Construction of micelles and hollow spheres via the self-assembly behavior of poly(styrene-alt-pHPMI) copolymers with poly(4-vinylpyridine) derivatives mediated by hydrogen bonding interactions.

This study describes the preparation of hydrogen bonding connected micelles, consisting of a poly(styrene-alt-(para-hydroxyphenylmaleimide)) [poly(S-alt-pHPMI)] core and a poly(4-vinylpyridine) (P4VP) derivative shell in a selective solvent. The aim was to modify hydrogen bonding interaction sites at the core/shell interface by synthesizing P4VP derivatives in three different sequences, namely, P4VP homopolymers, PS-co-P4VP random copolymers, and block copolymers. TEM images showed the successful self-assembly of poly(S-alt-pHPMI)/PS-co-P4VP inter-polymer complexes into spherical structures. To dissolve the core structures, 1,4-dibromobutane was used as a cross-linking agent to tighten the PS-co-P4VP shell. The morphologies, particle sizes, hydrogen bonding, cross-linking reaction, and core dissolution were confirmed by TEM, DLS, FTIR, and AFM analyses. Poly(S-alt-pHPMI)/PS41-r-P4VP59 hydrogen bonding connected micelles, cross-linked micelles, and hollow spheres were larger and more irregular than poly(S-alt-pHPMI)/P4VP inter-polymer complexes due to the random copolymer architecture and the decrease in intermolecular hydrogen bonds. However, poly(S-alt-pHPMI)/PS68-b-P4VP32 resulted in rod- or worm-like structures after core dissolution.

Intra- and Intermolecular Hydrogen Bonding in Miscible Blends of CO2/Epoxy Cyclohexene Copolymer with Poly(Vinyl Phenol).

In this study, we synthesized a poly(cyclohexene carbonate) (PCHC) through alternative ring-opening copolymerization of CO2 with cyclohexene oxide (CHO) mediated by a binary LZn2OAc2 catalyst at a mild temperature. A two-dimensional Fourier transform infrared (2D FTIR) spectroscopy indicated that strong intramolecular [C-H···O=C] hydrogen bonding (H-bonding) occurred in the PCHC copolymer, thereby weakening its intermolecular interactions and making it difficult to form miscible blends with other polymers. Nevertheless, blends of PCHC with poly(vinyl phenol) (PVPh), a strong hydrogen bond donor, were miscible because intermolecular H-bonding formed between the PCHC C=O units and the PVPh OH units, as evidenced through solid state NMR and one-dimensional and 2D FTIR spectroscopic analyses. Because the intermolecular H-bonding in the PCHC/PVPh binary blends were relatively weak, a negative deviation from linearity occurred in the glass transition temperatures (Tg). We measured a single proton spin-lattice relaxation time from solid state NMR spectra recorded in the rotating frame [T1ρ(H)], indicating full miscibility on the order of 2-3 nm; nevertheless, the relaxation time exhibited a positive deviation from linearity, indicating that the hydrogen bonding interactions were weak, and that the flexibility of the main chain was possibly responsible for the negative deviation in the values of Tg.

Meso/Microporous Carbons from Conjugated Hyper-Crosslinked Polymers Based on Tetraphenylethene for High-Performance CO2 Capture and Supercapacitor.

In this study, we successfully synthesized two types of meso/microporous carbon materials through the carbonization and potassium hydroxide (KOH) activation for two different kinds of hyper-crosslinked polymers of TPE-CPOP1 and TPE-CPOP2, which were synthesized by using Friedel-Crafts reaction of tetraphenylethene (TPE) monomer with or without cyanuric chloride in the presence of AlCl3 as a catalyst. The resultant porous carbon materials exhibited the high specific area (up to 1100 m2 g-1), total pore volume, good thermal stability, and amorphous character based on thermogravimetric (TGA), N2 adsoprtion/desorption, and powder X-ray diffraction (PXRD) analyses. The as-prepared TPE-CPOP1 after thermal treatment at 800 °C (TPE-CPOP1-800) displayed excellent CO2 uptake performance (1.74 mmol g-1 at 298 K and 3.19 mmol g-1 at 273 K). Furthermore, this material possesses a high specific capacitance of 453 F g-1 at 5 mV s-1 comparable to others porous carbon materials with excellent columbic efficiencies for 10,000 cycle at 20 A g-1.

Synthesis of Sodium Complexes Supported with NNO-Tridentate Schiff Base Ligands and Their Applications in the Ring-Opening Polymerization of L-Lactide.

A series of sodium complexes bearing NNO-tridentate Schiff base ligands with an N-pendant arm were synthesized and used as catalysts for the ring-opening polymerization of L-lactide (L-LA). Electronic effects of ancillary ligands coordinated by sodium complexes substantially influence the catalysis, and ligands with electron-donating groups increase the catalytic activity of the sodium complexes for catalyzing L-LA polymerization. In particular, a sodium complex bearing a 4-methoxy group has the highest activity with conversion up to 95% within 30 s at 0 °C and a low polydispersity index of 1.13, whereas the 4-bromo group showed the poorest performance with regard to the catalytic rate of L-LA polymerization in the presence of benzyl alcohol (BnOH). (1)H NMR pulsed-gradient spin-echo diffusion experiments and single-crystal X-ray analyses showed that sodium complexes [L(H)Na(THF)]2 and [L(4-Cl)Na(THF)]2 were dinuclear species in both solution and the solid state. The kinetic results indicated a first-order dependence on each of [[L(4-Cl)Na]2], [l-LA], and [BnOH].

IL-6 production stimulated by CD14(+) monocytes-paracrined IL-1ß does not contribute to the immunosuppressive activity of human umbilical cord mesenchymal stem cells.

BACKGROUND/AIMS: Human umbilical cord mesenchymal stem cells (hUC-MSCs) possess immunosuppressive activities but the mechanisms of such activities are not fully understood. Here, we investigated the role of IL-6, one of the characteristic factors of MSCs, in the immunoregulating effect of hUC-MSCs on CD4(+) T lymphocytes. METHODS: The condition media from human peripheral blood mononuclear cells (hPBMCs) or CD14+/- cell were tested if stimulating IL-6 production by hUC-MSCs. The related signaling pathway of IL-6, and the immunosuppressive activity of IL-6 on CD4(+) T lymphocytes were studied. RESULT: IL-6 production was dramatically increased by hUC-MSCs when co-culturing with resting or activated hPBMCs. CD14(+) monocytes-paracrined IL-1ß promoted the secretion of IL-6 by hUC-MSCs via JNK and NF-κB signaling pathway. Blocking of PGE2 synthesis did not affect the secretion of IL-6, anti-IL-6 antibody was not able to reverse hUC-MSCs-mediated inhibition on CD4(+) T lymphocytes. IL-6 did not mediate the suppressive activity of IL-1ß-hUC-MSCs- PGE2 on CD4(+) T cell. CONCLUSION: CD14(+) monocytes-paracrined IL-1ß promotes IL-6 secretion by hUC-MSCs through activating JNK and NF-κB signaling pathway. However, increased IL-6 production does not contribute to immunosuppressive activity of IL-1ß-hUC-MSCs- PGE2 on CD4(+) T cells.

Decreased expression of MBD2 and MBD4 gene and genomic-wide hypomethylation in patients with primary immune thrombocytopenia.

Genome-wide hypomethylation has been confirmed in patients with primary immune thrombocytopenia (ITP). Proteins containing methylcytosine-binding domain (MBD) are involved in promoter methylation as transcriptional repressors and promote the gene-silencing effect of DNA methylation. The purpose of this study was to investigate the methylation pattern of T cells and the relationship between genomic methylation and the expression of MBD2 and MBD4 in ITP patients. DNA deoxymethylcytosine content of CD4(+) cells from peripheral blood mononuclear cells was measured by enzyme-linked immunoassay. Real-time polymerase chain reaction was performed to quantify the transcription levels of MBD2 and MBD4 in peripheral blood mononuclear cells and CD4(+) cells. DNA dmC content in CD4(+) cells of ITP patients was significantly lower than in the controls (p = 0.001). The mRNA level of MBD2 and MBD4 in CD4(+) cells of ITP patients was statistically lower than those of the controls (p < 0.001). Positive correlations between methylation indexes and expression of each enzyme were observed in the control group (r(2) = 0.718, p = 0.004 for MBD2; r(2) = 0.608, p = 0.015 for MBD4). However, inverse correlations were found in ITP patients (r(2) = 0.604, p = 0.008 for MBD2; r(2) = 0.498, p = 0.027 for MBD4). Our results indicate that decreased expression of MBD2 and MBD4 might involve in the pathogenesis of ITP.

[Human coagulation factor IX gene therapy in murine hemophilia B by hydrodynamic delivery and site-specific genomic integration].

OBJECTIVE: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.

[Construction and identification of a novel adeno-integrase hybrid system for hemophilia B].

This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.

CD14+ monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells.

Here, the effect of CD14(+) monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-gamma (IFN-gamma) secretion capacities of CD4(+) and CD8(+) T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E(2) (PGE(2)) as an important soluble mediator. CD14(+) monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1beta, either exogenously added or produced by CD14(+) monocytes in culture, could trigger expression of high levels of PGE(2) by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE(2) expression, but also reversed the promotional effect of CD14(+) monocytes and partially restored CD4(+) and CD8(+) T cell proliferation and IFN-gamma secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.

Mesenchymal stem cells from bone marrow show a stronger stimulating effect on megakaryocyte progenitor expansion than those from non-hematopoietic tissues.

In order to evaluate whether mesenchymal stem cells (MSCs) from non-hematopoietic tissues are able to regulate megakaryocytopoiesis, we identified human MSCs from adult bone marrow (ABM), fetal pancreas (FPan) and umbilical cord (UC), and their abilities to support megakaryocyte (MK) differentiation from CD34(+) hematopoietic progenitor cells (HPCs) were comparatively studied. First, MSCs were isolated from ABM, FPan and UC then their growth kinetics, molecular characterization and mesodermal differentiation capacity were determined. ABM-MSCs, FPan-MSCs and UC-MSCs were irradiated and cocultured with human umbilical cord blood (UCB) CD34(+) cells, and the expansion efficiency of MK progenitor cells and MK formation were analysed and compared. Finally, SCF, IL-6 and GM-CSF expression by the three types of MSCs were also examined. Our results showed that FPan-MSCs and UC-MSCs shared most of the characteristic of ABM-MSCs, including morphology, immunophenotype, adipogenic and osteogenic differentiation potentials. Compared with ABM-MSCs, fetal MSCs had higher proliferative capacity. After 7 days' coculture, the maximal production of CD34(+)/CD41a(+) cells was obtained in a group of CD34(+) HPCs + ABM-MSCs. Furthermore, this group produced more MK colonies than other groups (p < 0.05). Surface antigen and ploidy analysis morphological observation demonstrated that a proportion of expanded cells in each group differentiated into mature MKs. ABM-MSCs, FPan-MSCs and UC-MSCs were revealed to express SCF, IL-6 and GM-CSF at mRNA level. We conclude that FPan-MSCs and UC-MSCs have the ability to promote megakaryocytopoiesis, while ABM-MSCs expand more MK progenitor cells from CD34(+) HPCs than MSCs from non-hematopoietic tissues and CD34(+) cells alone.

Human umbilical cord mesenchymal stem cells hUC-MSCs exert immunosuppressive activities through a PGE2-dependent mechanism.

Human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) constitute an attractive alternative to bone-marrow-derived MSCs for potential clinical applications because of easy preparation and lower risk of viral contamination. In this study, both proliferation of human peripheral blood mononuclear cells (hPBMCs) and their IFN-gamma production in response to mitogenic or allogeneic stimulus were effectively inhibited by hUC-MSCs. Co-culture experiments in transwell systems indicated that the suppression was largely mediated by soluble factor(s). Blocking experiments identified prostaglandin E(2) (PGE(2)) as the major factor, because inhibition of PGE(2) synthesis almost completely mitigated the immunosuppressive effects, whereas neutralization of TGF-beta, IDO, and NO activities had little effects. Moreover, the inflammatory cytokines, IFN-gamma and IL-1beta, produced by hPBMCs upon activation notably upregulated the expression of cyclooxygenase-2 (COX-2) and the production of PGE(2) by hUC-MSCs. In conclusion, our data have demonstrated for the first time the PGE(2)-mediated mechanism by which hUC-MSCs exert their immunomodulatory effects.

[Combined deficiency of factors V and VIII caused by a novel compound heterozygous mutation of gene Lman1].

Combined deficiency of factor V and VIII (F5F8D) is a rare, autosomal recessive disorder caused by mutations of either lman1 or mcfd2. To identify mutations of these two genes in a Chinese F5F8D family, the samples of peripheral blood were collected from the proband and her parents. Coagulation tests were carried out, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg) and coagulate activity of FV, FVIII (FV:C, FVIII:C). The genomic DNA was extracted, then all the exons and intron/exon boundaries of these two genes were amplified by polymerase chain reaction (PCR). The products were finally analyzed by direct sequencing. The results showed that the proband's APTT, PT, TT, Fg, FV:C and FVIII:C were 82.2 sec, 19.6 sec, 18.6 sec, 2.9 g/L, 7.1% and 18.7% respectively, while those parameters of the parents were all within the normal range. Two pathogenic mutations were identified in lman1 gene of the proband: one was the heterozygous c.912_913insA in exon 8 resulting in a frameshift of p.Glu305fsX20; the other was the heterozygous c.1366C > T in exon 11 resulting in p.Arg456X. The proband's father and mother were heterozygous for c.1366C > T and c.912_913insA respectively. It is concluded that F5F8D of the proband is caused by a novel compound heterozygous mutation of the lman1 gene, which has never been reported.

[Comparative study of in vitro hematopoietic supportive capability of human mesenchymal stem cells derived from bone marrow and umbilical cord].

The present study was aimed to isolate and identify human mesenchymal stem cells from adult bone marrow (BM-MSC) and umbilical cord (UC-MSC), and to compare their ability to support in vitro long-term hematopoiesis. MSC from bone marrow and umbilical cord were isolated by using density gradient centrifugation or enzyme digestion. MSC were further purified by adherent culture. Immunophenotype, adipogenic and osteogenic differentiation potential of BM-MSC and UC-MSC were detected. The hematopoietic supporting capacity of BM-MSC and UC-MSC was assessed by LTC-IC assay. Nonadherent cells in each group were collected for phenotypic analysis at 3, 5 and 7th week of culture. The results showed that BM-MSC and UC-MSC in culture shared a similar spindle-shaped morphology and adhered to the tissue culture substrate. They were both positive for CD90, CD105, CD73, CD29, CD54, CD166, HLA-ABC, and negative for HLA-DR, CD34 and CD45. BM-MSC and UC-MSC could differentiate into adipocytes or osteoblasts confirmed by oil red O staining and von Kossa staining, separately. LTC-IC assay showed that at 5th week of culture, the difference of the CFC yields between UC-MSC group and BM-MSC group was not statistically significant (p>0.05). At 6, 7, 9th week of culture, the CFC yields in the UC-MSC group were lower than those of BM-MSC (p<0.05). The phenotypic analysis of nonadherent cells at 3, 5, 7th week of culture indicated that along with prolongation of time, the percentages of CD34+ cells and CD117+ cells in each group decreased markedly, and the percentages of CD33+ cells, CD13+ cells and CD11b+ cells increased gradually. It is concluded that MSC from human adult bone marrow and umbilical cord can be successfully isolated and identified. UC-MSC are able to support long-term hematopoiesis in vitro, but its hematopoietic supportive capacity is weaker than those of BM-MSC.

[Genetic analysis of an inherited afibrinogenemia family caused by a novel frameshift mutation in FGA].

Inherited afibrinogenemia is a rare autosomal recessive bleeding disease characterized by complete absence of fibrinogen in blood. To identify the genotype in a Chinese family with inherited afibrinogenemia, the samples of peripheral blood were collected from 6 members of 3 generations. The activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (Fg, clauss) were tested. Fg was also analyzed by using immunoturbidimetry method. DNAs of six members were extracted by using a DNA extract kit. All the exons and exon-intron boundaries of the three fibrinogen genes were amplified by using PCR and analyzed by direct sequencing. The results showed that the parents of proband were 3 degree consanguinity. A homozygous c.934_935insA in FGA was found in proband which results in the change of protein p.Ser312fsX42. The parents, grandmother, maternal grandmother and father's sister were all detected with heterozygous mutation which was same as that in proband. In conclusion homozygous c.934_935insA in FGA is a cause of inherited afibrinogenemia and a novel mutation being reported.

[Reversal of leukemia multidrug resistance by sequence-specific short hairpin RNA].

This study was aimed to design and screen short hairpin RNA (shRNA) molecules targeting multidrug resistance gene (mdr1), as well as to investigate the effects of shRNA expression vector on K562/A02 cells. Mdr1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine 2000, and G418 was added to screen and establish the stable expression cell strain. The expressions of mdr1 mRNA and protein were detected by real-time RT-PCR and Western blot respectively. The sensitivity of cells to chemodrugs after interference were tested by CCK8 assay. The function of p-glycoprotein was determined by Rhodamine 123 efflux experiment. The results showed that all of 4 mdr1-shRNA expression vectors could significantly knockdown the expression of p-glycoprotein as compared with control vector, moreover, the vector targeting 508 - 526 sites of mdr1 gene was the best one. It is concluded that the mdr1-shRNA expression vector gained by screening can significantly knockdown the expression of mdr1 gene and reverse leukemia drug resistance, paving the way for the application of RNAi in the following animal experiments.

[Transfusion of allogeneic mesenchymal stem cells promotes progression of atherosclerotic plaque in rabbits].

The present study was purposed to evaluate the safety of mesenchymal stem cell (MSC)-based therapy impacting on atherosclerosis. Allogeneic MSCs were obtained from rabbit bone marrow aspirates and expanded in vitro. New Zealand white rabbits were divided into three groups: 24 rabbits with hypercholesterolemia receiving intravenous injection of either 5 x 10(7) MSCs (n = 12) or saline (n = 12) after 5 weeks on a high lipid diet and additional rabbits (n = 6) fed with standard rabbit diet were served as controls. Body weight and blood lipids were measured at weeks 0, 5, 9 and 13 during the study. All rabbits were sacrificed at week 13. Atherosclerotic lesion size and vasa vasorum were evaluated by using pathological analysis and immunocytochemical technique. The results showed that the aortic sinus lesion size significantly increased in rabbits infused with MSCs as compared with controls receiving saline (23.35 +/- 3.51% and 11.39 +/- 3.08% respectively). The lesion size in whole aortas of MSC-treated rabbits was 76.64 +/- 12.70% versus 57.61 +/- 9.00% in saline-treated animals (p < 0.05). Moreover, vasa vasorum networks in MSC-treated aortas were more numerous and had increased capillary density. It is concluded that the allogeneic MSC transfusion may result in an increase in atherosclerotic lesion size. In cell therapy with MSCs or cell populations containing MSCs a strategy to attenuate the high potential of MSCs involved in atherogenesis of atherosclerosis should be taken in account.

[Analysis of complicated anemia in 60 patients with systemic lupus erythematosus].

The study was aimed to explore the characteristics of clinical manifestation, laboratory indicators and bone marrow examination of SLE patients with anemia. 60 SLE patients with anemia were analyzed for their clinical manifestation, laboratory indicators and bone marrow examination in comparison with 40 contemporaneous SLE patients without anemia. The results indicated that there were significant differences in clinical manifestations of fatigue between the SLE patients with anemia and those without anemia. The detection rate of the decreased Plt and C4 and the percentages of eosinophils, early normoblast, polychromatic normoblast and orthochromatic normoblast in bone marrow were all higher than that in those without anemia. The ANA with titer 1:320 in SLE patients complicated by anemia was lower than that in those without anemia. In conclusion, the clinical manifestation, experimental examination and bone marrow findings were significantly different between the SLE patients with anemia and without anemia.

The role of T-cell immunoglobulin- and mucin-domain-containing molecule-3 polymorphisms in idiopathic thrombocytopenic purpura.

The family of T-cell immunoglobulin- and mucin-domain-containing molecules (TIMs) has an important role in immune regulation. TIM-3 is a transmembrane protein preferentially expressed on terminally differentiated Th1 cells and plays a role in T-helper (Th)-1-mediated autoimmune disease. Idiopathic thrombocytopenic purpura (ITP) is an acquired organ-specific autoimmune disease with a polarization of Th1. The purpose of this study was to investigate whether the -1516G>T, -574T>G, 4259G>T single-nucleotide polymorphisms within the TIM-3 gene contribute to the genetic susceptibility to ITP. Genotyping of TIM-3 -1516G>T, -574T>G, and 4259G>T was performed in 187 patients with ITP and 123 healthy individuals by polymerase chain reaction-restriction fragment length polymorphism assay. No significant differences existed in genotype and allele distributions between the patients with ITP and the controls in all three sites. There was strong linkage disequilibrium (LD; r(2) = 0.633) between -574T>G and 4259G>T, whereas -1516G>T was not in LD with -574T>G (r(2) = 0.007) or with 4259G>T (r(2) = 0.002). The -1516G>T, -574T>G, and 4259G>T of TIM-3 gene polymorphisms might not play an important role as a genetic risk factor in the pathophysiology of ITP.

[Neovascularization potential of mobilized peripheral mononuclear cells from diabetes patients].

OBJECTIVE: To determine whether mobilized peripheral blood mononuclear cells (M-PBMNCs) obtained from patients with diabetes was impaired in therapeutic neovascularization in limb ischemia, and to explore the pathological mechanisms of the impairment. METHODS: Endothelial progenitor cells (EPC) were cultured in EGM-2MV, and then characterized by uptake of 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Dil-AcLDL) and binding of ulex europaeus agglutinin (UEA). The number of EPC was compared between M-PBMNCs obtained from diabetic patients and those from normal subjects. M-PBMNCs obtained from diabetic patients, M-PBMNCs obtained from normal controls, or PBS were injected into the ischemic limbs of streptozotocin-induced diabetic nude mice. The limb blood perfusion was detected by laser Doppler blood perfusion imaging between these three groups in the following 1, 3, 7, 14, 21, and 28 days. Ambulatory score and ischemia damage were evaluated in the following 4 weeks. Capillary/fiber ratio was detected by CD31 or BS-1 lectin, and arteriole density was detected by alpha-smooth muscle actin (alpha-SMactin). RESULTS: The number of EPC from diabetic patients were positively correlated with the blood perfusion (R = 0.486, P < 0.05) and capillary density (R = 0.491, P < 0.05), and the EPC number in diabetic patient were negatively correlation with their disease courses (R = - 0.587, P < 0.05). Transplantation of diabetic M-PBMNCs augmented the blood perfusion of ischemia hindlimbs, increased the capillary and arteriole densities, and promoted the collateral vessel formation. However, all the improvements were less significant in the diabetic patients than in the non-diabetic patients (P < 0.05). CONCLUSION: Diabetes decreased the capability of M-PBMNCs to augment neovascularization in ischemia.