Effects of Synthetic Long-Chain Polyphosphate on Blood Coagulation and Platelet Aggregation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of synthetic long-chain polyphosphate on blood coagulation and platelet aggregation.</p><p><b>METHODS</b>The effect of artificial synthetic long chain poly phosphate on blood coagulation and platelet aggregation was detected by coagulation tests, coagulation factor activity detection and platelet aggregation test, and its mechanism was explored by ELISA, flow cytometry and high content imaging system.</p><p><b>RESULTS</b>The long chain polyphosphates prolonged activated partial thromboplastin time, decreased coagulation factor FⅧ, FⅨ, FⅪ and FⅫ activity, blocked ADP-induced platelet aggregation, and decreased the concentration of calcium and TXA2 in platelet.</p><p><b>CONCLUSION</b>The synthetic long-chain polyphosphate can inhibit endogenous coagulation and inhibit platelet aggregation, which may be related with the inhibition of intracellular calcium and TXA2.</p>

Interpretation of the Guidance for Immune Thrombocytopenia-Review / 中国实验血液学杂志

Since the American Medical Association published the 2011 guidelines for immune thrombocytopenia, China has been the first to update the guidelines for immune thrombocytopenia based on evidence-based medicine. Recently, there have been many breakthroughs in clinical research published, especially the Chinese medical workers have made a prominent contribution to the treatment of the immune thrombocytopenia. However, the references of systematic drug introduction for children, adults, aged and pregnant women are still insufficient, and the first or second line treatment for some patients were ineffective. Therefore, we tried to combine the references to interpret the guidelines, to explore the advantages and disadvantages of each treatment, to find out the bottleneck of clinical treatment, so as to facilitate the implementation and understanding of the guidelines, then update the next guideline.

Biomechanical properties of calcium silicate/calcium phosphate cement scaffolds with controllable porous structure for bone repair by 3D bioplotting / 医用生物力学

Objective To design and fabricate novel mesoporous calcium silicate/calcium phosphate cement (MCS/CPC) scaffolds for bone repair and investigate their in vitro biomechanical properties under different external forces. Methods MCS and CPC in certain proportion were mixed to form plotting material, and the composite MCS/CPC scaffolds with pore size of 350 μm and 500 μm were fabricated by 3D bioplotting technique, respectively. Surface topographies of the scaffolds were observed by scanning electron microscope (SEM). The compressive strength and mechanical properties of the scaffolds under dynamic cyclic loads at different frequencies were studied through universal mechanical testing machine and dynamic mechanical analysis instrument. Results MCS/CPC scaffolds with controllable macroporous structures could be fabricated by 3D bioplotting technique. Scaffolds with pore size of 350 μm had higher compressive strength ［(9.8±0.39) MPa］ and compressive modulus ［(132.5±4.3) MPa］. In addition, at the loading frequency of 1-100 Hz, scaffolds with pore size of 350 μm had a higher storage modulus. ConclusionsMCS/CPC scaffolds with pore size of 350 μm fabricated by 3D bioplotting technique possess not only regular pore connectivity and high compressive strength, but also structural stability under dynamic loads, which are promising as novel biomaterials for bone repair.

Tissue factor expression and methylation regulation in differentiation of embryonic stem cells into trophoblast

OBJECTIVE@#To explore tissue factor (TF) expression and methylation regulation in differentiation of human embryonic stem cells (hESCs) into trophoblast.@*METHODS@#Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein 4 (BMP4). Expression of gene, protein of TF and DNA methylation at different time points during induction process was detected by RT-PCT, Western blot, flow cytometry and MSP-PCR method.@*RESULTS@#The expression of mRNA, protein level of TF could be detected during directional differentiation of hESCs to trophoblast cells, semi methylation-semi non methylation expression appeared at TF DNA promoter region, and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression of TF.@*CONCLUSIONS@#It shows that during differentiation of hESCs into trophoblast, the differential expression of TF is related with DNA methylation level, and it is changed with the methylation or non methylated degree. It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.

Prognostic power of abnormal cytogenetics for multiple myeloma: a multicenter study in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China.</p><p><b>METHODS</b>All 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications.</p><p><b>RESULTS</b>The analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of β2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS.</p><p><b>CONCLUSIONS</b>Chinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.</p>

Screening and expression of CD34(+) cell-specific microRNA in acute myelogenous leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen and analyze CD34(+) cell specific microRNAs (miRNAs) from the patients with acute myelogenous leukemia (AML) and their expression.</p><p><b>METHODS</b>CD34(+) cells were sorted from AML patients or the mobilized peripheral blood of the donors of hematopoietic stem cell transplantation (normal control subjects) and followed by the extraction of the cell total RNAs. The differentially expressed microRNAs (miRNAs, miR) were selected after hybridizing with miRNA microarray, real time polymerase chain reaction (real-time PCR) was subsequently applied to confirm the expression of the selected miRs, and PCR products were further cloned and sequenced to check their specificity.</p><p><b>RESULTS</b>Of the differentially expressed miRNAs, 191 were found to be at least one-fold change in the CD34(+) cells between the AML patients and the normal control subjects. Of the 191 miRNAs, the expression difference of 94 was significant (P < 0.05). Among these 94 miRNAs, the expression of 44 miRNAs was increased and the other 50 miRNAs was decreased in the CD34(+) cells from the bone marrow of AML patients compared with the CD34(+) cells from the mobilized peripheral blood of the normal control subjects. Real time PCR verified that the expression level of miR-10a and miR-220c in the CD34(+) cells from the bone marrow of AML patients was 19.6% and 19.0% of that of CD34(+) cells from mobilized peripheral blood of the normal control subjects. DNA sequencing and BLAST DNA database searching results indicated that the PCR products were really miR-10a and miR-220c.</p><p><b>CONCLUSION</b>A variety of differentially expressed-miRNAs are existed between AML and normal control subjects CD34(+) cells, the expression of miR-10a and miR-220c was significantly down-regulated in the CD34(+) cells from the bone marrow of AML patients.</p>

Chimeric Ad5F35 adenoviral vector-mediated expression of mutant IκBα induces apoptosis of leukemia cells / 中国实验血液学杂志

Constitutive activation of nuclear transcription factor-κB (NF-κB) exists in a variety of leukemia, and induction of apoptosis through blocking NF-κB activation may be an alternative strategy for leukemia treatment. The aim of this study was to investigate the inducing effect of modified adenovirus 5-based adenovirus vector (i.e. chimeric Ad5F35 Vec)-mediated expression of mutant IκBα (IκBαDN) on apoptosis of HL-60 cells. The recombinant Ad5F35-IκBαDN Vec carrying IκBαDN cDNA which deleted the first 1-70 amino acids coding sequences at 5' terminal of human IκBα was transfected into HL-60 cells. The apoptosis, NF-κB DNA binding activity, the expressions of IκBα, cIAP-2 and xIAP in HL-60 cells were detected by DNA binding assay, flow cytometry, real-time quantitative polymerase chain reaction and Western blot respectively. The results showed that apoptosis rates were 22.53 ± 2.999%, 6.08 ± 2.464% and 4.86 ± 1.366% for Ad5F35-IκBαDN Vec-infected or blank vector of Ad5F35-EGFP Vec-transfected and untransfected HL-60 cells respectively, which showed a significant difference between Ad5F35-IκBαDN Vec-transfected and untransfected cells (p < 0.001) and between Ad5F35-IκBαDN Vec-transfected and Ad5F35-EGFP Vec-transfected cells (p < 0.001, p < 0.002), while NF-κB DNA binding activity was decreased, the truncated IκBα was expressed, and IκBα mRNA expression was up-regulated, but the expression of cIAP-2 and xIAP mRNA was down-regulated after transduction for 48 hours. It is concluded that the chimeric Ad5F35 Vec can effectively mediate the expression of IκBαDN cDNA in HL-60 cells, leading to the inhibition of NF-κB DNA binding activity and inducing apoptosis of HL-60 cells.

Screening and structure analysis of nucleic acid aptamers binding to surface of CD33(+)/CD34(+) cells from patients with acute myeloid leukemia subtype M₂ / 中国实验血液学杂志

A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M₂) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M₂ CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M₂ were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M₂ CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M₂ leukemia.

Expression of angiotensin-II type 1 receptor at1 mRNA in myeloid leukemia / 中国实验血液学杂志

This study was aimed to explore the expression level of angiotensin-II type 1 receptor (AT1) mRNA in bone marrow of myeloid leukemic patients, and its correlation with the proportion of leukemia cells in samples and Hb, WBC, Plt counting in peripheral blood. 51 samples, including 36 AML, 7 CML, and 8 samples of non-malignant hematological diseases as control group were collected. The expression of at1 mRNA was detected by real time-PCR; the expression levels of at1 gene in AML and CML groups were relatively quantitatively analyzed by using 2(-ΔΔCT) and were compared with control group. The results showed that the expression levels of at1 mRNA in AML, CML and control groups were 0.038 ± 0.076, 0.033 ± 0.039, 0.281 ± 0.366, respectively. at1 gene expression in the myeloid leukemic group was significantly lower than that in the control group. The expression level of at1 mRNA in AML was negatively correlated with the proportion of leukemia cells and positively with Hb level in peripheral blood. It is concluded that at1 gene may play a minor role in leukaemogenesis, however, may promote erythropoiesis.

Effect of acetyl-11-keto-β-boswellic acid on proliferation, apoptosis and cell cycle of human acute myeloid leukemia cell line HL-60 / 中国实验血液学杂志

The aim of this study was to investigate the effect of 3-O-acetyl-11-keto-β-boswellic acid (AKBA) on the proliferation, apoptosis and cell cycle of human acute myeloid leukemia (AML) cell line HL-60. HL-60 cells were treated by AKBA at various concentrations. The inhibitory effects of AKBA on the proliferation of HL-60 were analyzed by MTT assay. Morphologic changes of HL-60 cells were observed by fluorescence microscopy with Hochest33342 staining. Cell apoptosis rate was determined by flow cytometry with Annexin-V-FITC/PI double staining. The cell cycle was measured by flow cytometry with PI staining. The results showed that AKBA inhibited the proliferation of HL-60 and the apoptosis rate of HL-60 cells was gradually enhanced when AKBA dose increased. AKBA changed the cell cycle of HL-60, resulting in cell increase at G(1) phase and decrease at S phase. It is concluded that the AKBA has anti-proliferation and apoptosis-inducing effects on HL-60 cells, that seems a promising therapeutical approach for AML.

Establishment and evaluation of a rabbit model of arterial thrombosis by collagen encapsulated thread-drawing / 中南大学学报(医学版)

OBJECTIVE@#To establish and evaluate a rabbit model of arterial thrombosis by modified thread-drawing.@*METHODS@#Fifty-three rabbits were randomly divided into 6 groups: a normal group, a ligating group,a collagen encapsulated thread-drawing group,a aspirin group,a clopidogrel group, and an aspirin clopidogral group. The endovascular pathological changes in the rabbits were observed, and D-fructose-1,6-diphosphate trisodium salt octahydrate (FDP), D-Dimer and tissue factor (TF) were detected with enzyme linked immunosorbent assay.@*RESULTS@#In the thread-drawing group, thrombus was obvious, and the endovascular elastic membrane was injured seriously compared with the ligating group. After being treated with aspirin and clopidogrel, most arterial thrombus was softened, dissolved and absorbed. Compared with that in the modified thread-drawing group,wet and dry weight of thrombus increased,and the level of D-Dimer, FDP and TF also increased in the modified thread-drawing group (P<0.01). After being treated by aspirin and/or clopidogrel, the wet and dry weight of thrombus and the level of D-Dimer, FDP and TF decreased compared with the control (P<0.01). Aspirin plus clopidogral could obviously reduce the wet and dry weight of thrombus, and reduce the level of D-Dimer and FDP (P<0.01). Aspirin plus clopidogral could obviously inhibit the formation of TF compared with aspirin (P<0.05).@*CONCLUSION@#Arterial thrombosis model by collagen encapsulated thread-drawing which is visible, repeatable and effective is better than thread-drawing. It is suitable for screening anti-thrombosis drugs and evaluating their effect.

Novel frame-shift mutation of 540 A deletion in GP IIb gene from a patient with Glanzmann thrombasthenia / 中南大学学报(医学版)

OBJECTIVE@#To explore the molecular mechanism of Glanzmann thrombasthenia (GT).@*METHODS@#All 45 exons of alphaIIb and beta3 subunit genes as well as their splicing sites were amplified by polymerase chain reaction(PCR) with 40 primer pairs, and then the PCR products were used to screen the gene mutation by single strand conformation polymorphism-polyacrylamide gel electrophoresis (SSCP-PAGE). The mutation was further confirmed by direct DNA sequencing.@*RESULTS@#A DNA band alterated migration was detected after SSCP-PAGE. DNA sequencing showed that a base deletion within the band at the site of 540 in GPIIb gene(540A) was found.@*CONCLUSION@#The frame-shift mutation caused by the deletion of 540A in GPIIb gene is a novel mutation which is a genetic defect in patients with GT.

Therapeutic effect of priming induction regimen of CAG for newly diagnosed acute myeloid leukemia in elderly patients / 中南大学学报(医学版)

OBJECTIVE@#To evaluate the clinical efficacy and toxicity of priming induction regimen of CAG for newly diagnosed acute myeloid leukemia (AML) in elderly patients.@*METHODS@#Seventy-five patients with newly diagnosed AML were divided into 2 groups: 34 were treated with priming induction regimen CAG and the other 41 were treated with 2 classic routine chemotherapy regimens including pirarubicin+cytarabine (TA) and homoharringtonine+cytarabine (HA). All patients had a 14 day interval between the 2 courses of chemotherapy.@*RESULTS@#The complete remission rate after 2 courses of induction therapy in patients with the priming induction regimen CAG and the total efficacy rate was significantly higher than that of the routine chemotherapy patients(67.6% vs. 39%; 82.4% vs. 56.1%). Patients with unfavorable karyotypes had poor chemotherapy efficacy. The 3-year disease-free-survival (DFS) time was longer in patients with AML treated with priming induction regimen CAG than in patients treated with 2 classic routine chemotherapy regimens. Except for the muscular soreness, the hematological and non-hematological side effects in the CAG priming induction group were significantly fewer than those in the routine chemotherapy group.@*CONCLUSION@#The priming induction regimen of CAG has a significantly higher complete remission rate and an efficacy rate, fewer side effects, milder chemotherapy intensity and is more sensitive to chemotherapeutic drugs than those of the routine chemotherapy. It can shorten the duration of agranulocytosis and decrease infectious complications and increase the sensitivity of leukemia blast cells to chemotherapeutic drugs.

Effect of recombinant human interleukin 11 on the platelet after hematopoietic stem cell transplantation in patients with leukemia / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect and toxicity profile of recombinant human interleukin 11(rhIL-11) on the platelet after hematopoietic stem cell transplantation in patients with leukemia.@*METHODS@#Twenty-four patients with acute or chronic leukemia treated by allogeneic peripheral blood stem cell transplantation (PBSCT) were randomly divided into a test group and a control group. The patients in the test group were treated with rhIL-11 since the 13th day after PBSCT (1.5mg/d),while the control group were given symptomatic treatment.@*RESULTS@#The average time for the platelet to recover to the level of 20 x 10(9)/L was 20.8 days in the test group, and 26.0 days in control group respectively, there was significant difference (P<0.01). The average time for the platelet to recover to the level of 50 x 10(9)/L was 25.7 days in the test group, and 32.3 days in the control group respectively, there was also significant difference (P<0.01). The average time for the platelet transfusion was 2.2 in the test group, 4.1 in the control group, and there was significantly different (P<0.01). The average number of megakaryocytes was 12.2 in the test group, 4.8 in the control group on 30th day after the transplantation,and there was significant difference(P<0.01). The main side effects of rhIL-11 were nausea, vomit, debility, headache, dizzy and pain of injection site, and the degree was all Iapproximately II grade.@*CONCLUSION@#rhIL-11 has definite recuperative effect on the recovery of the platelet after PBSCT. There is little side effect, and it can be accepted.

Effects of Tongxinluo on cell viability and tissue factor in AngII induced vascular endothelial cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the effects of Tongxinluo on cell viability and tissue factor (TF) in AngII induced vascular endothelial cells and to investigate its mechanism.@*METHODS@#AngII(10(-6)mol/L) was added to human vascular endothelial cells (HUVECs) culture media alone or with various concentration of Tongxinluo drug containing plasma (5%,10%, and 20%) added 30 minutes before AngII. Cell viability was evaluated after 24-hour incubation with AngII in a dose manner. TF, AngII type 1 receptor (AT(1)) mRNA, NO synthase (NOS) and NO were observed after 24-hour incubation with AngII. In addition, NOS inhibitor nomega-nitro-larginine (L-NAME) was added 30 minutes before Tongxinluo and AngII. Cell viability, TF, AT(1)mRNA, the level of NOS and NO were evaluated after 24-hour incubation with Tongxinluo and AngII.@*RESULTS@#Tongxinluo significantly improved AngII induced endothelial cell viability and the effect was the most obvious at 10%. Tongxinluo (10%) decreased the TF and AT(1) mRNA while increased the NOS and NO levels. L-NAME obviously inhibited the effects of Tongxinluo on cell viability, TF, AT(1) mRNA, and NOS and NO levels.@*CONCLUSION@#Up-regulating NOS-NO signaling may be the mechanism of Tongxinluo on cell viability and TF in AngII induced vacular endothelial cells.

3 polymorphisms of gene GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship among 3 polymorphisms of GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness(PTR).@*METHODS@#The 26th exon, the 30th exon and the 21st intron of gene GP IIb in 110 individuals were amplified by polymerase chain reaction (PCR), and the PCR products were analyzed with single-strand conformation polymorphism(SSCP) and sequenced to investigate whether there was linkage among the polymorphisms of the gene. Human platelet antigen-3 (HPA-3) gene frequency was detected by Fok I enzyme in 147 patients with hematologic diseases, and was compared with that in 110 normal individuals. Forty-four patients who received apheresis platelet transfusion repeatedly were randomly divided into the HPA-3 homotype group and the control group. The antibodies of the platelet were detected after 3 times of platelet transfusion.@*RESULTS@#There were polymorphisms of gene GP IIb in the 26th, 30th exon and the 21st intron, and the mutations were: T changed into G in 13,959 th of the 26th exon; C changed into T in 16,997 th of the 30th exon; the 9 bps deletion occurred in 11,996-12,004 th of the 21st intron. The 3 polymorphisms had synchronization in the individuals. The results of Fok I enzyme indicated that the frequency of HPA-3a was 83.6% (92/110)and 81.9%(119/147), and that of HPA-3b was 16.4%(18/110) and 19.1%(28/147) in the normal individuals and the patients respectively. There was no significant difference between the patients and normal individuals (P>0.05). After the platelet transfusion, the antibodies of all the cases of the homotype platelet transfusion were negative, but the antibodies in 2 cases of the control group were positive, and there was antibody to HPA-3a in one of the antibodies positive cases.@*CONCLUSION@#(1)There is close linkage among the polymorphisms of gene GP IIb, which is T->G in 13 959 th of the 26th exon, C->T in 16,997 th of the 30th exon, and the 9 bps deletion in 11,996-12,004 th in the 21st intron. (2)The gene frequency of HPA-3a/3b is similar in the normal individuals and patients with hematologic diseases. (3) HPA-3 system may be one of the reasons for PTR in Chinese.

IkappaB alpha mRNA expression and its DNA sequence in nasopharyngeal carcinoma cell lines / 中南大学学报(医学版)

OBJECTIVE@#To examine the expression of the inhibitor alpha of nuclear transcription factor kappaB (IkappaBalpha) mRNA expression and its sequence characteristics in human nasopharyngeal carcinoma cell (NPC) lines CNE1, CNE2, HNE1 and HNE2.@*METHODS@#Reverse transcription was performed with the total RNAs isolated from the NPC cell lines CNE1, CNE2, HNE1 and HNE2, as well as the transplanted tumor tissues with HNE1 cells. Then IkappaBalpha cDNA was amplified by PCR, and the products were used to examine IkappaB alpha mRNA expression and DNA sequencing, or the DNA sequencing after the products were cloned into plasmid vector.@*RESULTS@#IkappaB alpha mRNA was expressed in all the 4 nasopharyngeal carcinoma cell lines. DNA sequencing showed that polymorphisms and 5 mutations (A825G, A975G, G576A, A655G and C653A) existed in IkappaB cDNA from the transplanted tumor tissues with HNE1 cells, CNE1 and CNE2 cells.@*CONCLUSION@#The expression of IkappaBalpha mRNA not only exists, but DNA polymorphisms and some additional mutations in IkappaBalpha cDNA are also detected in the nasopharyngeal carcinoma cells.

Effects of insulin like growth factor-1 on cell viability and tissue factor in vascular endothelial cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effects of insulin like growth factor-1 (IGF-1) on cell viability and tissue factor (TF) in angiotensin II (Ang II) induced vascular endothelial cells and to investigate its mechanisms.</p><p><b>METHODS</b>10(-6) mol/L Ang II was added to human vascular endothelial cells (HUVECs) culture media alone or 30 min after pretreatment with IGF-1 (0.1 microg/ml , 0.5 microg/ml, 2.5 microg/ml). Cell viability and AngII type 1 receptor (AT1-R) mRNA were evaluated after 24 h incubation with AngII. At the optimum concentration of IGF-1 affecting cell viability, the time dependent manner for 12 - 48 h incubation with Ang II was evaluated. TF, NOS and NO were investigated after 24 h incubation with Ang II. In addition, NO synthase inhibitor Nomega-nitro-1-arginine methylester(L-NAME) was added 30 min before addition of IGF-1 and Ang II, and cell viability, TF, AT1-R mRNA, NOS and NO were evaluated after 24 h incubation.</p><p><b>RESULTS</b>(1) Ang II induced a decrease in cell vitality, an upregulation of AT1-R mRNA, an increase in TF, and a decrease in the activity of NOS and content of NO. (2) Pretreatment with IGF-1 significantly inhibited the decreased cell viability and upregulation of AT1-R mRNA. IGF-1 at 0.5 microg/ml showed the most obvious effects. This effect of cell viability recovery was in a time dependent manner during 12 -48 h. (3) IGF-1 also inhibited the increased content of TF, the decreased activity of NOS and the decreased content of NO. (4) The beneficial effects of IGF-1 on cultured endothelial cells were completely abolished by L-NAME.</p><p><b>CONCLUSION</b>IGF-1 pretreatment could enhance the ang II injured cell viability and anti-thrombosis capacity, and the protective effects may be related to activation of NOS-NO signaling pathway which inhibited AT1-R.</p>

Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro / 中国实验血液学杂志

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.

CD34(+)/CD123(+) cell sorting from the patients with leukemia by Midi MACS method / 中国实验血液学杂志

The aim of this study was to sort the CD34(+)/CD123(+) cells from the bone marrow cells of patients with acute myeloid leukemia (AML) by Midi MACS method. Firstly, the bone marrow mononuclear cells (BMMNC) were isolated from the patients with AML with Ficoll Paque, CD34(+) cells were then isolated by Midi MACS method followed by the isolation of CD34(+)/CD123(+) cells from the fraction of CD34(+) cells. The enrichment and recovery of CD34(+) and CD34(+)/CD123(+) cells were assayed by FACS technique. The results showed that the enrichment of CD34(+) cells was up to 98.73%, its average enrichment was 95.6%, and the recovery of CD34(+) was 84.6%, its average recovery was 51% after the first round sorting, by the second round sorting, the enrichment of CD34(+)/CD123(+) cells was up to 99.23%, its average enrichment was 83%. With regard to BMMNCs before sorting, the recovery of CD34(+)/CD123(+) was 34%. But, on the CD34(+) cells obtained by the first round sorting, its recovery was 56%. In conclusion, these results confirmed that the method of Midi MACS sorting can be applied to sort CD34(+)/CD123(+) cells from the bone marrow cells of AML patients, which give rise to the similar enrichment and recovery of the sorted cells with that of literature reported by the method of FACS.