OBJECTIVE: Alveolar type II (AT II) cells play a crucial role in the maintenance of pulmonary surfactant homeostasis and pulmonary immunity. The effects of dexamethasone (Dex) on the ultrastructure of AT II cells after acute lung injury remain unknown. This study focused on the ultrastructural changes caused by acute lung injury and on the effects of Dex administration on these ultrastructural changes in young rats. METHODS: Seventy-two 21-day-old Sprague-Dawley rats were randomly divided into control, acute lung injury and Dex-treated groups. Rats in the lung injury group were intraperitoneally injected with 4 mg/kg lipopolysaccharide (LPS) in order to induce acute lung injury, while the control rats were injected with the same amount of normal saline (NS). The Dex-treated group was injected first with LPS followed 1hr later by Dex (5 mg/kg) injection. Eight rats in each group were sacrificed 24, 48 and 72 hrs after LPS or NS injection. Lung samples were obtained from the lower parts of left lungs and fixed with 2.5% glutaraldehyde for transmission electron microscope examination. RESULTS: Microvilli of AT II cells disappeared and the number of lamellar bodies (LBs) increased in the lung injury group 24 hrs after LPS injection. The ring-like arrangement of LBs around nuclei was present until 48 hrs after LPS injection. By 48 hrs after LPS injection, giant LBs with vacuole-like abnormalities appeared. The shape of nuclei became irregular and the border of the nuclei became blurred. By 72 hrs after LPS injection, the number of LBs was obviously reduced; nucleoli disappeared; and karyolysis occurred in some of the nuclei. In contrast, in the Dex-treated group, LBs crowded on one side of AT II cells and exocytosis appeared on the same side by 24 hrs after LPS injection. By 48 hrs, the number of LBs was reduced. The number of mitochondria increased, and some of them became swollen and enlarged. However, by 72 hrs, the number of LBs increased and the ring-like arrangement of LBs around the nucleus again appeared. CONCLUSIONS: Ultrastructural changes of AT II cells following lung injury induced by LPS were time-dependent in young rats. Dex may ameliorate AT II cell injury and promote functional restoration of AT II cells in LPS-induced acute lung injury. ［Chin J Contemp Pediatr, 2007, 9 (6):521-525］

OBJECTIVE: To detect plasma concentrations of vascular endothelial cell growth factor (VEGF) and tissue factor (TF) in children with acute lymphoblastic leukemia (ALL) and explore their clinical significance in ALL. METHODS: Thirty-three children with newly diagnosed ALL, including 18 cases of low risk, 7 cases of moderate risk and 8 cases of high risk, were enrolled in this study. Twenty-five patients received a complete remission and 8 cases were in non-remission after conventional remission induction chemotherapy. Plasma concentrations of VEGF and TF in the patients were detected using ELISA before and after treatment. Sixteen healthy children served as normal control group. RESULTS: Plasma concentrations of VEGF and TF in ALL patients before treatment were significantly higher than those in normal controls (P<0.01). Plasma concentrations of VEGF and TF in the non-remission group before treatment were significantly higher than those in the remission group (P<0.05) and the control group (P<0.01). After treatment the plasma concentrations of VEGF and TF in the non-remission group were not significantly reduced and higher than those in the remission and the control groups (P<0.01). There were significant differences in plasma concentrations of VEGF and TF among the low-risk, moderate-risk and high-risk groups before and after treatment (P<0.05)．Plasma concentrations of VEGF and TF in the high risk group were not significantly reduced after treatment and higher than those in the control group (P<0.01). A linear correlation was noted between plasma VEGF and TF concentrations in ALL patients before treatment (r=0.50, P<0.01). CONCLUSIONS: VEGF and TF play an important role in the development of ALL and may be useful to the evaluation of the severity and the outcome in ALL. ［Chin J Contemp Pediatr, 2007, 9 (6):526-528］

OBJECTIVE: To detect gene mutations of children with glucose-6-phosphorate dehydrogenase (G-6-PD) deficiency and of carriers of G-6-PD deficiency gene with the technique of polymerase chain reaction and denatured gradient gel electrophoresis (PCR-DGGE), and to explore the value of the technique in the diagnosis of G-6-PD deficiency and G-6-PD deficiency gene carrying. METHODS: cDNAs were harvested by reverse transcription method after RNAs had been extracted from peripheral blood of 43 children with G-6-PD deficiency and of their family members (36 lineages). Electrophoresis behaviors of the fragment from exons 11-12 of G-6-PD cDNA were detected with the technique of PCR-DGGE. Gene sequencing was then performed for the abnormal electrophoresis bands. RESULTS: Abnormal electrophoresis bands were found in the 1304-1520 fragment of G-6-PD cDNA in 33 out of 36 family lineages. The G-6-PD/6-PGD ratio was below 1.00 in 9 mothers of patients. Three of them had the G-6-PD/6-PGD ratio lower than 0.50. The PCR-DGGE bands were the same in the 3 mothers. Gene sequencing showed double heterozygote in the 3 mothers, but the maternal carriers of G-6-PD deficiency gene who had normal G-6-PD/6-PGD ratio showed mono-heterozygote in gene sequencing. Three mutational sites were found in the 1304-1520 fragment, i.e., C1311T, G1376T and G1388A. The electrophoresis behaviors were different among the 3 gene mutational sites. CONCLUSIONS: PCR-DGGE is a sensitive and reliable technique in the screening of gene mutations. It is useful in the diagnosis of G-6-PD deficiency, especially in the diagnosis of female G-6-PD deficiency gene carrying.［Chin J Contemp Pediatr, 2007, 9 (6):529-532］

OBJECTIVE: To study serum concentration and mRNA expression of interleukin-13 (IL-13) in children with steroid-responsive nephrotic syndrome (SRNS) and the effect of methylprednisolone pulse therapy (MPT) on IL-13 expression. METHODS: Twenty-eight children with SRNS were enrolled in this study. Serum protein level of IL-13 was measured using ELISA and IL-13 mRNA expression in peripheral blood mononuclear cells (PBMC) was detected with RT-PCR before MPT, 2 and 5 days after MPT, and 2 weeks after disappearance of proteinuria following MPT. Twenty-four urinary protein was measured with the biuret assay. Twenty healthy children were used as controls. RESULTS: Serum IL-13 levels (38.48±13.01 pg/mL vs 5.18±2.71 pg/mL) and PBMC IL-13 mRNA expression (1.31±0.23 vs 0.36±0.07) before MPT in SRNS patients were significantly higher than in the controls. After 5 days of MPT and 2 weeks after disappearance of proteinuria following MPT, serum IL-13 levels (15.33±7.81 and 5.35±2.12 pg/mL respectively) and PBMC IL-13 mRNA expression (0.89±0.26 and 0.33±0.08 respectively) were significantly reduced (P<0.01). Serum IL-13 levels and PBMC IL-13 mRNA expression in SRNS patients 2 weeks after disappearance of proteinuria following MPT were reduced to control levels, but remained at a higher level than controls 5 days after MPT. A positive correlation was found between serum levels of IL-13 and 24-hour urinary protein in SRNS patients before (r=0.75, P < 0.01) and after 2 and 5 days of MPT (r=0.68, r=0.71 respectively; P <0.05). ConclusionsSerum IL-13 levels and PBMC IL-13 mRNA expression in children with SRNS increase. MPT can inhibit the expression of protein and mRNA of IL-13 in these patients.［Chin J Contemp Pediatr, 2007, 9 (6):533-536］

OBJECTIVE: It has been reported that soluble intercellular adhesion molecule-1 (sICAM-1) participates in many immune and inflammatory reactions. Its expression and role in severe pneumonia has not fully been understood. This study aimed to investigate the changes of sICAM-1 expression in severe pneumonia and the relationship between sICAM-1 and severe pneumonia in children. METHODS: Serum sICAM-1 levels were determined by the double antibody sand using ELISA in 50 children with severe pneumonia and 56 children with mild pneumonia. Fifty-two healthy children served as control group. RESULTS: Serum sICAM-1 levels in children with severe pneumonia （402.36±31.24 μg/L）were remarkably higher than those in the mild pneumonia group (278.86±36.24 μg/L) at the acute stage and higher than in the control group (180.74±21.46 μg/L) (P<0.01). Serum sICAM-1 levels in children with severe pneumonia decreased significantly at the recovery stage (198.56±12.63 μg/L) (P<0.01), which were not statistically different from those in the mild pneumonia group at the recovery stage and the control group. There were no significant differences in serum sICAM-1 levels among the severe pneumonia subgroups caused by different pathogens (bacteria,virus or Mycoplasma) at the acute stage. Serum sICAM-1 levels at the acute stage in children with severe pneumonia who were treated successfully were not significantly different from those in patients whose symptoms were partly improved. Conclusions sICAM-1 might be involved in the inflammation course of severe pneumonia. It can severe as a marker of the diagnosis and the severity evaluation of severe pneumonia.［Chin J Contemp Pediatr, 2007, 9 (6):537-539］

OBJECTIVE: The incidence of childhood asthma is growing. This study aimed to find the changes of clinical features of childhood asthma over a 20-year period. METHODS: The medical data of 200 children with asthma between May 1986 and February 1988 (group 1) and of another 200 asthmatic children between June 2005 and May 2006 (group 2) were studied retrospectively. RESULTS: Sixty-seven patients (33.5%) in group 1 and 128 patients (64.0%) in group 2 had concomitant allergic rhinitis (P<0.01). The incidence of exercise-induced asthma, drug allergy and irritating smell allergy was significantly higher in group 2 than in group 1 (P<0.05). Twenty-nine patients (14.5%) in group 1 and 112 patients (56.0%) in group 2 re-visited to the clinic (P<0.01). The patients in group 1 received nonspecific immune and desensitizing therapy but those in group 2 received inhaled glucocorticoid therapy during the remission stage. After 3 months treatment, asthma was in under control in 11 patients (37.9%, 11/29) in group 1 and in 93 patients in group 2 (83.0%, 93/112) (P<0.01). CONCLUSIONS: The incidence of concomitant anaphylaxis increased in asthmatic children over a 20-year period. The compliance of re-exmination has increased in asthmatic children in recent years and this may be associated with a better efficacy of inhaled glucocorticosteroid therapy.［Chin J Contemp Pediatr, 2007, 9 (6):540-542］

OBJECTIVE: To understand the prevalence of sleep disorders and their influencing factors in primary school children from Urumqi. MethodsA total of 2034 children at the ages of 6-14 years were randomly sampled in 3 districts of Urumqi. The children's sleep states and their family and social environments were investigated through questionnaires. RESULTS: The prevalence of sleep disorders in the subjects was 55%. The prevalence of sleep inquietude was the highest (14.7%), followed by sleep talking (4.8%), sleep walking (1.5%), nocturnal enuresis (1.5%), sleep teeth grinding (5.7%), habitual snoring (12.9%), sleep apnea (0.5%), and waking up by choke (1.9%). Taking drugs to stimulate or inhibit the central nervous system, frequent colds, confined housing area, family history, and sleeping with parents were risk factors for the development of sleep disorders. CONCLUSIONS: The prevalence of sleep disorders within primary school children in Urumqi is higher than the reported data. The development of sleep disorders is multifactorial.［Chin J Contemp Pediatr, 2007, 9 (6):543-545］

OBJECTIVE: To study the cause of low immunologic function and insufficiency of immunoglubulin synthesis in neonates by detecting CD21 expression in B lymphocytes in different age group children.MethodsThis study consisted of three age group children: 2-26 days (n=18), 6 months-2 years (n=12) and 3-12 years (n=17). CD21 expression in B lymphocytes was detected with flow cytometry. Serum levels of immunoglobulins were measured by immunoturbidimetry. RESULTS: The percentage and the number of B lymphocytes expressing CD21 in the neonate group were significantly lower than in the other two age groups. The neonate group also showed lower mean fluorescence intension (MFI) of CD21. The percentage and the number of B lymphocytes expressing CD21 as well as the MFI of CD21 increased significantly with the age. The serum levels of IgA and IgM in the neonate group were noticeably lower than those in the other two age groups. The serum levels of IgA and IgM also increased significantly with the age. CONCLUSIONS: Low CD21 expression in B lymphocytes may be related to low function of humoral immunity in neonates.［Chin J Contemp Pediatr, 2007, 9 (6):546-548］

OBJECTIVE: Ventilator-associated pneumonia (VAP) is a common nosocomial infection and is responsible for a very high mortality in neonatal intensive care unit (NICU) patients. This study was designed to investigate the etiology and high risk factors of neonatal VAP. METHODS: The clinical data of 106 critical neonates who were treated with mechanical ventilator between 2003 and 2005 were studied retrospectively. ResultsOf the 106 neonates, 84 received mechanical ventilation for ≥ 48 hrs. Thirty-five (41.7%) out of the 84 patients developed VAP. Univariate analysis showed that gestational age, duration of mechanical ventilation, reintubation, birth weights, primary lung disease and gamma globulin administration were associated with the development of VAP (P<0.05). Multivariate stepwise logistic regression analysis showed that primary lung disease (OR=3.671, 95% CI=1.0-13.45, P<0.05), duration of mechanical ventilation (OR=4.945, CI=1.51-16.21, P<0.01), reintubation (OR=7.721, 95% CI=2.31-25.85, P<0.01) and high-dose gamma globulin administration (OR=5.520, 95%CI=2.08-16.26, P<0.01) were predicted factors for the development of VAP. The detection rate of gram negative bacilli (76.9%) was the highest, followed by gram positive coccus (17.9%) in VAP patients. CONCLUSIONS: Opportunistic drug-resistant bacteria are common pathogens for neonatal VAP. The risk of VAP is multifactorial, including external medical environments and patients' internal agents.［Chin J Contemp Pediatr, 2007, 9 (6):549-552］

OBJECTIVE: B cell multiplication plays a key role in infections mononucleosis. The present study was designed to detect the expression of B-lymphocyte stimulator (BLyS) mRNA in peripheral blood using real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) in children with infectious mononucleosis in order to explore the role of BLys in this disorder. METHODS: Specific primers and TaqMan probes of BLyS were designed, and fluorescence of the PCR products were detected continuously during amplification. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples were calculated using Stata Software version 8.0, and the results were presented as the ratio of copies of target gene mRNA to β2 microglobulin (β2M) mRNA copies. BLyS mRNA expression in peripheral blood was measured by RFQ-PCR in 18 children with infectious mononucleosis and the results were compared with those measured in 15 healthy controls. RESULTS: The range of target gene mRNA detected by REQ-PCR was from 109 ng/L to 101 ng/L. The coefficient of variation for intra-experimental and inter-experimental reproducibility ranged from 1.88% to 5.89% and 6.32% to 12.34%, respectively. BLyS mRNA expression in peripheral blood in children with infectious mononucleosis were significantly higher than that in controls (1.65±0.10 vs 0.56±0.08; P<0.01). CONCLUSIONS: RFQ-PCR has a high sensitivity and reproducibility for the measurement of BLyS mRNA expression. BLyS may be involved in the development of infectious mononucleosis.［Chin J Contemp Pediatr, 2007, 9 (6):553-556］

OBJECTIVE: The development of recurrent respiratory tract infection (RRTI) is related to vitamin A deficiency and immune function abnormality in children. This study examined serum levels of IgG subclasses and vitamin A in children with recurrent respiratory tract infection.MethodsSerum IgG subclasses levels (IgG1, IgG2, IgG3, IgG4) were detected using ELISA and serum vitamin A levels were detected using high performance liquid chromatography-Miller method in 80 children with RRTI (ranged from 2-10 years old). The values were compared with those from 80 aged-matched healthy children.ResultsSerum levels of IgG2 (1.52±0.18 g/L) and IgG4 (0.22±0.12 g/L) in children with RRTI were significantly lower than controls (IgG2: 2.23±0.08 g/L; IgG4: 0.28±0.01 g/L) (P<0.05). Serum vitamin A levels in children with RRTI were also significantly lower than controls (1.16±0.22 μmol/L vs 1.56±0.12 μmol/L; P<0.05). IgG2 and IgG4 deficiency (27%) was the most common in 22 RRTI children with vitamin A deficiency.ConclusionsSerum levels of IgG subclasses, IgG2 and IgG4, and vitamin A decrease in children with RRTI. There might be some relationship between the decreased IgG2 and IgG4 levels and vitamin A deficiency. ［Chin J Contemp Pediatr, 2007, 9 (6):557-558］

OBJECTIVE: Salvia miltorrhiza Bunge (SMB) is a traditional Chinese herb, which is considered to promote blood flow and remove blood stasis. This study examined whether SMB can alleviate injury induced by hypoxia/reoxygenation (H/R) in human kidney proxiamal tubular cells-2 (HK-2 cells).MethodsThere were 3 experimental groups: control, H/R injury and SMB-treated H/R injury. H/R injury of HK-2 cells was induced by first covering the cells with and then removing liquid paraffin wax. Different concentrations of compound SMB solution (0.05%, 0.10%, 0.15% or 0.20%) were administered to the SMB-treated H/R injury group before the hypoxic injury. After 4, 12 and 24 hrs of hypoxia and 4, 12, 24 and 48 hrs of reoxygenation, morphologic changes of HK-2 cells were observed under an inverted microscope. Cell viability was measured by the MTT method. Lactate dehydrogenase (LDH) activity in the culture supernatants was assayed using biochemical methods; TNF-α levels were determined by radioimmunoassay (RIA). RESULTS: The number of HK-2 cells was significantly reduced in the H/R injury group after hypoxia, and reached a nadir 24 hrs after hypoxia treatment. Various concentrations of SMB-treated groups showed significantly greater number of HK-2 cells than the H/R injury group. SMB solution (0.10%) produced the best effect. The levels of LDH and TNF-α in the H/R injury group were significantly increased, and reached a peak between 24 hrs of hypoxia and 4 hrs of reoxygenation when compared to the control group. Pre-treating with 0.10% SMB resulted in significantly lower levels of LDH and TNF-α than in the untreated H/R injury group at various time points of H/R.CONCLUSIONS: SMB has protective effects against H/R injury of HK-2 cells, possibly through inhibition of inflammatory cytokines.［Chin J Contemp Pediatr, 2007, 9 (6):559-562］

OBJECTIVE: To study the effects of heat shock preconditioning on the expression of heat shock protein-70 (HSP70) and apoptosis of the neuron in experimental autoimmune encephalomyelitis (EAE) rats. METHODS: Thirty-six Wistar rats were randomly divided into control, EAE and heat shock preconditioning groups (n=12 each). The EAE animal model was induced with guinea pig myelin basic protein. Heat shock preconditioning was performed 24 hrs prior to the EAE model inducement. No treatment was done in the control group. The neurological signs were observed after immunization. The spinal cords were removed and stained with hematoxylin and eosin. HSP70 was detected by immunohistochemistry. Apoptosis of the neuron was measured by TUNEL. RESULTS: Heat shock preconditioning significantly alleviated clinical signs and neuronal injury. HSP70 expression in the heat shock preconditioning group was significantly higher than in the untreated EAE group (21.08±0.87 vs 10.17±0.51; P<0.01). Heat shock preconditioning suppressed apoptosis of the neuron compared with the EAE group (apoptosis rate: 21.92±1.00% vs 58.92±1.67%; P<0.01). CONCLUSIONS: Heat shock preconditioning might improve the neurological outcome in EAE rats, possibly through the induction of HSP70 synthesis and the reduction of apoptosis of the neuron in spinal cords.［Chin J Contemp Pediatr, 2007, 9 (6):563-566］

OBJECTIVE: To study the relationship between copper-induced apoptosis and reactive oxygen species (ROS) in BRL cells and the effect of curcumin, a plant-derived polyphenol, on copper-injured BRL cells. METHODS: BRL cells were treated with CuSO4 (100 μmol/L) or curcumin + CuSO4. The BRL cells without any treatment were used as controls. Flow cytometry was applied to detect the production of ROS with fluorescent probe DCFH-DA. MTT colorimetry was used to evaluate cell activity. Apoptosis was measured using Hoechst 33258 staining and Annexin V-FITC and propidiumiodide (PI) staining. JNK/SAPK protein level was detected using Western blot. RESULTS: ROS levels (711.70±68.33 vs 87.22±7.58) and apoptosis rate (45.08±1.87% vs 8.23±2.56%) of BRL cells reached to a peak after 6 hrs of CuSO4 treatment, which were significantly higher than controls (P<0.01). JNK/SAPK levels increased significantly after 6 hrs of CuSO4 treatment and peaked at 24 hrs of CuSO4 treatment compared with controls (P<0.01). Curcumin pretreatment decreased significantly ROS and JNK/SAPK levels as well as the apoptosis rate when compared with the CuSO4-treated alone group (P<0.01).ConclusionsCopper may induce apoptosis of BRL cells. ROS participated in apoptosis induced by copper. Curcumin produced protections on copper-injured BRL cells possibly by anti-oxidation and inhibition of p-JNK expression.［Chin J Contemp Pediatr, 2007, 9 (6):567-570］

Objective To explore the effect of mouse bone marrow mesenchymal stem cells (MSCs) on the expression of chemokine receptors in T lymphocytes in vitro.METHODS: Mouse bone marrow MSCs were separated with Percoll, cultured and expanded in low glucose DMEM. C57BL/6 mouse slpeenocytes were cultured in the 24-hole flasks by the density of 1×106/hole. Phytohemagglutinin (PHA) was then added to the holes and cultured for 72 hrs. This study consisted of three groups. Groups A and B were co-cultured by adding MSCs as the ratio of 0.1 and 0.01 to slpeenocytes respectively. The control group was cultured without MSCs. Three days later the suspended spleenocytes were harvested for detecting the expression of three chemokine receptors CXCR3, CCR5 and CCR7 in T lymphocytes by the flow cytometry. RESULTS: The expression of CD3+CCR5+ and CD3+CCR7+ were statistically different among the three groups. Group A had the strongest expression, followed by group B and the control group. The expression of CD3+CXCR3+ in group A was statistically higher than that in group B and the control group. ConclusionsMSCs could up-regulate the expression of chemokine receptors CXCR3, CCR5 and CCR7 in T lymphocytes stimulated by PHA.［Chin J Contemp Pediatr, 2007, 9 (6):571-573］

OBJECTIVE: The mechanism of high pulmonary blood flow-induced pulmonary hypertension remains unclear. The aim of this study was to explore the effect of proadrenomedullin N-terminal 20peptide (PAMP) on pulmonary hypertension, through examining the alterations of pulmonary PAMP expression and plasma PAMP concentration in rats with pulmonary hypertension induced by high pulmonary blood flow. METHODS: Sixteen male Sprague-Dawley rats were randomly divided into control (n=8) and shunt groups (n=8). Aortocaval shunting was produced in the shunt group. After 11 weeks of shunting, systolic pulmonary artery pressure (sPAP), diastolic pulmonary artery pressure (dPAP) and mean pulmonary artery pressure (mPAP) were evaluated by using a right cardiac catheterization procedure. The ultrastructural changes in intra-acinar pulmonary arteries were observed. The concentration of plasma PAMP was measured by radioimmunoassay. The expression of PAMP in pulmonary arteries was detected by immunohistochemical assay.RESULTS: sPAP, dPAP and mPAP were significantly increased in shunt rats compared with controls (P<0.01). Ultrastructural changes, such as hyperplasia and swelling of endothelial cells, irregularity of internal elastic laminar, and hypertrophy and increased number of synthetic phenotype of smooth muscle cells, were found in intra-acinar pulmonary muscularized arteries in the shunt group. Plasma PAMP concentration (616±195 pg /mL vs 427±90 pg /mL) and PAMP expression in endothelial cells (0.62±0.09 vs 0.38±0.12) and in smooth muscle cells (0.24±0.07 vs 0.14±0.05) of pulmonary arteries increased significantly in the shut group compared with controls. CONCLUSIONS: The up-regulation of pulmonary and plasm PAMP expression might be involved in the development of high pulmonary blood flow-induced pulmonary hypertension.［Chin J Contemp Pediatr, 2007, 9 (6):574-576］

OBJECTIVE: The loss of casepase-8 expression correlates with unfavorable survival outcomes in neuroblastoma (NB). Casepase-8 gene inactivation is caused by methylation. This study aimed to explore the effect of the demethylation agent 5-azacytidine on casepase-8 expression and whether 5-azacytidine can increase the sensitivity of chemotherapy drug doxorubicin to NB cells. METHODS: Caspase-8 mRNA expression in NB cell lines (SH-SY5Y cells) was examined by RT-PCR before and after 5-azacytidine treatment. Survival rates of SH-SY5Y cells were detected using MTT analysis and compared among the doxorubicin alone treatment, 5-azacytidine along with doxorubicin treatment, and caspase-8 inhibitor+5-azacytidine+doxorubicin treatment groups. RESULTS: Caspase-8 mRNA was not expressed in untreated SH-SY5Y cell lines. Caspase-8 mRNA expression in SH-SY5Y cells was detectable 3 days after 5-azacytidine treatment, and increased significantly 5 days after 5-azacytidine treatment (P<0.05). Survival rates of SH-SY5Y cells treated with 5-azacytidine along with different concentrations of doxorubicin (0.05, 0.1,0.25, 0.5 μg/mL) were (77.61±7.30)%, (57.35±6.64)%, (46.25±4.46)% and (35.59±5.12)%, respectively, which were significantly lower than those treated with doxorubicin alone (94.89±4.15%, 80.60±8.50%, 64.48±4.92% and 52.32±6.71%) (P<0.01). Caspase-8 inhibitor pretreatment resulted in an increased survival rate of SH-SY5Y cells (92.95±3.48%, 78.39±4.28 %, 62.31±6.50% and 49.92±5.77%)compared with the 5-azacytidine+doxorubicin treatment group. CONCLUSIONS: 5-azacytidine may enhance anti-tumor efficacy of doxorubicin to NB cell lines, possibly through an up-regulation of caspase-8 mRNA expression.［Chin J Contemp Pediatr, 2007, 9 (6):577-579］

OBJECTIVE: To study the influence of human cytomegalovirus (HCMV) infection on cell cycle and the expression of replication licensing factor Cdt1 in human embryonic lung fibroblastic (HEL) cells and to explore the pathogenesis of HCMV infection. MethodsHEL cells were synchronized in the G0/G1 phase by the serum starvation method. The synchronized HEL cells were infected with HCMV, and those that were not subjected to HCMV infection were used as the control group. The HEL cells were harvested at 12, 24, 48, 72 and 96 hrs of HCMV infection. The cell cycle of HEL cells was detected by the flow cytometry. The expression of Cdt1 mRNA in HEL cells was determined by reverse transcription-ploymerase chain reaction (RT-PCR). ResultsThe cells in the G1 phase in the control group was significantly more than in the HCMV-infected group 12 and 24 hrs after infection (P<0.01). The expression of Cdt1 mRNA in the HCMV-infected group was significantly lower 12 and 24 hrs after infection but increased significantly 48 hrs after infection compared with the control group (P<0.05). The expression of Cdt1 mRNA reached a peak at 12 hrs of infection in the control group, but at 48 hrs of infection in the HCMV-infected group, which markedly lagged behind the control group.ConclusionsHCMV infection arrests the cell cycle of HEL cells at the G1 phase. HCMV infection makes Cdt1 expression delay. HCMV infection can interfere cell cycle of HEL cells possibly through affecting the expression of Cdt1.［Chin J Contemp Pediatr, 2007, 9 (6):580-582］

OBJECTIVE: Human cytomegalovirus (HCMV) displays genetic polymorphisms. Nineteen open reading frames (ORFs, UL133-UL151) found in the Toledo strain of HCMV and other low-passage clinical isolates may be essential for viral infection. This study aimed to analyze the polymorphism of HCMV UL134 gene in clinical isolates and explore the relationship between the polymorphism and HCMV infection. METHODS: PCR was performed to amplify entire UL134 region in 32 clinical isolates, which had been proven as HCMV-DNA positive by FQ-PCR. PCR products were sequenced. RESULTS: All of the 32 isolates were amplified and sequenced successfully. HCMV UL134 gene was highly conserved in the clinical isolates. UL134 ORF and its predicted protein in the clinical strains displayed 96.4%-98.3% nucleotide identity and 92.7%-94.9% amino acid identity respectively compared to those in the Toledo strain. A new posttranslational modification site, sulfationcamp (SUL) site, was found in UL134 protein of all of the clinical isolates except 35j. CONCLUSIONS: HCMV UL134 gene in clinical isolates was highly conserved. No substantial relation was found between UL134 gene and HCMV infectious diseases. ［Chin J Contemp Pediatr, 2007, 9 (6):583-586］

OBJECTIVE: This study examined the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), on eukaryotic initiation factor (eIF- 4E) expression in rat myocardial fibroblasts infected by Coxsackievirus B3 (CVB3) in order to identify the drug target for treatment of viral myocarditis. METHODS: Primary cultured rat myocardial fibroblasts were treated with CVB3 with multiplicity of infection (MOI=0.5 PFU/cell). The experiment consisted of four groups in which the cultured rat fibroblasts cells were treated with CVB3, rapamycin (10 nM) and CVB3 + rapamycin or placebo (control). Experimental model of CVB3-infected myocardial fibroblasts was confirmed by detection of CVB3 mRNA expression with RT-PCR and observation of morphological changes of the infected cells with microscopy. eIF-4E expression was determined by both RT-PCR and Western Blot methods. RESULTS: Morphological changes were found in the fibroblasts treated with MOI 0.5 PFU/cell of CVB3 by transmission electron microscope and the viral particles were found in the cytoplasm. CVB3 mRNA was expressed in CVB3-infected fibroblasts after 1, 2, and 3 days after infection and 2 days after passage. The gray scale values of the eIF- 4E /β-actin in the control, the CVB3, the rapamycin and the CVB3+rapamycin groups were 0.73±0.07, 0.87±0.03, 0.32±0.03 and 0.56±0.04 respectively detected by RT-PCR, and were 0.79±0.09, 1.35±0.12, 0.55±0.04, and 0.62±0.07 respectively detected by Western blot. EIF- 4E expression in the CVB3 group was higher than that in the control group. Both the rapamycin and the CVB3+rapamycin groups had lower eIF- 4E expression than the control and the CVB3 groups. CONCLUSIONS: CVB3 can infect myocardial fibroblasts and up-regulate the eIF- 4E expression in rat myocardial fibroblasts. Rapamycin can inhibit eIF- 4E expression and may be a potential medicine for treatment of viral myocarditis. It was suspected that mTOR/eIF- 4E signal pathway in rat myocardial fibroblasts might play an important role in the pathogenesis of viral myocarditis.［Chin J Contemp Pediatr, 2007, 9 (6):587-590］

OBJECTIVE: In addition to regulating blood pressure, angiotensin II is involved in lung fibrogenesis. This study aimed to explore the effect of losartan, an angiotensin II type 1 receptor antagonist, on lung fibrosis in neonatal rats with hyperoxia-induced chronic lung disease (CLD) and its possible mechanisms. METHODS: Neonatal Wistar rats were randomly divided into four groups within 24 hrs after birth: room air exposure, hyperoxia exposure (85%-90% O2), hyperoxia exposure + losartan, and hyperoxia exposure + placebo. Losartan (5 mg/kg?d) or placebo was administered beginning on the 6th day after birth. After 7, 14 and 21 days of exposure, 8 rats in each group were sacrificed. Lung histological changes were evaluated by hematoxylin-eosin staining. Levels of hydroxyproline (HYP), superoxide dismutase (SOD) and malondialdehyde (MDA) in lung tissues were determined by spectroscopy. RESULTS: Hyperoxia exposure resulted in decreased alveolar septation, enlarged terminal air space, increased collagen deposition, pulmonary hemorrhage, and pulmonary consolidation. In the hyperoxia exposure + losartan group, the alveolar septum became thinner and lung fibrosis was alleviated, but the alveolar space was not obviously deflated and the number of secondary septum was not increased. Hyperoxia exposure increased significantly the HYP contents in lung tissues 14 and 21 days after exposure. Addition of losartan to the hyperoxia exposure resulted in decreased HYP contents (471.46±30.63 μg/kg vs 545.15±34.90 μg/kg for hypoxia alone; P<0.01) after 21 days of exposure. SOD activity increased 7 days after hyperoxia exposure and then decreased to levels similar to the air exposure group. MDA levels increased to a peak at 7 days and remained at higher levels through 21 days of exposure when compared with the air exposure group (P<0.01). Losartan treatment significantly increased SOD activities (82.94±4.62 U/mg protein vs 67.78±8.02 U/mg protein; P<0.01) and decreased MDA levels (30.54±5.89 nmol/mg protein vs 48.75±8.09 nmol/mg protein, P<0.01) compared with the hyperoxia exposure group 21 days after exposure. CONCLUSIONS: Losartan attenuated lung fibrosis in neonatal rats with hyperoxia-induced CLD, possibly through an increase of antioxidase enzyme activity and reduction of lipid peroxidation.［Chin J Contemp Pediatr, 2007, 9 (6):591-594］

OBJECTIVE: The pathogenic mechanisms of chronic lung disease (CLD) of premature infants are not fully understood. Its final pathological changes have been shown to relate with lung cell proliferation. The aim of the study was to explore the relationship of the expression of cyclin dependent kinase 4 (CDK4) mRNA and cyclin dependent kinase inhibitor (CDKI) p21 mRNA with lung cell proliferation. METHODS: Eighty premature rats were randomly assigned to a hyperoxia group and a control group (n=40 each). The hyperoxia group was exposed to high concentration of oxygen (FiO2 >0.90）and developed CLD. The control group was exposed to room air (FiO2 =0.21). The rats were randomly subdivided into groups sacrificed at 1, 3, 7, 14 and 21 days of exposure. Lung tissues were collected and made into 5 μm sections. Expression of proliferating cell nuclear antigen (PCNA) in lung tissues was detected by immunohistochemistry. Dynamic expression of CDK4 mRNA and p21 mRNA were detected by in situ hybridization. Lung histomorphology and fibrosis were observed by microscopy. RESULTS: Expression of PCNA and CDK4 mRNA of lung cells in the hyperoxia group increased significantly after 7 days of exposure, and further increased after 14 and 21 days compared with controls. p21 mRNA expression in the hyperoxia group significantly increased after 1 and 3 days of exposure. After 7 days, p21 mRNA expression progressively decreased, but remained at a higher level than control through 21 days of exposure. PCNA expression was positively correlated to CDK4 mRNA expression (r=0.83, P<0.05) and negatively correlated to p21 mRNA expression (r=-0.81, P<0.05) in the hyperoxia group between 7 and 21 days of exposure. CONCLUSIONS: Hyperoxia can induce lung cell proliferation in premature rats. The over-expression of CDK4 gene and the decreased expression of p21 gene in lung tissues may be associated with lung cell proliferation induced by hyperoxia.［Chin J Contemp Pediatr, 2007, 9 (6):595-600］