Acute Disease ; Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Female ; Liver Diseases ; pathology ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley

Objective To establish a HPLC-MS/MS method for deter-mination of human plasma concentrations of Bezafibrate , and for the study of pharmacokinetic characteristics of bezafibrate ( two formulations ) . Methods Chromatographic separation was using Welch Ultimate C 18 column (5 μm, 2.1 mm ×50 mm) with gradient elution ( A: acetoni-trile, B:water with 0.02% formic acid).Quantification was performed using Q-TRAP MS with multiple-reaction monitoring .Six health sub-jects were divided into three groups and received single 400 mg dose of test preparation or reference preparation in three period .Plasma samples were collected at different time.The concentrations of bezafibrate were determined by LC -MS/MS. Pharmacokinetic parameters and related statistics analysis were calculated by DAS 3.2.7.Results The linear range is 50.0-1.5 ×10 4 ng? mL-1 , and had a good linear relationship ( r=0.996 9 ).The lower limit of quantitation of bezafibrate was 50 ng? mL-1 .The intra-and inter-day precisions were 1.21%-6.36%and 5.73%-7.62%, respectively.And the absolute recoveries of beza-fibrate were range from 67.56%to 77.42%.The main pharmacokinetic parameters of single oral test preparation were:Cmax was (14.52 ±4.78 )μg? mL-1 , tmax was ( 3.00 ±0.89 ) h, t1/2 was ( 2.49 ±0.84 ) h, AUC0-t was ( 52.36 ±6.23 )μg? mL-1? h, AUC0-∞ was (52.48 ±6.19 )μg? mL-1? h.According to the coefficient of variation to estimate sample size was 18 cases.Conclusion This study established a rapid , efficient and sensitive method for the analysis of the concentration of bezafibrate in plasma and also suitable for pharmacokinetic studies of bezafibrate .

OBJECTIVETo evaluate the expression of CD133 and its clinical significance in acute leukemia (AL) patients.

METHODSThe expression of CD133 and CD133 mRNA in leukemic blasts from 76 AL patients were detected by three-color flow-cytometry and hemi-quantitative RT-PCR respectively.

RESULTS(1) CD133 mRNA expression was highly correlated with CD133 expression in both of normal donors and AL patients groups. The expression of CD133 in AL patients was significantly higher than that in control group (P < 0.01). (2) The positive rates of CD133 and CD133 mRNA in AL group were 42.1% (32/76) and 46.1% (35/76) respectively. There was no significant difference in CD133 expression between AML-M(3) and normal control, AML and ALL, as well as T-ALL and B-ALL. The expression of CD133 in AML-M(4) were significantly higher than those in other AML subtypes (81.8% vs 43.7% and 81.8% vs 46.9% at CD133 and CD133 mRNA level, respectively, P < 0.01). (3) The expression of CD133 in AML was significantly correlated with the expression of CD34 and HLA-DR (P < 0.001). (4) The expression of CD133 had no relationship with the clinical prognostic factors such as cytogenetic or molecular aberrations, WBC counts, LDH, mdr1 expression and age. (5) There was a trend toward lower CR rate in CD133(+) cases, but only CD34/CD133(+) double positive cases had significant lower CR rate than that of negative ones (44.4% vs 71.4%, P < 0.05).

CONCLUSIONSAL had significantly higher CD133 expression compared to normal control. The detection of CD133 expression might help to identify AL type and predict therapeutic outcomes. Co-expression of CD133/CD34 might convey adverse prognosis of AL.

AC133 Antigen ; Acute Disease ; Adolescent ; Adult ; Aged ; Antigens, CD ; genetics ; Antigens, CD34 ; genetics ; Child ; Female ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; genetics ; HLA-DR Antigens ; genetics ; Humans ; Leukemia ; genetics ; pathology ; Male ; Middle Aged ; Peptides ; genetics ; Prognosis ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo study the effect of ex vivo expansion on the migration ability and the CXCR4 expression of umbilical cord blood (CB) hematopoietic stem/progenitor cells (HSPC).

METHODSCD(34)(+) cells isolated from fresh CB samples were cultured in a serum-free and stroma-free culture system. On day 7, 10 and 14, CD(34)(+) cells were re-selected from the expanded cells, and the expression of CXCR4 and the transmigration ability of these CD(34)(+) cells were evaluated respectively and compared with those of the precultured fresh CD(34)(+) cells.

RESULTS(1) SDF-1 induced a higher migration percentage of fresh or expanded CB CD(34)(+) cells than that of uninduced ones. (2) Both of the uninduced and SDF-1-induced migrations were slightly reduced in the first week and then much more reduced in the second week expansion (P < 0.05). (3) The number of the CD(34)(+)CXCR4(+) cells were significantly increased during the culture period, but there was a downtrend of CXCR4 expression on CD(34)(+) subset; the expression levels on day 10 and 14 were lower than that on day 0.

CONCLUSIONSThe expanded HSPC would sustain the chemotactic activity during one-week-culture, but with further extended culture time their intrinsic homing potential would be partly impaired.

Antigens, CD34 ; genetics ; metabolism ; Cell Culture Techniques ; Cell Movement ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Male ; Pregnancy

BACKGROUNDThe self-consciousness and practicality of preferentially prescribed essential medicines (EMs) are not high enough in county hospitals. The purposes of this study were to use the information-motivation-behavioral skills (IMB) model to identify the predictors of essential medicines prescribing behavior (EMPB) among doctors and to examine the association between demographic variables, IMB, and EMPB.

METHODSA cross-sectional study was carried out to assess predictive relationships among demographic variables and IMB model variables using an anonymous questionnaire administered in nine county hospitals of Anhui province. A structural equation model was constructed for the IMB model to test the instruments using analysis of moment structures 17.0.

RESULTSA total of 732 participants completed the survey. The average age of the participants was 37.7 ± 8.9 years old (range: 22-67 years old). The correct rate of information was 90.64%. The average scores of the motivation and behavioral skills were 45.46 ± 7.34 (hundred mark system: 75.77) and 19.92 ± 3.44 (hundred mark system: 79.68), respectively. Approximately half (50.8%) of respondents reported that the proportion of EM prescription was below 60%. The final revised model indicated a good fit to the data (χ2 /df = 4.146, goodness of fit index = 0.948, comparative fit index = 0.938, root mean square error of approximation = 0.066). More work experience (β = 0.153, P < 0.001) and behavioral skills (β = 0.449, P < 0.001) predicted more EMPB. Higher income predicted less information (β = -0.197, P < 0.001) and motivation (β = -0.204, P < 0.001). Behavioral skills were positively predicted by information (β = 0.135, P < 0.001) and motivation (β = 0.742, P < 0.001).

CONCLUSIONThe present study predicted some factors of EMPB, and specified the relationships among the model variables. The utilization rate of EM was not high enough. Motivation and behavior skills were crucial factors affecting EMPB. The influence of demographic variables, such as income and work experience, on EMPB should be fully appreciated. Comprehensive intervention measures should be implemented from multiple perspectives.

Adult ; Aged ; China ; Cross-Sectional Studies ; Female ; Hospitals, County ; statistics & numerical data ; Humans ; Male ; Middle Aged ; Practice Patterns, Physicians' ; statistics & numerical data

OBJECTIVETo study the effect of human umbilical blood (UB) mesenchymal stem cells (MSC) on the CD34(+) cells transplantation in NOD/SCID Mice.

METHODSUmbilical blood CD34(+) cells (3.5 x 10(5) cells) alone or combined with umbilical cord MSC cells were transplanted into NOD/SCID mice that had been irradiated with (137)Cs (3.0 Gy) before transplantation. Changes in peripheral blood cells within 6 post-transplantation weeks were detected. The mice were sacrificed 6 weeks after transplantation. The human hematopoietic cells (hCD45(+)) and multi-lineage engraftment cells (CD3/CD19, CD33, CD14, CD61, and CD235a) in NOD/SCID recipients bone marrow, spleen, and peripheral blood were analyzed by flow cytometry.

RESULTSIn the 3rd post-transplantation week, white blood cells (WBC), platelets (PLT), and red blood cells (RBC) began to increase in both two groups. In the 6th post-transplantation week, WBC and PLT counts in CD34(+) + MSC group reached peak levels and were significantly higher than CD34(+) alone group (P < 0.05), while RBC level was not significantly different between these two groups P > 0.05). hCD45(+) cell levels in bone marrow and peripheral blood were (42.66 +/- 2.57) % and (4.74 +/- 1.02) % in CD34(+) + MSC group, which were significantly higher than those in CD34(+) alone group [(25.27 +/- 1.67) % and (1.19 +/- 0.54) %, respectively, P = 0.006]. Also in the 6th post-transplantation week, the proportions of CD19(+), CD33(+), CD14(+), CD61(+), and CD235a(+) in CD34(+) + MSC group were significantly higher than those in CD34(+) alone group (P < 0.05), while the proportion of CD3(+) T lymphocyte in CD34(+) + MSC group was significantly lower than that in CD34(+) alone group (P = 0.003). The amplification of CD19(+) B lymphocyte was significantly higher than other blood cell lineages (P < 0.05).

CONCLUSIONThe co-transplantation of MSC cells and CD34(+) cells can promote hematopoietic stem cell transplantation and hematopoietic recovery in vivo.

Animals ; Antigens, CD34 ; Cord Blood Stem Cell Transplantation ; Hematopoiesis ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

OBJECTIVETo study the effect of ex vivo expansion on the adhesion activities of umbilical cord blood hematopoietic stem and progenitor cells (HSPC).

METHODSFresh UCB CD(34)(+) cells were cultured in a serum and stroma-free culture system. At day 7, day 10 and day 14, CD(34)(+) cells were re-selected from the expanded products. The expression of adhesion molecules (CAMs) such as VLA-4, VLA-5, LFA-1, ICAM-1, HCAM, L-selectin and PECAM-1, and the adhesion activity of the expanded CD(34)(+) cells were evaluated and compared with those of precultured fresh CD(34)(+) cells.

RESULTS(1) The CD(34)(+) cells expressing homing-related CAMs were increased (from 15-fold increase for CD(34)(+) CD(54)(+) subset to 72-fold increase for CD(34)(+) CD(49e)(+) subset at day 14). (2) The expressions of CD(49d), CD(44), CD(11a) and CD(49e) on the expanded CD(34)(+) cells were increased or sustained the same levels as those on fresh UCB CD(34)(+) cells, while the expression of CD(62L), CD(54) and CD(31) on expanded CD(34)(+) cells declined with the cultivating. (3) Spontaneous adhesion and SDF-1-induced adhesion tended to be increased in the course of the first 10 day's culture.

CONCLUSIONSThe culture system used in this study could substantially support the expansion of HSPCs expressing the above CAMs, and the expanded HSPCs would sustain their intrinsic adhesion potentials.

Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Cell Adhesion ; Cell Adhesion Molecules ; biosynthesis ; Cell Division ; Fetal Blood ; cytology ; immunology ; metabolism ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; metabolism ; Humans ; Receptors, Lymphocyte Homing ; biosynthesis

OBJECTIVETo identify the mutation of low density lipoprotein receptor(LDLR) gene in a large Chinese family with familial hypercholesterolemia(F H) and make a discussion on the pathogenesis of FH at the molecular level.

METHODSInvestigations were made on a patient with the clinical phenotype of homozygous FH and his parents for mutations of promoter and all 18 exons of LDLR gene. Screening was carried out using Touch down PCR and a g arose gel electrophoresis, combined with DNA sequence analysis. The results were compared with the normal sequences in GenBank and FH database (www.ucl.uk/fh) t o find the mutation. Then the mutation was identified in other members of the family. In addition, the authors screened the apolipoprotein B(100) (apoB(100)) gene f or known mutations (R3500Q) that cause familial defective apoB(100) (FDB) by PCR-RFLP.

RESULTSA novel homozygous IN III 5' GT --> AT mutation in the splice donor of LDLR intron 3 was detected in the homozygote propositus with FH. The mutation was also identified in four heterozygous carriers in his family. No mutations R3500Q of apoB(100)were observed.

CONCLUSIONA homozygous G --> A splice mutation in LDLR gene was first reported. The change of the splice donor in LDLR intron 3 may cause skipping of exon 3, which is responsible for FH. Perhaps it is a particular pathogenesis for Chinese people.

Adolescent ; Adult ; Alternative Splicing ; genetics ; Base Sequence ; Child ; China ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Female ; Homozygote ; Humans ; Hyperlipoproteinemia Type II ; blood ; genetics ; pathology ; Lipids ; blood ; Male ; Middle Aged ; Mutation ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Receptors, LDL ; genetics

OBJECTIVETo explore the role of stromal cell-derived factor (SDF-1) and its specific receptor CXCR4 in the G-CSF-induced hematopoietic stem/progenitor cells (HSPCs) mobilization in human healthy donor.

METHODSThe changes of SDF-1/CXCR4 in bone marrow (BM) and peripheral blood (PB) of healthy donors during G-CSF-induced mobilization were detected by enzyme-linked immunosorbent assay (ELISA), immunohistological staining and flow cytometry. SDF-1 neutralizing antibody wes injected into BALB/c mice to further test its effect on mobilization.

RESULTSSDF-1 concentration in mobilized BM (mBM), steady state BM (ssBM) and PB were(7.23 +/- 0.66) microg/L, (5.43 +/- 0.35) microg/L and (5.42 +/- 0.52) microg/L, respectively. SDF-1 protein levels were decreased in the BM (P < 0.05) after 5-day G-CSF injection, and its concentration gradient between BM and PB disappeared (P > 0.05). Significant up-regulation of CXCR4 expression was observed on mBM CD34 cells in healthy donors. The rate of CXCR4 expression on CD34 cells in ssBM, mBM and mobilized PB were (40.98 +/- 21.56)%, (65.80 +/- 24.68)% and (27.54 +/- 26.03)%, respectively. Comparing with that in ssBM and mBM, CXCR4 expression on mobilized PB CD34+ cells were significantly decreased (P < 0.05). Inhibition of SDF-1 signal by blocking monoclonal antibodies significantly reduced G-CSF-induced mobilization in BALB/c mice. This resulted in significant decrease of white blood cell count and progenitors mobilized into peripheral circulation.

CONCLUSIONG-CSF induces HSPCs mobilization by decreasing bone marrow SDF-1 and down-regulating CXCR4 expression on HSPCs.

Animals ; Chemokine CXCL12 ; metabolism ; physiology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Mice ; Mice, Inbred BALB C ; Receptors, CXCR4 ; metabolism ; physiology

OBJECTIVETo investigate the effect and the potential mechanism of umbilical cord (UC) derived mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) derived CD34+ cells in vivo homing in xenotransplanted NOD/SCID mice model.

METHODSCD34+ cells and MSCs were derived from fresh UCB and UC, respectively. CD34+ cells (5 x 10(5) per mice) and MSC cells (5 x 10(6) per mice) were co-transplanted into irradiated NOD/SCID mice intravenously. CD34+ cells (5 x 10(5) per mice) alone were transplanted into the mice as control group. CD34+ cells home in bone marrow and spleen of recipient mice were detected 20 hours after transplant by FACS and RT-PCR, and the homing efficiencies were calculated. The effect of MSCs on CD34+ cells chemotactic function was investigated after co-cultured UCB CD34+ cells with UC MSCs in vitro. After 4 and 7 days coculture, the homing related adhesion molecules (the CD49e, CD31, CD62L, CD11a) expressed on CD34+ cells were detected by FACS.

RESULTS1) The homing efficiencies in bone marrow in experimental and control group were (7.2 +/- 1.1)% and (5.4 +/- 0.9)%, respectively (P < 0.05). 2) Human GAPDH gene was detected in bone marrow in experimental group and in spleen in both groups. 3) The migration efficiency of CD34+ cells was significantly higher in experimental group (35.7 +/- 5.8)% than in control group (3.5 +/- 0.6)% (P < 0.05). 4) The expression of CD49e, CD31, CD62L on CD34+ cells kept higher level in MSCs cocultured group than in CD34+ cells alone group.

CONCLUSIONSMSCs can efficiently increase homing of CD34+ cells to bone marrow and spleen in vivo by keeping a high level of homing adhesion molecules expression and improving migration efficiency of UCB CD34+ cells.

Animals ; Antigens, CD34 ; Cell Movement ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; metabolism ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred NOD ; Mice, SCID