone marrow implantation for critical hand ischemia in patients with peripheral arterial disease: a pilot study

ABSTRACT

Objective: Implantation of bone marrow mononuclear cells (BM-MNCs), including endothelial progenitor cells, into ischemic lower limbs has been shown to improve symptoms in patients with peripheral arterial diseases (PAD). This study investigated whether BM-MNC implantation (BMI) is also effective for the ischemic hands of these patients.

Methods: Seven PAD patients with hand ischemia were enrolled: six patients had thromboangiitis obliterans and one had collagen disease. All seven had symptoms involving either resting pain or non-healing ischemic ulcers of the hand. Approximately 600?mL of MNCs were separated from BM and concentrated to a final volume of 40–50?mL, which were injected into ischemic hands. Ischemic status was evaluated by measuring the digital/brachial pressure index (DBI), visual analog pain scale, and the healing of ulcers before and 6?months after BMI.

Results: The mean number of implanted MNCs, CD34-positive cells, and CD34,133-positive cells was 3.67 ± 0.53 × 10⁹, 4.94 ± 2.45 × 10⁷, and 2.52 ± 1.57 × 10⁷, respectively. Mean DBI in those patients was 0.15 ± 0.30 before BMI and significantly increased to 0.67 ± 0.19 at 6 months after BMI (?p = 0.004). All patients also showed improvement of pain scale and ischemic ulcers. There was no significant correlation between the number of implanted cells and improvement in the degree of DBI or the pain scale.

Conclusion: Autologous BMI could be a promising and safe method of therapeutic angiogenesis for critical hand ischemia in PAD patients.     

银杏叶提取物对外周血内皮祖细胞数量和功能的影响

目的观察银杏叶提取物对外周血内皮祖细胞(endothelial progenitor cell, EPC)数量和功能的影响。方法密度梯度离心法获取外周血单个核细胞，培养7 d后，收集贴壁细胞并加入银杏叶提取物(10, 25和50 mg·L^-1)干预一定时间(6,12,24和48 h)。激光共聚焦显微镜和流式细胞仪鉴定EPC，分别观察EPC的增殖、迁移、粘附和体外血管生成能力。结果银杏叶提取物促进外周血EPC扩增，25 mg·L^-1银杏叶提取物作用24 h对EPC数量的影响最为显著(较对照组增加了1倍， p<0.01)。银杏叶提取物也显著改善了外周血EPC的粘附、迁移、增殖和体外血管生成能力。结论银杏叶提取物可增加EPC数量并改善其功能。     

米诺环素治疗慢性牙周炎的临床疗效及其对外周血T细胞亚群、PD-1、PD-L1表达的影响

目的 探究米诺环素治疗慢性牙周炎的临床疗效及其对外周血T细胞亚群及程序性死亡分子-1（PD-1）、程序性死亡分子-1配体（PD-L1）表达的影响。方法 选择2015年1月-2017年1月在榆林市星元医院口腔科接受治疗的96例慢性牙周炎患者为研究对象。根据随机数字表法将患者分为对照组和观察组,每组各48例。对照组患者采用龈下刮治及根面平整进行治疗。观察组患者在对照组治疗的基础上在牙周袋底部缓慢注入盐酸米诺环素软膏,0.2 g/次,1次/周,边注射边后退。两组均治疗4周。观察两组患者的临床疗效,同时比较两组治疗前后的牙龈指数（GI）、菌斑指数（PLI）、牙槽骨吸收、附着丧失（CAL）以及牙周袋深度（PD）、C反应蛋白（CRP）、白细胞介素-10（IL-10）、龈沟出血指数（SBI）、外周血T淋巴细胞亚群和表面PD-1、PD-L1的表达情况。结果 治疗后,对照组患者临床总有效率为72.92%,显著低于观察组的91.67%（P<0.05）。治疗后,两组患者GI、PLI、PD、CAL和牙槽骨吸收等指标均显著降低（P<0.05）;且观察组以上各指标均显著低于对照组（P<0.05）。治疗后,两组患者龈沟液中CRP水平和SBI评分均显著降低,而龈沟液中IL-10水平显著升高（P<0.05）;且观察组龈沟液中CRP、IL-10水平和龈沟出血指数均优于对照组（P<0.05）。治疗后,两组患者外周血T淋巴细胞亚群中CD4⁺和CD4⁺/CD8⁺均显著降低,CD8⁺显著升高（P<0.05）;且观察组外周血T淋巴细胞亚群中CD4⁺和CD4⁺/CD8⁺均显著低于对照组,CD8⁺比例高于对照组（P<0.05）。治疗后,两组患者外周血CD4⁺和CD8⁺T淋巴细胞表面的PD-1和PD-L1表达水平均显著降低（P<0.05）;且观察组外周血CD4⁺和CD8⁺T淋巴细胞表面的PD-1和PD-L1表达水平均显著低于对照组（P<0.05）。结论 米诺环素治疗慢性牙周炎可有效改善患者各项牙周指标,改善牙周组织炎症反应抑制作用,在慢性牙周炎临床治疗中具有重要价值。     

Effect of interleukin 8 and ICAM-1 on calcium-dependent outflow of K+ in erythrocytes from subjects with essential hypertension

SUMMARY

Introduction: The pathogenic mechanisms underlying the increase in peripheral resistance and the contraction of smooth muscular fibre cells in essential hypertension are not yet clearly understood. However, it is now known that immune system activation plays a role in the pathogenesis of some forms of arterial hypertension, and recent data show that the Ca²⁺ influx in some cells (i.e. red blood cells, leukocytes, platelets, smooth muscular fibre cells) is increased in subjects with essential hypertension, thus revealing a possible alteration in cellular membrane. The end-points of this study were therefore to ascertain whether red blood cells used as a cellular membrane model have a greater Ca²⁺ dependent K⁺ flow (Gardos effect) in hypertensive patients than in normotensive controls, to point out a different regulation of ionic channels, and whether IL-8 and the adhesion molecule ICAM-1 influence the membranous outflow.

Material and methods: The study was conducted on 87 Caucasian subjects. Of these, 50 (25 men, 25 women; mean age 43?±?3 years, mean body mass index (BMI) 27?±?0.5 and 22.3?±?0.3?kg?m², respectively) had mild-to-moderate hypertension (mean arterial blood pressure 120±8mmHg).The other 37 (18 men, 19 women; mean age 39?±?3 years; BMI 23.8?±?0.5?kg?m² and 22.8?±?0.5?kg?m², respectively were normotensive healthy volunteers (mean arterial blood pressure 89?±?2?mm?Hg).All the patients and subjects were untreated for at least 4 weeks before blood sampling.

Results: Ca²⁺-dependent K⁺ outflow was found to be greater in samples from patients with essential hypertension than in those from normotensive controls. lL-8 and ICAM-1 significantly enhanced the Ca²⁺-dependent K⁺ outflow in red blood cells from hypertensive subjects but had an inhibitory effect on cells from controls. In the experimental model, the presence

of TMB-8, a membrane calcium antagonist, significantly reduced the Ca²⁺-dependent K⁺ efflux.

Conclusion: Vasoconstriction in subjects with essential hypertension may therefore depend on a different regulation of ionic flow that probably supports an increased Ca²⁺ inflow in smooth

muscle fibre cells. Under certain pathological conditions, some immune system components (i.e. interleukins, adhesion molecules) may directly enhance membrane permeability to Ca²⁺, thus inducing vasoconstriction in the smooth muscle cells.     

Impact of prior chronic statin therapy and high-intensity statin therapy at discharge on circulating endothelial progenitor cell levels in patients with acute myocardial infarction: a prospective observational study

Background

Endothelial progenitor stem cells (EPCs) are mobilized to the peripheral circulation in response to myocardial ischemia, playing a crucial role in vascular repair. Statins have been shown to stimulate EPCs. However, neither the impact of previous statin therapy on EPC response of acute myocardial infarction (AMI) patients nor the effect of post-AMI high-intensity statin therapy on the evolution of circulating EPC levels has yet been addressed. Therefore, we aimed to compare circulating EPC levels between patients receiving long-term statin therapy before the AMI and statin-naive patients and to assess the impact of high-intensity statin therapy at discharge on the evolution of circulating EPCs post-AMI.

Methods

This is a prospective observational study of 100 AMI patients. Circulating EPCs (CD45dimCD34?+?KDR?+?cells) and their subpopulation coexpressing the homing marker CXCR4 were quantified by the high-performance flow cytometer FACSCanto II in whole blood, in two different moments: within the first 24 h of admission and 3 months post-AMI. Patients were followed up clinically for 2 years.

Results

Patients previously treated with statins had significantly higher levels of EPCs coexpressing CXCR4 (1.9?±?1.4 vs. 1.3?±?1.0 cells/1,000,000 events, p?=?0.031) than statin-naive patients. In addition, the subanalysis of diabetics (N?=?38) also revealed that patients previously on statins had significantly greater numbers of both CD45dimCD34?+?KDR?+?CXCR4+ cells (p?=?0.024) and CD45dimCD34?+?KDR?+?CD133+ cells (p?=?0.022) than statin-naive patients. Regarding the evolution of EPC levels after the AMI, patients not on a high-intensity statin therapy at discharge had a significant reduction of CD45dimCD34?+?KDR?+?and CD45dimCD34?+?KDR?+?CXCR4+ cells from baseline to 3 months follow-up (p?=?0.031 and p?=?0.005, respectively). However, patients discharged on a high-intensity statin therapy maintained circulating levels of all EPC populations, presenting at 3 months of follow-up significantly higher EPC levels than patients not on an intensive statin therapy. Moreover, the high-intensity statin treatment group had significantly better clinical outcomes during the 2-year follow-up period than patients not discharged on a high-intensity statin therapy.

Conclusion

Chronic statin therapy prior to an AMI strongly enhances the response of EPCs to myocardial ischemia, even in diabetic patients. Furthermore, high-intensity statin therapy after an AMI prevents the expected decrease of circulating EPC levels during follow-up. These results reinforce the importance of an early and intensive statin therapy in AMI patients.     

Immune biomarkers in older adults: Role of physical activity

ABSTRACT

Aging is associated with a decline in the normal functioning of the immune system. Several studies described the relationship between immunological alterations, including immunosenescence and inflammation, and aging or age-related outcomes, such as sarcopenia, depression, and neurodegenerative disorders. Physical activity is known to improve muscle function and to exert a number of benefits on older adult health, including reduced risk for heart and metabolic system chronic diseases. However, the positive influence of physical activity on the immune system has not been elucidated. In order to shed light on the role of physical activity in immune responses of older individuals, a number of immunological parameters comprising % lymphocyte subsets (CD3⁺, CD4⁺, CD8⁺, CD19⁺, and CD16⁺56⁺) and serum levels of neopterin and tryptophan metabolism products were evaluated in peripheral blood samples of older adults performing normal (N = 170) or reduced (N = 89) physical activity. In addition, the potential influence of other clinical and epidemiological factors was also considered. Results showed that subjects with reduced physical activity displayed significantly higher levels of CD4⁺/CD8⁺ ratio, kynurenine/tryptophan ratio, and serum neopterin, along with lower %CD19⁺ cells and tryptophan concentrations. Further, some immunological biomarkers were associated with cognitive impairment and functional status. These data contribute to reinforce the postulation that physical activity supports healthy aging, particularly by helping to protect the immunological system from aging-related changes.     

T cell regulation by Phlomis lanata protein extracts in mice

Context: Phytopharmacology is a complex but very promising research area. The different plant parts and extraction methods may result in opposed effects. Phlomis species have been reported for both anti-inflammatory and tonic properties.

Objective: The effect of Phlomis lanata Willd. (Lamiaceae) protein extracts on immune cell reactivity was studied in the experimental mouse model.

Materials and methods: Protein extracts from P. lanata aerial parts were fractionated by Q-sepharose ion-exchange chromatography and applied to whole spleen cells or T-cell subsets at 5?μg/ml. Cell growth and cytokine production were evaluated after 4 and 2?d of culture using ³H-thymidine-uptake and ELISA techniques, respectively.

Results: Among the protein fractions tested, column wash proteins (W1) and the fraction eluted using 600?mM NaCl (F6) reduced by 76% and increased by 78% spleen cell proliferation, respectively. W1 suppressed proliferation of effector T-cells, but stimulated the growth of suppressor/regulatory cells by 62–148%. Although W1 stimulated IL-2 and IL-10 production from total spleen cells, it significantly increased IL-10 (50%) and reduced IL-2 (30–50%) production from T-cells, while TNF-α release was enhanced in CD25⁺CD4⁺ by 92% and reduced by 50% in CD25⁺CD8⁺ cells. F6 stimulated whole spleen cell growth, reduced proliferation of CD8⁺ and CD25⁺ cells by approximately 50%, while decreasing by 60–80% TNF-α production from CD25^? and CD25⁺CD8⁺ cells.

Discussion and conclusion: The suppressive activity of W1 could be attributed to IL-10 and TNF-α, while the stimulatory effect of F6 could be attributed to the inhibition of T-regulatory cells. In the same plant, coexisting protein fractions induce both immunostimulatory and immunosuppressive activities.     

Effects of ox-LDL on number and activity of circulating endothelial progenitor cells

BACKGROUNDS: Endothelial dysfunction is thought to play a crucial role in the pathogenesis of atherosclerosis induced by ox-LDL. Recently, a variety of evidence suggested that endothelial progenitor cells (EPCs) participated in neovascularization and reendothelialization. However, effects of ox-LDL on EPCs number and activity are ill understood. METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with ox-LDL (to make a series of final concentrations: 25 microg/mL, 50 microg/mL, 100 microg/mL, 200 microg/mL), native LDL (100 microg/mL) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. RESULTS: Incubation of isolated human EPCs with ox-LDL decreased the number of EPCs in concentration-dependent manner, maximum at 200 microg/mL (approximately 70% reduction, P < 0.001). In time-course experiments performed with an ox-LDL concentration of 100 microg/mL, decrease of EPCs number became apparent at 12 hours and reached the maximum at 24 hours (approximately 50% reduction, P < 0.01). In addition, ox-LDL dose and time dependently impaired EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. CONCLUSION: The results of the present study defined a novel mechanism of action of ox-LDL: the reduction of EPCs with decreased functional activity.     

双歧杆菌三联活菌胶囊联合乌司他丁对脓毒症机械通气患者肠道菌群及外周血NLRP3、Caspase-1的影响

目的 观察双歧杆菌三联活菌胶囊联合乌司他丁对脓毒症机械通气患者肠道菌群及外周血核苷酸结合寡聚化结构域样受体3（NLRP3）、半胱氨酸天冬氨酸酶-1（Caspase-1）的影响。方法 回顾性收集2017年1月—2020年12月重庆市急救医疗中心收治的98例脓毒症机械通气患者为研究对象,按照治疗方法不同分为对照组和试验组,每组各49例。所有患者均予以常规基础治疗,对照组采用乌司他丁注射液治疗,每次静脉滴注1.0×10⁵ U,每天3次;试验组在对照组基础上加用双歧杆菌三联活菌胶囊治疗,口服,每次0.42 g,每天2次。两组均在治疗7 d后评估治疗效果。比较两组疗效、机械通气时间、症状改善时间、ICU入住时间、住院时间,分别于治疗前、治疗7 d后检测肠道菌群（双歧杆菌、乳酸杆菌、葡萄球菌）菌含量、黏膜屏障功能指标［二胺氧化酶（DAO）、D-乳酸、内毒素（ET）］、免疫功能相关指标（CD3^＋、CD4⁺、CD8⁺、CD4⁺/CD8⁺）水平及外周血NLRP3、Caspase-1蛋白水平,观察两组治疗期间的不良反应发生情况。结果 试验组总有效率为91.84%,较对照组的75.51%显著升高（P＜0.05）。试验组患者机械通气时间、症状改善时间、ICU入住时间、住院时间均显著短于对照组（P＜0.05）。治疗前两组患者肠道双歧杆菌、乳酸杆菌、葡萄球菌含量比较,差异无统计学意义（P＞0.05）;治疗7 d后两组患者肠道双歧杆菌和乳酸杆菌含量均显著升高（P＜0.05）,葡萄球菌含量较治疗前显著降低（P＜0.05）;治疗7 d后试验组患者肠道双歧杆菌、乳酸杆菌含量显著高于对照组,葡萄球菌含量显著低于对照组（P＜0.05）。治疗前两组患者血清DAO、D-乳酸、ET水平比较,差异无统计学意义（P＞0.05）;治疗7 d后两组患者血清DAO、D-乳酸、ET水平均较治疗前显著降低（P＜0.05）,试验组治疗后血清DAO、D-乳酸、ET水平均显著低于对照组（P＜0.05）。治疗前两组患者外周血CD3＋、CD4+、CD8+、CD4+/CD8+水平比较,差异无统计学意义（P＞0.05）;治疗7 d后两组外周血CD3^＋、CD4⁺、CD8⁺、CD4⁺/CD8⁺水平均较治疗前显著升高（P＜0.05）,CD8+水平较治疗前显著降低（P＜0.05）;治疗后试验组外周血CD3^＋、CD4⁺、CD8⁺、CD4⁺/CD8⁺水平均显著高于对照组（P＜0.05）,CD8⁺显著低于对照组（P＜0.05）。治疗前两组外周血NLRP3、Caspase-1蛋白水平比较,差异无统计学意义（P＞0.05）;治疗7 d后两组外周血NLRP3、Caspase-1蛋白水平均较治疗前显著降低（P＜0.05）,治疗后试验组外周血NLRP3、Caspase-1蛋白水平均显著低于对照组（P＜0.05）。两组不良反应总发生率比较,差异无统计学意义（P＞0.05）。结论 双歧杆菌三联活菌胶囊联合乌司他丁治疗脓毒症机械通气患者效果显著,可有效缩短患者机械通气时间、症状改善时间、ICU入住时间、住院时间,恢复肠道菌群平衡,提高黏膜屏障功能及免疫功能,调节外周血NLRP3、Caspase-1水平,且安全性高。     

Effects of electroacupuncture on endothelial function and circulating endothelial progenitor cells in patients with cerebral infarction

This study evaluated the effects of electroacupuncture (EA) on endothelial function and endothelial progenitor cells (EPC) in patients with cerebral infarction. In a randomized, placebo‐controlled, crossover study, 20 patients with cerebral infarction were randomized into two treatment groups: EA or placebo. Before and after each intervention, pulse amplitude tonometry (PAT) was used to assess endothelial function and peripheral blood was analyzed for the number of EPCs. Circulating EPCs were quantified by flow cytometry as CD45^lowCD34⁺KDR2⁺ cells. Plasma vascular endothelial growth factor (VEGF) and interleukin (IL)‐10 levels were measured. Seven days later, crossover was performed on each group, with each group receiving the other treatment using the same protocol. The PAT hyperemia ratio ranged from 1.57 ± 0.41 to 2.04 ± 0.51 after EA, representing a significant improvement (P = 0.002); however, there was no improvement in the placebo group (P = 0.48). Circulating EPCs, as measured by flow cytometry, increased to 110.6 ± 74.3/100 μL in the EA group (P = 0.001) but did not change in the placebo group (45.9 ± 35.3/100 μL, P = 0.08). The increases in the number of EPCs and the PAT ratio after treatment were correlated (r = 0.78, P < 0.001). Plasma VEGF levels increased with EA compared to baseline (261.2 ± 34.0 vs 334.9 ± 80.5 pg/mL, P = 0.003). The number of circulating EPCs was positively correlated with plasma levels of VEGF (r = 0.50, P = 0.02). In conclusion, EA induced improvement of EPC levels and the PAT ratio in patients with cerebral infarction.     

阻塞性睡眠呼吸暂停患者外周血内皮祖细胞及 促血管生成因子水平研究

摘要： 目的 观察阻塞性睡眠呼吸暂停 （OSA） 患者外周血内皮祖细胞 （EPC） 不同亚族和促血管生成因子水平的变化, 探讨不同程度 OSA 患者外周血 EPC 对血管修复的可能性。方法 选取 90 例 OSA 患者和 30 例健康志愿者 (对照组), 根据睡眠呼吸暂停低通气指数(AHI)将 90 例 OSA 患者均分为轻、 中、 重度 OSA 组。密度梯度离心法提取单个核细胞, 依据乙醛脱氢酶(ALDH)活性对 EPC 进行分选, 流式细胞仪联合 CD133、 CD34、 含激酶域插入片段受体 (PE-KDR)相应细胞表面标志物测定 CD133+ KDR+ EPC 及 CD133+ CD34+ EPC、 CD34+ KDR+ EPC、 ALDHlo CD34+ KDR+ EPC 的水平。酶联免疫吸附试验(ELISA)测定患者外周血低氧诱导因子-1α(HIF-1α), 血管内皮生长因子(VEGF)及基质细胞衍生因子-1α(SDF-1α)的水平。结果 对于外周血 CD133+ KDR+ EPC、 CD133+ CD34+ EPC、 CD34+ KDR+ EPC 水平, 重度 OSA 组>中度 OSA 组>轻度 OSA 组>对照组 （均 P < 0.05）; 轻、 中度 OSA 组的外周血 ALDHlo CD34+ KDR+ EPC 水平高于对照组, 重度 OSA 组低于其他 3 组 （均 P < 0.05）; 血清 HIF-1α、 VEGF 均是重度 OSA 组>中度 OSA 组>轻度 OSA 组>对照组, SDF-1α水平为重度 OSA 组<中度 OSA 组<轻度 OSA 组<对照组 （均 P < 0.05）。结论 OSA 患者可能都会诱导动员并招募大量无效 EPC, 其数量庞大, 但直接参与修复内皮的 ALDHlo CD34+ KDR+ EPC 并未增加, 尤其对于重度 OSA 患者甚至有可能减少, OSA 减弱了修复内皮的可能性, 加重了内皮损伤, 从而增加心血管事件的发生风险。