Effects of IL-6 and IL-11 on differentiation of cord blood CD34+ cells towards megakaryocytes

AIM: To investigate the effects of interleukin-6 (IL-6) and interleukin-11 (IL-11) on differentiation of cord blood CD34+ cells towards megakaryocytes and platelet production in vitro.METHODS: The CD34+ cells from fresh umbilical cord blood samples were cultured in serum-free culture medium with thrombopoietin (TPO) 50 μg/L,IL-3 10 μg/L,stem cell factor (SCF) 50 μg/L as control groups,then 10 μg/L IL-6 or IL-11 or IL-6＋IL-11 respectively was added as treatment groups.Mononuclear cells (MNCs) in cultured cells were detected by cell counter,megakaryocytes (CD41+ cells) and platelets were measured by flow cytometry,respectively.Platelet agglutination after thrombin induced was observed by microscopy and flow cytometry.RESULTS: Compared with the control group,the number of MNCs was not significantly different（P>0.05）,but the numbers of CD41+ cells and platelets were increased significantly (P<0.05) in treatment groups.There were more platelet particles in treatment groups than those in control group by microscopy and the results also showed that the cytoplasmic fragments from the cultures responded to thrombin induction.CONCLUSION: It is concluded that both IL-6 and IL-11 induce the cord blood CD34+ cells to differentiate towards megakaryocytes and produce platelets.     

Effect of trichosanthes injection on expression of proliferating cell nuclear antigen of vascular smooth muscle cell in rabbit

AIM: To observe the effect of thichosanthes injection on the expression of proliferating cell nuclear antigen (PCNA) of vascular smooth muscle cell (SMC). METHODS: The expression of PCNA of cultured rabbit aortic SMC was examined with LSAB immunohistochemical technique, and [³H]-thymidine( [³H]-TdR) incorporation data of SMC and the contents of superoxide dismutase (SOD), lipid peroxide (LPO), prostacyclin (PGI₂) and cyclic AMP (cAMP) in medium were simultaneously determined. RESULTS: Thichosanthes injection has an effects of increasing SOD activity, decreasing LPO, elevating PGI₂ and cAMP, reducing [³H]-TdR incorporation and expression of PCNA (all P＜0.05,P＜0.01). CONCLUSION: Thichosanthes could inhibit SMC proliferation.     

Effect of hypoxia on the expression of proliferating cell nuclear antigen and phenotype of cardiac fibroblasts

AIM:To investigate effect of hypoxia on the expression of proliferating cell nuclear antigen(PCNA) and phenotype of cardiac fibroblasts(CFs). METHODS:The purified cardiac fibroblasts were cultured and divided randomly into there groups :control group, moderate hypoxia(MH) group and severe hypoxia(SH) group. After 72 h, MTT method was used to investigate the proliferation of CFs, and the ultrastructure of fibroblasts were observed with transmission electron microscopy. The expression of PCNA and α-actin in cardiac fibroblasts were measured by the means of immunohistochemistry and laser scanning confocal microscopy, respectively. RESULTS: MTT A_{490 nm}value of MH group was significantly higher than that of control group by (18.4±25.0)% (P＜0.05), whereas MTT A_{490 nm} value of MH group was significantly lower than that of control group by (15.8±25.0)% (P＜0.05).The immunohistochemistry experiment showed that there was basal PCNA expression in control CFs, and it was increased in MH CFs(P＜0.05 vs control group), but there was no significant difference between SH and control CFs. Observed with transmission electron microscopy, the control CFs had typical phenotype of fibroblasts: shuttle-shaped, abundant rough endoplasmic reticulums (RER) and golgi complexes, but few mitochondria or micromyofilaments in cytoplasm. After MH for 72 h, there appeared lots of micromyofilaments in cytoplasm, but there were little micromyofilaments in SH CFs. Observed with laser confocal scanning microscopy, there was lower expression of α-actin in CFs of control group, while many regular bundles of micromyofilaments and the expression of α-actin were signifiantly increased (P＜0.05 vs control group) in CFs treated with MH. The α-actin expression in severe hypoxia CFs was not significantly different from control group. CONCLUSION:MH made CFs have the characteristics of myofibroblasts phenotype and enhanced PCNA expression.     

Effect of protein kinase C-α antisense oligonucleotide on cell cycle of CNE-2Z cells

AIM:To observe the effect of protein kinase C-α(PKCα)antisense oligonucleotide on cell growth, cell cycle and the expression of cyclin E in human poor-differentiated nasopharyngeal carcinoma(NPC) cell line CNE-2Z. METHODS:Antisense PKCα was transfected by cationic liposome(LP) in CNE-2Z cells to analyze the cell growth and cell cycle by MTT colorimetric assay and flow cytometry, respectively. Moreover, the expression of cyclin E was determined by immunocellularchemistry and scanning the result of dot-blotting. RESULTS:①With the concentration of antisense PKCα increasing, the relative cell growth index was decreased gradually(P＜0.01). ②After treated with antisense PKCα, the percentage of cells in G₁ phase enhanced(P＜0.05). ③Compared with the control group, the expressing intensity of cyclin E reduced in antisense PKCα group, and the expression of cyclin E decreased to 66.5%±18.4％(P＜0.05) of the control by scanning quantitative analysis. CONCLUSION:These results indicated that antisense PKCα may inhibit cell growth in CNE-2Z via suppressing the expression of cyclin E and hindering cell process in G₁ phase.     

Effects of aplastic anemia patient serum on the expression of the crucial cyclin D isoform in cord blood CD34+ cells

AIM:To explore the pathogenesis of aplastic anemia (AA), we identified the crucial isoform of cyclin D that determine the proliferation of the cord blood CD34+ cells and observed effects of AA serum on the expression of crucial cyclin D isoform in umbilical cord blood CD34+ cells. METHODS:The CD34+ cells were isolated with MIDI-MACS system. The isoforms of cyclin D were detected by RT-PCR and Western blotting. Methylcellulose culture system was used to measure the formation of CFU-GM. The expression level of crucial cyclin D isoform was assayed by RT-PCR and Western blotting after the CD34+ cells were incubated in AA serum. RESULTS:The crucial cyclin D isoform in CD34+ cells was cyclin D3. The AA serum inhibited the formation of CFU-GM and down-regulated expression level of the cyclin D3 from the mRNA to protein level, respectively. CONCLUSION:The AA serum inhibits the proliferation of CD34+ cells and down-regulates level of cyclin D3, this may be one of hematopoiesis inhibition mechanisms in AA.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

Effect of CLCN2 antisense oligonucleotide on U251 cells injury by cisplatin

AIM: To investigate the effect of chloride channel CLCN2 antisense oligonucleotide on the cell injury of malignant U251 glioma cells induced by cisplatin (DDP). METHODS: The experiment was divided into 4 groups: control group (nonsense oligonucleotide), CLCN2 antisense oligonucleotide group, DDP group (DDP＋nonsense oligonucleotide), DDP＋CLCN2 antisense oligonucleotide group. The viability of U251 cells was measured by MTT assay, CLCN2 mRNA level was determined by RT-PCR, cell apoptosis was measured by TUNEL assay. RESULTS: Compared to the control group, the cell viability, CLCN2 and cyclinD1 mRNA decreased in CLCN2 antisense oligonucleotide group, DDP treated group and CLCN2 antisense oligonucleotide with DDP treated group, cells apoptosis increased. Compared to DDP group, the cell viability (P<0.05) and CLCN2 mRNA decreased in CLCN2 antisense oligonucleotide with DDP treated group, and cells apoptosis increased (P<0.01). Compared to CLCN2 antisense oligonucleotide group, CLCN2 mRNA significantly decreased (P<0.01) in CLCN2 antisense oligonucleotide with DDP treated group. CONCLUSION: CLCN2 antisense oligonucleotide inhibits the expression of CLCN2 mRNA in U251 cells. Inhibition of CLCN2 mRNA facilitates the cell injury of U251 cells induced by DDP. The decrease in CLCN2 mRNA is involved in the mechanism of cell injury by DDP.     

Differentiation of CD34+ cells in human umbilical blood to eosinophils under the condition of cell culture in vitro

AIM: To investigate differentiation of CD34+ cells in human umbilical blood into eosinophils under the condition of cell culture in vitro. METHODS: CD34+ cells were separated and purified from human umbilical blood. The cells were divided into negative group, IL-5 group and allergic rhinitis serum group. The differentiation ability of the cells was measured by flow cytometry, HE staining and electron microscope at the first day, second day, 7th day, 14th day and 28th day culture. RESULTS: The proportion of CD34+ cells in IL-5 group and allergic rhinitis serum group were decreased at the second day. The proportion in allergic rhinitis serum group was lower than that in IL-5 group significantly. The typical structure of eosinophils was observed at the second day. CONCLUSION: The allergic patient serum and IL-5 induce differentiation of CD34+ cells in human umbilical blood to eosinophils.     

Effects of tetramethylpyrazine on calcinerin and proliferating cell nuclear antigen gene expression in the proliferation of vascular smooth muscle cells

AIM：To evaluate the effects of tetramethylpyrazine (TMP) on calcineurin (CaN) and proliferating cell nuclear antigen (PCNA) gene expression in the proliferation of vascular smooth muscle cells (VSMCs) treated by angiotensin Ⅱ (AngⅡ).METHODS：A cell proliferating model of VSMCs induced by AngⅡ was established.PCNA gene exprersion was observed by immunocytochemical staining and image analysis technique;Calcineurin (CaN) activity was detected by enzyme reaction phosphorus measurement.RESULTS：AngⅡ significantly stimulated the proliferation of VSMCs,cell proliferation activity,CaN activity and the expression levels of PCAN were higher than those in control (P<0.01).While treated with TMP,the CaN activity and PCNA expression were obviously lower than those in AngⅡ group (P<0.01).CONCLUSION：The VSMCs proliferation induced by AngII can be inhibited by tetramethylpyrazine significantly,and the inhibiting mechanism of TMP may be related to inhibiting CaN activity and restraining the expression of PCNA in a dose and time-dependent manner.     

Mechanisms underlying the induction of IL-2 secretion by PDB pl us ionomycin in CD4+CD25+ T cells from cord blood and adult peripheral blood

AIM:To confirm that CD4+CD25+ regulato ry T cells don't have an instinctive defection in IL-2 secretion,and to have an insight into the maturation state of CD4+CD25+ T cells in cord blood.METHODS:CD4+CD25+ and CD4+CD25- T cells were purified f rom cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS,and stimulated with PDB plus ionomycin.After 45 hours of culture,cells were d etected for expression of CD69 and CD25 by flow cytometry,and the supernatants were measured for 7 kinds of cytokines by Luminex.RESULTS:CD4+CD25+ T cells from both CB and PB proliferated comparably with CD4+CD25- T cells when stimulated with PDB plus ionomycin.A fter 45 hours of culture,however,the CD4+CD25+ T cells underwent a tendenc y of cell death.Expression of CD25 was further upregulated when CD25+ cells w ere activated.Under stimulation of PDB plus ionomycin,both CD4+CD25+ and C D4+CD25- T cells in PB secreted high levels of IFN-γ,IL-2 and TNF-α,with CD25+ cells secreted much higher level of IL-5,IL-4 and IL-10 than those in CD25- cells;CD4+CD25+ and CD4+CD25- T cells in CB also secreted high level of IL-2 and TNF-α but much lower level of IFN-γ than those in PB,and no secretion of IL-5,IL-4 and IL-10 was observed.CONCLUSION:CD4+CD25+ regulatory T cells don't have an i nstinctive defection in IL-2 secretion,otherwise there may be a different TCR signaling pattern in CD4+CD25+ T cells from traditional T cells.The CD4+C D25+ T cells in cord blood have not fully matured in function.     

Effect of N-copine antisense oligonucleotide on primary culture dcortical neuron in mice

AIM: To study the function of N-copine on cell level by investigating the effect of N-copine antisense on the cultured corti cal neuron of mouse embryos. METHODS: Neuronal cultures were prepared from cerebral cortices of 16-day old mouse (BALB/C) embryos. N-copine expression was blocked by using an tisense oligonucleotides. The effect of inhibition of N-copine expression on the cultured neuronal viability and development was observed. RESULTS: With oligonucleotides treatment for 72 h, the length of neuronalaxon and the area of neurosoma in antisense group were decreased marke dly as compared with those in control group. The number of trypan-blue-stained n eurons and the LDH activity of the culture media were increased significantly as compared with th ose in control groups. CONCLUSIONS: Inhibition of N-copine expression affects the deve lopment of cortical neuron cultured in vitro, and induces the neuron demaged severely. The results suggest that N-copine is a functional protein in neurons, and it may play an important role in the regeneration of nervous system and preventing neuronal degeneration.     

Effects of lipopolysaccharide on proliferating cell nuclear antigen expression on alveolar macrophages and Fas/FasL system expression on alveolar type Ⅱ epitelial cells in smoking rats

AIM:To study the effect of proliferating cell nuclear antigen (PCNA) expression on alveolar macrophages (AM) and Fas/FasL expression on alveolar type Ⅱ epithelial cells induced by lipopolysaccharide (LPS) in smoking rats. METHODS:Immunohistochemistry SABC and immunofluorescence techniques were used to examine PCNA expression on AM and Fas/FasL system expression on alveolar type Ⅱ epithelial cells in smoking rats of different stages induced by LPS. RESULTS:The AM PCNA expression in smoking rats reached the highest level after 3 or 4 months. The AM PCNA expression in every groups stimulated by LPS significantly increased ( P＜0.01). The Fas/FasL system expression on alveolar type Ⅱepithelial induced by LPS were higher than control groups ( P＜0.01). Both the AM PCNA expression and Fas/FasL system expression on alveolar type Ⅱ epithelial cells were parallel. CONCLUSION:Smoking caused the increase in proliferous rate of AM and it may play an important role in the regulation of the injury and repair of alveolar type Ⅱ epithelial cells.     

Effects of gemcitabine on inhibiting proliferation and inducing apoptosis of CD34+CD38- myeloid leukemia stem cells

AIM：To investigate the effects of gemcitabine (GEM), a novel analog of deoxycytidine and nucleoside reductase inhibitor similar to cytarabine (Ara-C) in structure, on the proliferation and apoptosis of myeloid leukemic stem cells (LSCs), CD34+CD38-KG1a cells. METHODS：The expression of CD34 and CD38 on the surface of acute myeloid leukemia KG1a cells was detected by flow cytometry. The effects of GEM at various concentrations for 24 h and sustained medication for 14 d and 21 d on the proliferation and colony-forming ability of KG1a cells were analyzed by soft agar colony-forming experiment. The changes of the cell cycle of KG1a cells treated with various concentrations of GEM were tested by flow cytometry. The apoptosis of KG1a cells was determined by flow cytometry with the staining of Annexin V-FITC and propidium iodide (PI). RESULTS：The percentage of CD34+CD38- cells in acute myeloid leukemia KG1a cells was (98.02±0.72)%.Treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell cycle distribution of the KG1a cells, whereas KG1a cells treated with 0.5 mg/L GEM for 24 h were arrested at G0/G1 phase. After treatment with 0.1 mg/L and 0.5 mg/L GEM for 24 h, the colony numbers at 14 d and 21 d were lower than that in saline control group. No difference of the colony numbers between the cells treated with normal saline and 0.05 mg/L GEM for 14 d and 21 d was observed. After sustained medication with 0.05 mg/L, 0.1 mg/L and 0.5 mg/L GEM and Ara-C for 14 d and 21 d, the colony numbers decreased as compared to saline control group. Treatment with 0.5 mg/L GEM for 24 h increased the apoptotic rate of KG1a cells compared with saline control group, while treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell apoptosis. CONCLUSION：GEM inhibits the proliferation and colony-forming ability, arrests the cell cycle at G0/G1 phase and induces apoptosis of CD34+CD38- acute myeloid leukemia cells.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

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

Effects of dendritic cell stimulation on cytotoxic activity of cord blood derived cytokine-induced killer cells,natural killer cells and CD45RO expression in CIK cells

AIM: To investigate the efficacy of dendritic cells (DCs) that augments the cytotoxic activity of cytokine-induced killer (CIK) cells, natural killer (NK) cells from a same donor and the CD45RO expression on CIK cells. METHODS: The expanded killer cells were divided into two groups: group A was pre-cocultured with DCs for 6 days, group B was the control that without any stimulation. Cytotoxicity of CIK and NK cells was measured at different effect-target ratio against K562 and HL-60. CD45RO and CD45RA expression on CIK cells in different groups were detected by flow cytometry. RESULTS: Cytotoxicity of CB derived killer cells was positive correlation with effect-target ratio. The cytotoxicity of group A against HL-60 was higher than that of group B significantly. At 20∶1 effector-target ratio, the lytic activity of group A CIK, NK cells against K562 was higher than that of group B significantly, but no significant difference between them at 10∶1 effector-target ratio. The CD45RO expression on CIK cells in groups A was significantly higher than that in groups B. CONCLUSION: CIK and NK cells cocultured with DCs can augment the killer's cytotoxicity against tumor cells and promote the CD45RO expression on CIK cells.     

Effects of glutamine and recombinant human growth hormone on intestinal mucosal barrier and proliferating cell nuclear antigen in postoperative portal hypertension patients

AIM:To investigate morphologic and functional changes of small intestinal mucosa and proliferating cell nuclear antigen in postoperative portal hypertension patients with single or combined administration of Gln and rhGH.METHODS:Twenty-nine portal hypertension patients with surgical treatment were prospectively randomized to four groups as follows: ① Gln group (n=6);② rhGH group (n=8);③ Gln+rhGH group (n=7) and ④ control group (n=8).A standard solution for TPN was given three days after operation for a week.The concentration ratio of urinary lactulose and mannitol (L/M),the villus height and crypt depth and PCNA index of small intestinal mucosa were compared.RESULTS:A week after TPN postoperation,the increased ratios of L/M in Gln+rhGH group were less than those in control group (P<0.05).The villus height and crypt depth increased in Gln+rhGH group compared with preoperation (P<0.05) or control group (P<0.05).PCNA index increased in Gln+rhGH group compared with preoperation (P<0.05) or other three groups (P<0.05).The villus height of control group decreased (P<0.05),whereas the crypt depth had no significant difference (P＞0.05).CONCLUSION:This study suggest that Gln together with rhGH reduce the intestinal permeability and protect the mucosa integrality in postoperative portal hypertension patients,but not in single treatment.     

Effect of CD151 on biological characteristics of human umbilical cord mesenchymal stem cells

AIM：To study the effect of CD151 on the biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS：CD151 expression on hUC-MSCs was interfered by siRNA. The cells were divided into siRNA-CD151 group and negative control group (treated with siRNA-NC). The efficiency of interference after 72 h and the changes of other surface markers were detected by flow cytometry. The ability of differentiation was assessed by oil red O and von Kossa staining. The cell cycle was analyzed by flow cytometry. The mRNA expression of CD151, hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), cyclooxygenase 2 (COX-2) and indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs was detected by real-time PCR. The secretion of HGF by hUC-MSCs was measured by ELISA. RESULTS：The results of flow cytometry showed that the expression of CD151 (11.97±2.63 vs 95.66±1.56, P<0.01) and CD105 (93.66±0.21 vs 83.37±0.71, P<0.05) on hUC-MSCs in siRNA-CD151 group was lower than that in negative control group. The consistent results were also achieved by using the method of real-time PCR. Treatment with siRNA-CD151 down-regulated the progress of the cell cycle as the G1 phase increased and the S phase decreased. The mRNA expression levels of HGF and TGF-β1 in hUC-MSCs in siRNA-CD151 group were lower than those in negative control group, and opposite result of COX-2 mRNA expression was observed. The IDO mRNA in hUC-MSCs was unchanged with IFN-γ stimulation for 24 h. HGF concentration in siRNA-CD151 group was decreased as compared with negative control group. CONCLUSION：Interfering CD151 expression on hUC-MSCs doesn’t change other surface markers except CD105, and maintains the capacity of adipogenic differentiation. However, it changes the osteogenic differentiation, proliferation and the expression of immunomodulatory cytokines.