Physiological function of plasma membrane Ca2+-ATPase isoform 2 in inner ear hair cells of mouse

AIM: To investigate the auditory and balanceable function of hair cell plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) channel in mouse inner ear.METHODS: Hearing threshold and endocochlear potential (EP) were measured in the PMCA2 homozygous mutants mice,heterozygous mice and wild-type mice by auditory brainstem response (ABR),distortion product otoacoustic emission (DPOAE) and EP recordings,respectively.RESULTS: The auditory function in PMCA2 wild-type mice was normal,the mean value for ABR thresholds in response to click sound stimulus was (13.75±11.08)dB SPL,EP was (91.3±11.0)mV.The mean value for ABR thresholds in response to click sound stimulus was (63.89±12.90)dB SPL in PMCA2 heterozygous mice and significantly increased relative to that observed in PMCA2 wild-type mice (P<0.01).EP in PMCA2 heterozygous mice was about (80.7±9.0) mV and lower than that in PMCA2 wild-type mice but no statistics significance was observed (P>0.05). DPOAE thresholds increased and reduced amplitudes at high frequencies in PMCA2 heterozygous mice compared to wild-type mice.PMCA2 homozygous mutant mice were completely deaf,the ABR wave form was not observed even 100 dB SPL sound stimuli was used,the value of EP was (56.6±13.0) mV lower than that in PMCA2 wild type mice (P<0.05).PMCA2 homozygous mutants mice did not produce DPOAEs,PMCA2 homozygous mutant mice were deaf and demonstrated difficulties in maintaining their balance.CONCLUSION: These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major Ca²⁺ pump used in endolymph Ca²⁺ maintenance.     

Role of Kv1.3 channel of CD4+T lymphocytes in the formation of atherosclerosis in rat spleen

AIM: To investigate the function of voltage-gated potassium channel Kv1.3 and its possible role in CD4⁺ T lymphocytes in the formation of atherosclerosis (AS) in rat spleen. METHODS: The rat atherosclerosis model was established by feeding high-fat diet. The proportion of lymphocytes was determined by flow cytometry. The CD4⁺ T lymphocytes were separated using immunomagnetic bead. The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was detected. The concentrations of intracellular calcium and cytokines were also measured. RESULTS: (1) The proportion of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (74.93%±2.15% vs 67.80%±2.54%, P<0.05). (2) After stimulated with concanavalin A (ConA), the proliferation of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (1.1321±0.1750 vs 0.7971±0.0955, P<0.05). (3) After stimulated with ConA, the concentration of intracellular calcium in AS group was higher than that in control group. (4) In AS group, the releases of cytokines of IL-2 and TNF-α in AS group were significantly higher when stimulated with ConA for 48 h than that for 24 h. (5) The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was greatly higher in AS group than that in control group (3.670±1.579 vs 1). CONCLUSION: In AS rats, the increase in CD4⁺ T lymphocytes as well as the augmentation of Kv1.3 mRNA expression in the cells suggest that up-regulation of Kv1.3 mRNA expression in CD4⁺ T lymphocytes may be involved in the mechanism of atherosclerotic formation in rat spleen.     

Effects of salvianolic acid B on the energy metabolism and hydrocephalus in mice with cerebral ischemia

AIM: To explore the effects of salvianolic acid B (SalB) on the energy metabolism and hydrocephalus in mice with cerebral ischemia.METHODS: NIH mice were randomly divided into four groups: sham-operated group,cerebral ischemia group,SalB-treated group and nimodipine-treated group.The brain tissue energy charge (EC),phosphocreatine (PCr),the activity of ATPase,excitability amino acid (EAA) content and water content of brain were measured when cerebral ischemia for 30 min.RESULTS: EC (0.520±0.034),PCr content ［(98.344±13.249) μmol/g］,the activity of Na⁺-K⁺-ATPase ［(0.593±0.013)×10³ U/g］ and Ca²⁺-ATPase ［(0.484±0.053)×10³ U/g］ in SalB-treated group were significantly higher than those in cerebral ischemia group {EC (0.465±0.037),PCr content ［(81.614±9.919) μmol/g］ ,the activity of Na⁺-K⁺-ATPase ［(0.244±0.065)×10³ U/g］,the activity of Ca²⁺-ATPase ［(0.321±0.086)×10³ U/g］} (P<0.01).The glutamate (Glu) content ［(0.405±0.110) μmol/g］,aspartate (Asp) content ［(0.141±0.020) μmol/g］ and water content of brain ［(38.1±0.1)%］ in SalB-treated group were markedly lower than those in cerebral ischemia group ［ Glu content (0.550±0.140) μmol/g,Asp content (0.287±0.050) μmol/g,water content of brain (44.1±0.1)%］ (P<0.05,P<0.01).CONCLUSION: The increase in cerebral energy metabolism and the activity of ATPase,and decrease in EAA content in brain tissue are the mechanism of SalB alleviating hydrocephalus at the early stage of cerebral ischemia in mice.     

Changes of RyR-mediated Ca2+ release in VSMCs is involved in the development of vascular hyporeactivity in hemorrhagic shock rats

AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca²⁺ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca²⁺ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca²⁺ concentration in VSMCs were evaluated by fura3-AM and the role of IP₃R and RyR mediated Ca²⁺ release from calcium store was further observed. The role of IP₃R and RyR mediated Ca²⁺ release from Ca²⁺ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca²⁺, NE upregulated by mobilizing Ca²⁺ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca²⁺ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca²⁺ releasing activator caffeine. However, the increase in triggered by IP₃R-mediated Ca²⁺ release agonist adenophostin A (10^-5 mol/L) and ATP-Na₂ (10^-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP₃R mediated Ca²⁺ release with ATP-Na₂ (10^-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP₃R mediated Ca²⁺ release with heparin (10⁴ U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca²⁺-free K-H solution, RyR antagonist ryanodine (10^-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10^-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca²⁺ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.     

Effect of G-CSF on dendritic cell subsets in donors mobilized peripheral blood cells

AIM: To explore the effect of granulocyte colony stimulating factor (G-CSF) on donor’s dendritic cell （DC）subsets in peripheral blood cells (PBC). METHODS: The subsets of dendritic cells in PBC were analyzed by CD34^-Lin^- HLA-DR⁺ cells and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in the serum were tested by ELISA before (25 cases) or after G-CSF mobiling. The relationship between the ratio of DC₁/DC₂ (CD11c⁺CD123^-/CD11c^-CD123⁺) and CD34⁺/MNC was explored. RESULTS: CD34⁺/MNC in PBC harvests was above 0.4% in 23 cases, and the ratio of DC₁/DC₂ was lower, the HLA-DR expression of DC₂ was enhanced after G-CSF mobiling than before, but the levels of IL-12p40, IL-10, IFN-γ, IL-4 in donors serum and CD83 expression of DC₂ were not changed by G-CSF. CONCLUSION: DC₂ increased accompanied by the increase in CD34⁺ cells in the PBC harvests. Although the expression of HLA-DR in DC₂ was up-regulated by G-CSF, these DC₂ did not regulate Th2 cells to excrete inhibitor cell factors and kept on the precursor characters.     

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.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

Modulatory roles of muscarinic cholinergic receptor 3 in proliferation and migration of small cell lung cancer

AIM: To study the expression and roles of muscarinic cholinergic receptor 3 (M3R) in human small cell lung cancer (SCLC) cells. METHODS: Human SCLC cell lines SBC3 and H82 were cultured in vitro. RT-PCR and Western blotting were used to investigate the expression of M3R. MTT assay and Boyden chamber assay were carried out to determine the roles of cholinergic receptor agonist acetylcholine iodide (ACh) and M3R antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) in the proliferation and migration of SBC3 cells. RESULTS: M3R was expressed in SBC3 and H82 cells. The relative protein expression of M3R normalized with β-actin in SBC3 was 2.65-fold higher than that in H82. ACh stimulated SBC3 cell proliferation in a dose-dependent manner. Treatment with ACh at concentrations of 10^-4 and 10^-3 mol/L significantly stimulated SBC3 cell growth at 48 h and 72 h (P＜0.01). SBC3 cell proliferation induced by ACh was inhibited by 4-DAMP in a dose-dependent manner. Pretreatment of the cells with 10^-5 mol/L 4-DAMP suppressed the effect of ACh at 48 h (P＜0.05). Pretreatment with 4-DAMP at concentrations of 10^-6, 10^-7 (P＜0.05) and 10^-5 mol/L (P＜0.01) inhibited the effect of ACh at 72 h. Treatment with 10^-5 or 10^-6 mol/L 4-DAMP alone inhibited the cell proliferation at 48 h (P＜0.01) and the inhibitory effect of 4-DAMP at concentration of 10^-5 mol/L was stronger than that of 4-DAMP at concentration of 10^-6 mol/L at 72 h. ACh increased the cell migration towards fibronectin (Fn) in a dose-dependent manner and ACh at concentration of 10^-4 mol/L enhanced the cell migration by about 3 folds. The cell migration stimulated by 10^-4 mol/L ACh was almost completely blocked by pretreatment with 4-DAMP at concentration of 10^-6 or 10^-5 mol/L (P＜0.01). CONCLUSION: M3R is expressed in human SCLC cells. The M3R antagonist inhibits SBC3 cell proliferation and migration.     

Harmful factors affect glycine receptor α1 subunit mRNA expression in the neonatal rat cardiomyocytes

AIM: To investigate the effects of lipopolysaccharide, hypoxia/reoxygenation,isoproterenol and high concentration of glucose on glycine receptor α₁ subunit mRNA expression in the neonatal rat cardiomyocytes. METHODS: Isolation of cardiomyocytes from Sprague-Dawley rats aging 1～3 d were performed. Cardiomyocytes （1×10⁵～5×10⁵ cells·L^-1）were cultured in DMEM medium containing 15% fetal bovine serum at 37 ℃ in 5%CO₂ atmosphere for 72 h. Then, cultured rat cardiomyocytes were treated with lipopolysaccharide, isoproterenol or high concentration of glucose for 24 h, respectively, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell survival rate was measured using CCK-8 reactant and RT-PCR was applied to monitor the expression of glycine receptor α₁ subunit mRNA. RESULTS: Compared with the control group, no significant difference in the cell survival rate was observed （P＞0.05）. The expression of glycine receptor α₁ subunit mRNA was increased （P＜0.01） in lipopolysaccharide（5,10,20,40,80 mg/L）,isoproterenol（20,100,500 μmol/L） or hypoxia/reoxygenation, hypoxia groups, but decreased（P＜0.01）in the group treated with high concentration of glucose（25, 50 mmol/L）. CONCLUSION: Lipopolysaccharide, isoproterenol, hypoxia/reoxygenation or hypoxia upregulates the expression of glycine receptor α₁ subunit mRNA,but high concentration of glucose down-regulates the expression of glycine receptor α₁ subunit mRNA in cultured neonatal rat cardiomyocytes.     

Relationship between Th1/Th2 balance and CD28/CTLA-4 expression on peripheral blood T cells in patients with systemic lupus erythematosus

AIM: To investigate the pattern of Th1/Th2 balance in systemic lupus erythematosus(SLE) patients and the relationship between CD28/CTLA-4(cytotoxic T-lymphocyte antigen-4) molecule expression and Th1/Th2 balance.METHODS: Eighteen SLE patients met the ARA 1997 updated SLE criteria were selected in the study. According to Bombardier's SLEDAI criteria, all patients were classified into two groups: active group(12 cases) and static group(6 cases). Fourteen normal individuals, matched for age and sex of the patients, served as controls. The peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation and cultured in RPMI-1640 culture medium. After treated with PMA(5 μg/L) and ionomycin(500 μg/L) for 72 h, the PBMCs were collected, the contents of IFN-γ and IL-10 in the supernatant of cultured PBMCs were detected using enzyme linked immunosorbent assay(ELISA). The expression of CD28 and CTLA-4 molecules on T cells were detected by flow cytometric technique with double staining by FITC or PE labeled monoclonal antibodies. RESULTS: The level of IL-10 was higher in the PBMCs of active and static SLE patients(351.29 ng/L±153.31 ng/L and 319.37 ng/L±153.39 ng/L) than that in controls(254.48 ng/L±120.69 ng/L), but the difference did not reach statistical significance(P>0.05). The level of IFN-γ was significantly lower in the PBMCs of active SLE patients(25.76 ng/L±16.09 ng/L) than that in controls(50.71 ng/L±27.92 ng/L, P＜0.05). The ratio of IL-10/IFN-γ was significantly higher in active SLE patients(18.74±13.77) than that in controls(6.66±4.95, P<0.05). Either before or after culture, the expression of CD28 molecule on CD3⁺and CD8⁺ T cells from all SLE patients was not remarkably different from that in the cells of controls. Before culture, the expression of CTLA-4 molecule on CD3⁺T cells of active SLE patients(0.79%+0.37%) was significantly lower than that in the cells of controls(1.31%+0.61%, P<0.05). After culture, the expression of CTLA-4 molecule on CD3⁺ T cells of SLE patients was still lower than that in the cells of normal controls without statistical significance(P＞0.05).The expression level of CD28 molecule on CD3⁺ or CD8⁺ T cells in active SLE patients and controls was not correlated with the levels of IFN-γ and IL-10 in the supernatants(P＞0.05). The level of CTLA-4 molecule expression on CD3⁺ T cells of active SLE patients was positively correlated with IFN-γ level(r=0.681, P<0.05), while was negatively correlated with IL-10 levels(r=-0.624,P＜0.05) and the ratio of IL-10/IFN-γ(r=-0.738, P<0.01). The level of CTLA-4 molecule expression on CD3⁺ or CD8⁺ T cells of controls showed no correlation with IFN-γ levels, while showed negative correlations with IL-10 level(r=-0.587, P<0.05; r=-0.563, P<0.05, respectively).CONCLUSION: There is a bias in the differentiation of Th0 cells towards Th2 in SLE patients. CTLA-4 probably plays an important role in this mechanism through suppressing the signal transmitted by CD28.     

Tissue irradiation injury and past-transplantation distributing diversity of Sca-1 positive cells from murine fetal liver

AIM: To study the relationship between tissue irradiation injury and past-transplantation distributing diversity of Sca-1 positive cells from murine fetal liver and their offspring cells in multi-organs after syngeneic sex mismatch hematopoietic remodeling.METHODS: The Sca-1 positive cells from the livers of C57BL/ 6j male mouse fetus aged 14.5 day were identified and separated by quick PCR and magnetic cell sorting (MACS) techniques. In order to reconstruct hematopoiesis of the adult female mice, which were lethally irradiated with 8G of ［Co⁶⁰］, the 2×10⁴ of Sca-1+ cells were transplanted through caudal vein into each of them. After 6 months, these recipient mice were sacrificed, and their kidneys, livers, lungs, stomachs, and small intestines were taken out, fixed and slices were made. Fluorescence in situ hybridization was performed and observed by fluorescent microscope. Images were captured and analyzed through appropriative cool CCD and software. RESULTS: After 2×10⁴ of Sca-1+ cells were transfused, the hematopoietic function in the lethally irradiative female mice was completely restored. The cells containing Y chromosome were observed 6 months latter in multi-organs, including kidney, liver, lung, stomach, and small intestine. The frequency of the cells containing Y chromosome respectively was kidney (1.65±0.18)%, liver (0.90±0.10)%, lung (1.90±0.60)%, stomach (6.10±0.50)%, and small intestine (7.61±2.30)%, presented the trend of small intestine>stomach>lung>kidneys>liver. Combined informational analysis showed that the presenting frequency of the cells containing Y chromosome was consistent with the irradiative sensitivity of the organ. CONCLUSION: These findings suggest that the capability of differentiation of Sca-1 positive cells from murine fetal liver was potentially connected to the extent of damage in these organs when transferred in vivo.     

RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice, and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group, and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group, ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method, and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1, p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method, and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG, TC and LDL-C, and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner, inhibited the intimal plaque formation and vascular medial proliferation, and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG, TC, HDL-C and LDL-C, and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group, ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs, while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）, but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice, and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Protective effect of HSF1 on mice with LPS-induced acute lung injury and screening of relevant differentially-expressed genes

AIM: To study the protective effect of heat shock factor1 (HSF1) on the mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI), and to screen the relevant differentially-expressed genes. METHODS: ALI mouse model was established by LPS intracheal instillation. The macroscopic and pathological changes of the lung tissue were observed, and the concentrations of total protein, TNF-α, IL-β, IL-6 and VEGF in the bronchoalveolar lavage fluid (BALF) were analyzed. Differentially-expressed genes in the lung tissues of HSF1^+/+ mice and HSF1^-/- mice with ALI induced by LPS were screened by gene chips. The key gene was verified by real-time qPCR. RESULTS: The macroscopic and pathological changes of the lung injury in HSF1^-/-+LPS mice were more serious than those in HSF1^+/++LPS mice. The concentrations of total protein, VEGF, TNF-α, IL-1β and IL-6 in the BALF of HSF1^-/-+LPS mice were significantly higher than those of HSF1^+/++LPS mice (P<0.05). Compared with the HSF1^+/+ mice, a total of 918 differentially-expressed genes were indentified in the HSF1^-/- mice, among which the expression levels of 65 genes had obvious diffe-rence, with 28 genes up-regulated, including Atg7, ccr1, cxcr2, Tbl1xr1, Mmp9, Pparg, Plcb2, Arrb2, Cntn1, Col4a6, etc, and 37 genes down-regulated, including Fgfr1, Fgfr2, Map4k4, Ddx58, Tfg, Stat3, Smad4, Lamc1, Sdc3, etc. The results of real-time qPCR showed that the mRNA level of CXCR2 in HSF1^-/-+ LPS mice was significantly higher than that in HSF1^+/++ LPS mice, which was consistent with the results of gene chips. CONCLUSION: HSF1 has protective effect on the mice with LPS-induced ALI. CXCR2 may be involved in the protective effect of HSF1 on this process.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Effects of intravenous immunoglobulin on IL-4 levels and CD20+ cells in peripheral blood and recurrent rate of infants with wheezing

AIM: To investigate the effects of intravenous immunoglobulin on IL-4 levels and CD20⁺ cells in peripheral blood and the recurrence rate of infants with wheezing. METHODS: IL-4 levels and CD20⁺ cells in peripheral blood of 30 normal infants and 66 infants with wheezing were tested by flow cytometry and ELISA, respectively. The relief time of wheezing and recurrent rate were also recorded. RESULTS: The IL-4 levels and CD20⁺ cells in the wheezing infants were higher than those in controls(P<0.01). The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after routine treatment but were still higher than those in control infants after treatment. The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after immunoglobulin treatment and were almost the same as controls after treatment. The relief time of wheezing in the infants with immunoglobulin treatment was shorter than that in the infants with routine treatment(P<0.01), and recurrent rate of immunoglobulin treatment was lower than that of routine treatment(P<0.05). CONCLUSION: The IL-4 levels and CD20⁺ cells in peripheral blood are increased more significantly in infants with wheezing than those in control infants. The mechanisms of wheezing relief and decreasing the recurrent rate by intravenous immunoglobulin are associated with the down-regulation of IL-4 levels and CD20⁺ cells.     

Effects of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 induced by H2O2 in myocardial cells

AIM: To observe the effect of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 expression induced by hydrogen peroxide (H₂O₂) in cultured cardiomyocytes. METHODS: Changes of cellular morphology were detected under microscope. Apoptosis rates of the cells were analyzed by PI staining with flow cytometry. Expressions of caspase-3 and Bcl-2 proteins in the cells were determined by immunofluorescence with flow cytometry. RESULTS: In the concentrations used, more severe morphological changes with higher apoptosis rate of the cultured myocardial cells were seen in each H₂O₂ group than that in control group. When treated with 1×10-4 mol·L^－1 H₂O₂, the caspase-3 was increased and Bcl-2 protein decreased remarkably in the cells. But each dosage of crocetin, especially the highest one (5×10^－5 mol·L^－1, P<005 compared with 5×10^－7 mol·L^－1 group), seemed efficient in maintaining the cell morphology, reducing the cell apoptosis rate and improving the changes in caspase-3 and Bcl-2 protein expression in the cells exposed to 1×10^－4 mol·L^－1 H₂O₂. CONCLUSION: Crocetin obviously inhibits the apoptosis induced by H₂O₂ in the cultured myocardial cells. The mechanisms may involve the balance of the functions of the apoptosis-related regulating proteins, caspase-3 and Bcl-2 protein.     

Renal function and pathological changes in Niemann-Pick disease type C1 mice

AIM: To investigate the renal function and pathological changes in Npc1 mutant (Npc1^-/-) mice. METHODS: Different genotypes of Niemann-Pick disease type C1 (Npc1) mice were identified by PCR. Subsequently, the renal function of Npc1^-/- and Npc1^+/+ mice at postnatal day 60 (P60) was evaluated by measuring the activity and content of important indicators in the serum including ALT, AST, LDH, urea, UA and Cr. Furthermore, β-galactosidase staining and Masson staining were performed to examine the aging and fibrosis of the renal tissues, respectively. RESULTS: Compared with the Npc1^+/+ mice, the body weight and kidney weight had a significant reduction (P<0.01) in the Npc1^-/- mice. The results of hepatic and renal functions showed that the activities of ALT, AST and LDH, and contents of urea, UA and Cr had marked increases (P<0.05) in the Npc1^-/-mice. Moreover, the results of senescence-associated β-galactosidase staining in the renal tissues demonstrated accelerated aging in the Npc1^-/- mice (P<0.01), and these results were confirmed by Masson staining, which clearly showed the formation of collagen fibers (P<0.01).CONCLUSION: Mutation of the Npc1 gene results in abnormal lipid metabolism, which accelerates kidney senescence by promoting fibrosis in the renal tissue and subsequently causes reduction in renal function.