Anti-tumor effects of enterovirus 71 on human malignant glioma cell line U251 in vitro / 中华实用儿科临床杂志

Objective To investigate the effects of enterovirus 71 (EV71) on growth,apoptosis and human telomerase reverse transcriptase (hTERT) expression of human malignant glioma cell line U251 in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In the experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously for 72 hours with U251 cells,but in the control group,EV71 were not put.The morphological features and growth of cells were observed under inverted microscope at 0 hour,24 hours,48 hours and 72 hours of EV71 treatment,respectively.At the same time,cell apoptosis was measured by flow cytometry using Annexin V/PI double staining method.hTERT expression was detected with semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR).Results The growth of U251 cells was inhibited significantly at 24 h of EV71 treatment under inverted microscope in experimental group when compared with control group,and at 48 h and 72 h,the morphological features of these cells became significantly shrunken,smaller and irregular shape in experimental group,but which were uniform size in control group.At 24 h,48 h,72 h of EV71 treatment,flow cytometry showed the apoptosis rates were significantly higher in experimental group than that in control group [24 h:(12.55 ± 2.38) ％ vs (1.42 ± 0.21) ％,48 h:(65.60 ± 8.48)％ vs (1.42 ±0.17)％,72 h:(87.52 ±3.05)％ vs (1.41 ±0.16)％,all P =0.000],and there was upward trend day by day,but no change in control group.At the same time,hTERT mRNA expression levels of U251 cells were significantly lower in experimental group than control group [24 h:(0.58 ± 0.05) vs (0.89 ± 0.05),48 h:(0.23 ± 0.04) vs (0.89 ± 0.03),72 h:(0.10 ± 0.03) vs (0.90 ± 0.06),all P =0.000],and the downward trend was observed day by day,but there was no this trend in control group.Conclusions EV71 can effectively inhibit the growth of U251 cells,induce the cell apoptosis,and has the potential anti-tumor effect.Its mechanisms may be partly related to down regulation the hTERT expression of U251 cells.

Study of enterovirus 71-induced autophagy in human malignant glioma cell line U251 / 中华实用儿科临床杂志

Objective To show evidences that autophagy is induced in human malignant glioma cell line U251 by enterovirus(EV71) and its effect on the expression of microtubule-associated protein 1 lightchain 3 (LC3) in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously.After 12 hours post infection of U251 cells with EV71,autophagic vacuoles of U251 cells were marked by monodansylcadaverine staining and observed under fluorescence microscope.After 24 hours post infection,expression and intracellular distribution of LC3 protein in U251 cells were observed under fluorescence microscope by immunofluorescence.Expressions of LC3-I and LC3-Ⅱ protein were measured by Western blot and analysis of LC3-Ⅱ protein expression was performed with semi-quantitative calculation at 2,4,8,12,24 and 48 hours post infection of U251 cells with EV71,respectively.Results Autophagic vacuoles stained by MDC in U251 cells appeared as distinct dot-like structures distributed under fluorescence microscope.The number of autophagic vacuoles were increased significantly at 12 hours post infection of U251 cells with EV71 when compared with control group.The morphological features of these cells became significantly shrunken,smaller and irregular shape at 24 hours post infection of U251 cells,and the expressions of LC3 protein were significantly higher in experimental group than those in control group.Under a fluorescence microscope,LC3 protein distributed within the cytoplasm or localizing in the perinuclear regions.At 4 hours post infection of U251 cells with EV71,the expression of LC3-Ⅱ protein started to increase,and was significantly higher than that in control group (P ＜ 0.01).Conclusion These results indicate that EV71 can effectively induce autophagy of human malignant glioma cell line U251,and play its oncolytic effect.

Effect of nuclear transcription factor kappaB pathway on proliferation in rat pulmonary artery smooth muscle cells induced by platelet-derived growth factor / 中华实用儿科临床杂志

Objective To explore the effect of nuclear transcription factor kappaB(NF-κB) pathway on proliferation of rat pulmonary artery smooth muscle cells (PASMC) induced by platelet-derived growth factor (PDGF).Methods PASMC isolated from rats and cultured in vitro were divided into 3 groups according to the randomization principle:control group(cultured by M199),PDGF treatment group(cultured by M199 and stimulated by PDGF),PDGF + parthenolide treatment group (cultured by M199 and stimulated by PDGF,and intervented by the NF-κB inhibitors parthenolide).MTT colorimetric assay and flow cytometry were performed to detect cell proliferation and cell cycle distribution.Immunohistochemistry was performed to detect the expressions of NF-κB and COX-2 protein.Fluorescence quantitative RT-PCR was performed to detect NF-κB and COX-2 mRNA expressions.One-way ANOVA was used for statistical analysis,multiple comparisons were analyzed by LSD.Results Compared with the control group,MTT value of PASMC was increased significantly when induced by PDGF at each time points(all P ＜ 0.05).MTT value decreased dramatically after the intervention of NF-κB inhibitor parthenolide(P ＜0.05).Data from flow cytometry detection showed that cell proportion of S phase and G2 + M phase increased significantly in PDGF treatment group,which had statistical difference compared with control group (all P ＜ 0.05).Compared with PDGF induced group,after the intervention of parthenolide,cell proportion of S phase and G2 + M phase ratio decreased dramatically (P ＜ 0.05).The expressions of NF-κB and COX-2 protein and mRNA were promoted in the PDGF induced group compared with the control group (all P ＜ 0.05).Compared with PDGF induced group,after the intervention of parthenolide,the expressions of NF-κB and COX-2 protein and mRNA decreased dramatically(all P ＜ 0.05).Conclusions PDGF can induce proliferation of PASMC,promote cell cycle process and enhance the expressions of NF-κB and COX-2 protein and mRNA.NF-κB pathway involves in the proliferation of PASMC induced by PDGF.

Risk factors of heart and lung failure in children with severe hand, foot and mouth disease and treatment experience / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study risk factors for severe hand, foot and mouth disease (HFMD) complicated by heart and lung failure and treatment experience.</p><p><b>METHODS</b>A total of 198 children with severe HFMD between March and August in 2011 were enrolled. Univariate analysis and logistic regression model were used to analyze the risk factors severe HFMD complicated by heart and lung failure. The effects of combination therapy with immunoglobulin+dexamethasone+ribavirin were observed.</p><p><b>RESULTS</b>Univariate analysis indicated that HFMD patients with heart and lung failure had higher proportions of consciousness, tachypnoea, abnormal hemodynamics, increased troponin and EV71 infection than HFMD patients without heart and lung failure (P<0.05).Multivariate logistic regression analysis indicated that tachypnoea, abnormal hemodynamics and EV71 infection were the main risk factors for heart and lung failure. Compared with combination therapy with dexamethasone+ribavirin, combination therapy with immunoglobulin+dexamethasone+ribavirin was more effective for preventing hemodynamic changes in children with severe HFMD (P<0.01). Compared with HFMD patients with heart and lung failure, the effect of the combination therapy with immunoglobulin+dexamethasone+ribavirin was better in HFMD patients without heart and lung failure (P<0.01).</p><p><b>CONCLUSIONS</b>The main risk factors for heart and lung failure in children with severe HFMD include tachypnoea, abnormal hemodynamics and EV71 infection. Early combination therapy with immunoglobulin+dexamethasone+ribavirin can reduce the incidence of heart and lung failure in children with severe HFMD.</p>

hTERT gene expression in children with beta-thalassemia major / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Human telomerase reverse transcriptase (hTERT) is a rate-limiting enzyme which dictates the activity of human telomerase and thus decides the life span of cells. The aim of this study was to explore the expression of hTERT in bone marrow from children with beta-thalassemia major and the relationship between the expression of hTERT and hemoglobin levels.</p><p><b>METHODS</b>Multiple allele specific polymerase chain reaction (MASPCR) was used for targeted DNA amplification and gene mutation analysis of beta-thalassemia. hTERT mRNA expression in bone marrow was examined using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis in 29 children with beta-thalassemia major, in 10 children with agranulocytosis and in K562 cell line. The hemoglobin levels in peripheral blood were measured. The relationship between hTERT expression and hemoglobin levels was evaluated by the Spearman test in the beta-thalassemia major group.</p><p><b>RESULTS</b>hTERT mRNA expression significantly increased in bone marrow from children with beta-thalassemia major compared with that from children with agranulocytosis (0.2928+/- 0.0838 vs 0.0993+/- 0.0336; P<0.01), but was significantly lower than that in K562 cell line (0.8291+/- 0.0908) (P<0.01). A significantly inverse correlation was found between hTERT mRNA expression and hemoglobin levels (r=-0.841, P<0.01).</p><p><b>CONCLUSIONS</b>A low hemoglobin concentration might contribute to the up-regulation of marrow hTERT expression in children with beta-thalassemia major.</p>

Administration of low-dose heparin in total nutrient admixture prevents central venous catheter-related infections in neonates / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Peripherally inserted central catheter (PICC) is widely used to provide a long-term access for the administration of total parenteral nutrition and medications. Catheter-related infections (CRI) are common complications of PICC. The purpose of this retrospective study was to investigate the role of low-dose heparin added to the total nutrient admixture (CTNA) in the prevention of CRI.</p><p><b>METHODS</b>Eighty-three neonates who underwent PICC received TNA with (heparin group, n=43) or without heparin (0.5 U/mL) (control group, n=40). The incidence of CRI was compared between the two groups.</p><p><b>RESULTS</b>The incidences of catheter obstruction (5% vs 20%) and the catheter-tip colonization (2% vs 18%) in the heparin group were significantly lower than those in the control group (p<0.05). None of the neonates in the heparin group had clinical evidence of catheter-related bloodstream infection, but 5 cases in the control group (p<0.05).</p><p><b>CONCLUSIONS</b>The administration of low-dose heparin in TNA may decrease the incidences of catheter obstruction and CRI.</p>

Comparative study on clinical features between TEL-AML1 positive and negative childhood acute lymphoblastic leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the incidence of TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia (ALL) and to compare the clinical features between TEL-AML1 positive and negative patients.</p><p><b>METHODS</b>Samples of bone marrow or peripheral blood were collected from 95 newly diagnosed ALL children and the TEL-AML1 fusion gene was detected using nested reverse transcription-polymerase chain reaction (RT-PCR). The ALL patients were stratified into TEL-AML1 positive and negative groups and the clinical features were compared.</p><p><b>RESULTS</b>Among 95 patients, 20 (21.05%) were TEL-AML1 positive. The median age of TEL-AML1 positive patients was 5.9 years old and M/F ratio was 1.22:1. There were significant differences between TEL-AML1 positive and negative patients in hepatomegaly (2.75 cm vs. 4 cm below costal arch, P=0.006), splenomegaly (0 cm vs. 3 cm below costal arch, P < 0.001), initial white blood cell count (median 7.40 x 10(9)/L vs.18.70 x 10(9)/L, P=0.011), initial peripheral blood blast (median 2.45 x 10(9)/L vs.11.66 x 10(9)/L, P=0.013), hemoglobin level [(61.45 +/- 13.46) g/L vs. (75.89 +/- 23.11) g/L, P=0.003] and serum lactate dehydrogenase [(621.47 +/- 335.85) U/L vs.(1566.64 +/- 1720.45) U/L, P=0.020], while no differences were found between two groups in age, gender ratio, initial platelet count, percentage of blast in bone marrow, immunophenotypes and the expression of myeloid antigen CD13, CD33 and CD34. The prednisone sensitivity test showed that all 12 TEL-AML1 positive patients were good responders, while there were 11 prednisone poor responders among 40 negative patients (27.50%, P < 0.05). Bone marrow examination on day 15 showed no difference in the rate of complete remission between TEL-AML1 positive and negative patients.</p><p><b>CONCLUSION</b>The incidence of TEL-AML1 fusion gene in cases of ALL is 21.05%. The load of leukemia cells in TEL-AML1 positive patients is significantly smaller than its counterparts, and the blast cells in TEL-AML1 positive patients are more sensitive to prednisone, indicating childhood ALL with TEL-AML1 fusion gene has a favorable prognosis.</p>

Study of Human Telomerase Reverse Transcriptase Gene Expression in Acute Non - Lymphocytic Leukemia Children / 实用儿科临床杂志

Objective To explore the gene expression of human telomerase reverse transcriptase (hTERT)in childhood acute non- lymphocytic leukemia (ANLL) and its clinical implication. Methods Expression of hTERT mRNA was detected in HL - 60 leukemia cell line, 14 ANLL children and 11 healthy children by reverse transcriptase- polymerase chain reaction (RT- PCR). Results hTERT gene was expressed in HL-60 cell line, ANLL children (11/14) and healthy children (2/11). The expression of hTERT gene was observed in all subtypes of ANLL. Furthermore, there were statistically significant differences in expression levels of hTERT gene between children with ANLL and healthy children (t = 5.034 P = 0). Conclusions The up - regulation of gene expression of hTERT may play a very important role in the progression of children ANLL. The detection of hTERT expression may be a useful additional method for monitoring the state of illness.

Expression of Hypoxia-Inducible Factor-1? in Cultured Cortical Neurons after Hypoxia-Ischemia / 实用儿科临床杂志

Objective To explore the expression of hypoxia-inducible factor-1?(HIF-1?) protein and its mRNA in cultured cortical neurons after hypoxia,ischemia or hypoxia-ischemia(HI) and explore the possibilities of HIF-1? gene therapy in HI neurons.Methods The in vitro models of HI,pure hypoxia and pure ischemia were established using embryonic day 16-18 rats cortical neurons.Immunohistochemical and in-situ hybridization were performed to examine the expression of HIF-1? protein and its mRNA at different reperfusion time points in neurons.Results The expression of HIF-1? protein was very week in normoxic cultured neurons,but was up-regulated while treated with hypoxia and(or) ischemia.HIF-1? expression reached peak at 4 to 8 h after reperfusion with HI,which were statistically significant higher than that at other time points(Pa=0),and decreased gradually at 12 h.Furthermore,HIF-1? protein expression was significantly higher in HI group compared with that in the pure hypoxia or ischemia group(Pa=0).HIF-1? mRNA reached peak immediately after HI,decreased gradually at 2 h,and returned to the baseline at 8 h after reperfusion.Conclusions HIF-1? expression on cortical neurons is regulated differently with hypoxia,ischemia or HI treatment,HIF-1? gene therapy for HI neurons maybe a useful method in the future studies.

Preliminary analysis on human telomerase reverse transcriptase gene expression in acute leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the relationship between gene expression of human telomerase reverse transcriptase (hTERT) and its clinical characteristics in leukemia.</p><p><b>METHODS</b>The protocol of RT-PCR was used to detect the hTERTmRNA expressing levels in peripheral blood samples from leukemic patients under primary treatment(n=42), in complete remission(n=21), with recurrent leukemia (n=4); and from normal subjects (n=5), respectively.</p><p><b>RESULTS</b>The positive percentage of hTERTmRNA expression was 73.81% for the primary treatment cases, and 19.05% for the complete remission cases. All of the recurrent cases gave positive results. One of the normal controls presented low level of hTERTmRNA expression. The expressing level of hTERTmRNA in primary treatment cases was 0.64+/-0.21, in complete remission leukemia 0.31+/-0.16, in recurrent cases 0.84+/-0.09, and in normal controls 0.10.</p><p><b>CONCLUSION</b>The activation of telomerase may be an essential factor in the development of leukemia and usually be the late event in its progression. As an indicator of leukemia cell, the detection of hTERT mRNA may be used in clinical analysis, disease monitoring and prognosis judgement.</p>