Deficiency in Na-K-2Cl co-transporter impaired hearing and balance in mice

AIM: We generated transgenic mice of NKCC1^-/- (homozygous mutant),NKCC1^+/- (heterozygous) and NKCC1^+/+ (wild-type) that have a targeted disruption in the NKCC1 gene to investigate the role of Na-K-2Cl (NKCC1) channel in auditory function of the inner ear.METHODS: Hearing threshold and endocochlear potential (EP) were measured in the NKCC1^-/-,NKCC1^+/- and NKCC1^+/+ mice by auditory brainstem response (ABR) and EP recordings,respectively.The inner ears of the mice were removed and examined morphologically with the light microscope.RESULTS: The auditory function of NKCC1^+/+ mice was normal,the mean value for ABR thresholds in response to click sound was ［（23.13±3.78）dB,SPL］,EP was （98±16）mV.The mean value for ABR thresholds to click sound was elevated in NKCC1^+/- mice ［（38.49±12.29） dB,SPL］,relative to that significantly increased in NKCC1^+/+ mice (P<0.01).EP in NKCC1^+/- mice was about （78±7） mV,significantly decreased than that in NKCC1^+/+ mice (P<0.05).NKCC1^-/- mice were completely deaf,the ABR waveform was not observed for even 100 dB SPL sound stimuli used and EP was nearly disappeared (EP,4 mV±6 mV).NKCC1^-/- mice were deaf and demonstrated difficulties in maintaining their balance.NKCC1^-/- mice exhibited a marked atrophy of the stria vascularis,contraction of the endolymphatic compartments and collapse.CONCLUSION: NKCC1 channel plays a critical role in potassium homeostasis of endolymph in the inner ear.Mice lacking of NKCC1 can lead to changing K⁺ concentration in endolymph and influence auditory function subsequently.NKCC1 knockout mice exhibit marked structural abnormalities of the cochlea as well.     

Role of intracellular free Ca2+ concentration in the regulation of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

AIM: To investigate the role of intracellular free Ca²⁺ concentration (［Ca²⁺］_i) in the regulation of calcium-activated chloride (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe ［Ca²⁺］_i of rat PASMCs in normal and chronic hypoxic condition. The influences of Cl_Ca channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The Cl_Ca channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca²⁺］_i was increased. In normoxic condition, ［Ca²⁺］_i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca²⁺］_i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca²⁺］_i had no significant change and no effect on Cl_Ca channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca²⁺］_i which opened Cl_Ca channels. The NFA and IAA-94 blocked them and decreased ［Ca²⁺］_i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca²⁺］_i which opened Cl_Ca channels and had a positive-feedback to ［Ca²⁺］_i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, Cl_Ca channel may play a role in the regulation of PASMCs proliferation.     

Vasodilatatory effect and mechanism of CH2Cl2 extract of flos magnoliae on isolated rat thoracic aorta

AIM: To characterize the vasodilatatory effect of CH₂Cl₂ extract of flos magnoliae (CEF) on isolated rat thoracic aorta and to elucidate its possible mechanism. METHODS: The thoracic aorta was isolated from male Sprague-Dawley (SD) rats and the isometric tension of aortic rings with or without endothelium was measured. RESULTS: CEF (0.1-1 000 mg/L) produced concentration-dependent, endothelium-independent relaxations in phenylephrine (PE)-contracted aortic rings. The maximum relaxation induced by CEF was 78.68%±6.03% in endothelium intact rings and 64.98%±13.90% in endothelium removed rings while the forskolin (1 μmol/L)-induced vasodilation was obtained as 100%. The vasodilatatory effect of CEF was not statistically inhibited by 10 μmol/L glibenclamide (Glib), 3 mmol/L tetraethylammonium (TEA), 100 μmol/L BaCl₂ and 10 μmol/L 1H- -oxadiazole- -quinoxalin-1-one (ODQ) in the preparations without endothelium. The CEF pre-treatment significantly inhibited vasoconstrictions to angiotensin Ⅱ (AngⅡ), prostaglandin F_2α, (PGF_2α), dopamine (Dopa), vasopressin (Vaso), 5-hydroxytryptamine (5-HT) and PE by 91.31%, 82.11%, 95.32%, 90.53%, 72.22% and 83.63%, respectively (P<0.01). In Ca²⁺-free medium treated endothelium removed aortic ring, incubation with CEF at concentration of 82 mg/L significantly attenuated intracellular Ca²⁺ release by PE. In Ca²⁺-free + high potassium medium incubated aortic rings without endothelium, CEF (82 mg/L) markedly inhibited potassium-stimulated Ca²⁺-dependent contraction which was mainly due to Ca²⁺ influx (P<0.01).CONCLUSION: CEF induced vasorelaxation is mainly related to interfering intracellular calcium homeostasis by blocking Ca²⁺ influx and intracellular Ca²⁺ release.     

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.     

Neuropeptide Y induced redistribution of intracellular free calcium in rat cardiomyocytes

AIM: To investigate the effect of neuropeptide Y (NPY) on intracellular free calcium(［Ca²⁺］_i) and Ca²⁺ sarcoplasmic reticulum of cardiomyocytes in rats.METHODS: Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY at concentration of 100 nmol/L for 24 h. Fluorescent indicator Fluo-4 AM was used to detect ［Ca²⁺］_i and Fluo-5N AM was used to detect Ca²⁺ in sarcoplasmic reticulum (SR). Calcium image was recorded by laser scanning confocal microscope. The SR Ca²⁺ load was estimated by caffeine-induced Ca2+ transient (CCT). RESULTS: 24 h after incubation with NPY, compared with control group, the concentration of ［Ca²⁺］_i was significantly elevated (P<0.05), and the concentration of free Ca2+ in SR (［Ca²⁺］_SR) was significantly decreased (P<0.05), and the peak of CCT was attenuated.CONCLUSION: Stimulation with NPY for 24 h causes redistribution of free calcium in rat cardiomyocytes, namely the elevation in ［Ca²⁺］_i and decline in ［Ca²⁺］_SR.     

Effects of chronic hypoxia on intracellular calcium concentration in pulmonary arterial smooth muscle cells

AIM:To study the effect of chronic hypoxia (CH) on the intracellular calcium (［²⁺］_i) in pulmonary artery smooth muscle cells (PASMCs) and the role of L-type calcium channel and calcium store. METHODS:The rat chronic hypoxia model was set up and intervene the PASMCs with normal PSS, calcium-free PSS, nifedipine, and heparine respectively. The resting ［Ca²⁺］_i was determined with the Fura-2/AM calcium imaging technique. RESULTS:(1) The ［Ca²⁺］_i in CH group in normal PSS was higher than that in control group in normal PSS (P<0.05). The ［Ca²⁺］_i in CH group in normal PSS was higher than that in calcium-free PSS (P<0.05). (2) No obvious change of ［Ca²⁺］_i before and after application of nifedipine in PASMCs of CH groups was observed. (3) No difference of ［Ca²⁺］_i before and after application of heparine in PASMCs of CH groups was detected. CONCLUSION:Chronic hypoxia increased the ［Ca²⁺］_i in PASMCs. Chronic hypoxia induced increase in ［Ca²⁺］_i may relate to the influx of extracellular calcium, but not due to the L-type calcium channel or the IP3R modulation only.     

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.     

Influences of Vit-E on the mtDNA damage and Ca2+ homostasis of hippocampus and antioxidative ability in brain of aging mice induced by D-galactose

AIM: To study the influences of vitamin E (Vit-E) on the mtDNA damage and Ca²⁺ homeostasis in hippocampus and antioxidative ability in aging brain induced by D-galactose.METHODS: D-galactose (1 000 mg·k^-1·d^-1 ) was injected into mice hypodermically for 8 weeks to induce aging animal model, and Vit-E (100 mg·kg^-1; 250 mg·kg^-1) was administered for 6 weeks by ig at the 3rd week of making model. After Vit-E treatment for 8 weeks, water maze test was used to determine the ability of mice’s learning and memory. The activities of glutathione peroxidase (GSH-Px) and succinate dehydrogenase (SDH), the content of nitric oxide (NO) and activity of nitric oxide synthase (NOS) in the brain tissue were detected separately. Fura-2/AM, double-wave-length fluorospectrophotometer and PCR method were used to measure the concentration of calcium ion and mtDNA mutation in the hippocampus cells.RESULTS: Administration of Vit-E improved significantly the ability of learning and memory in model mice, inhibited the activity of NOS and decreased the amount of NO, and increased the activities of GSH-Px and SDH respectively in brain tissues, decreased the concentration of calcium ion (P<0.01, P<0.05), and prevented the damage of mtDNA in hippocampus.CONCLUSION: Vit-E can enhance the antioxidative ability, regulate the homeostasis of Ca²⁺ and inhibit the damage of mtDNA caused by oxidative stress in aging brain, and improve the ability of learning and memory in aging mice.     

Effects of FKBP12.6 gene on intracellular Ca2+ concentration in mouse H9c2 (2-1) myocardial cells transfected by ultrasound-mediated destruction of microbubbles

AIM:To investigate the changes of intracellular calcium ion (Ca²⁺) concentration in mouse H9c2 (2-1) cells transfected with or without FK506 binding protein 12.6(FKBP12.6) gene by ultrasound mediated destruction of microbubbles. METHODS:The pcDNA3.1-FKBP12.6 plasmid, mingled with albumin-coated microbubbles agents, was transfected into H9c2 (2-1) cells by ultrasound-mediated destruction of microbubbles. The H9c2 (2-1) cell growth state was investigated by inverted microscope. The changes of intracellular Ca²⁺ concentration was determined by laser scanning confocal microscope. The FKBP12.6 protein expression was checked by immunohistochemistry. RESULTS:As compared with control cells, the H9c2 (2-1) cells, transfected with FKBP12.6 gene, grew better, had higher gross intracellular Ca²⁺ concentration. CONCLUSION:FKBP12.6 gene augments Ca²⁺ concentration in mouse H9c2 (2-1) cells, enhances the contractibility of the myocardial cell, which may be helpful to improve the myocardial dysfunction.     

Changes of ［Ca2+］i and the activity of ACE in alveolar macrophages of rabbits with high-fat diet

AIM: To investigate the effects of high-fat diet on the level of intracellular free calcium (［Ca²⁺］_i) and the activity of angiotensinⅠconverting enzyme (ACE) in alveolar macrophages (AMs) of rabbits.The association between asthma and high-fat diet was also observed.METHODS: Twelve male New Zealand rabbits were medially divided into normal diet group and 1.2% high-cholesterol diet group randomly.8 weeks later,bronchial alveolar lavage was performed in vitro.［Ca²⁺］_i was determined by Fluo-2/am.The activity of ACE was detected with ultraviolet method.RESULTS: The levels of ［Ca²⁺］_i in AMs greatly increased (P＜0.01).The activity of ACE both in BALF and in culture supernatants of AMs was all greatly increased compared with normal diet group (P＜0.01).In hypercholesterolemic group the number of macrophages in BALF showed a positive correlation with the content of cholesterol in serum,such as the level of ［Ca²⁺］_i in AMs and the activity of ACE in the culture supernatants of AMs (all P＜0.05).CONCLUSION: The results suggest that AMs of rabbits may be activated by hyperlipoidemia and become ease to be stimulated.     

Calcineurin signal transduction pathway involves in cardiomyocyte hypertrophy induced by prostaglandin F2α

AIM: To study the role of prostaglandin F₂α (PGF₂α) in cardiac hypertrophy and its relation with calcineurin (CaN) signal transduction pathway in vitro. METHODS: The cultured neonatal rat cardiomyocyte was used to observe the hypertrophic effect of PGF2α, and the hypertrophic response was assayed by measuring the cell diameter, protein content and atrial natriuretic factor (ANF) mRNA expression. For mechanism studies, the intracellular free calcium concentration (［Ca²⁺］_i) in cultured cardiomyocytes was measured by using Fura-2/AM as a fluorescent indicator. ANF and CaN mRNA expressions, and the expressions of CaN and its downstream effectors, NFAT₃ and GATA₄ proteins were assayed by RT-PCR and Western blotting, respectively.RESULTS: In cultured cardiomyocytes, PGF₂α induced profound hypertrophic morphology change, the significant increase in cell diameter and protein content in a concentration-dependent manner compared with those in vehicle control (P<0.01). The same result was found in measuring the ［Ca²⁺］_i in cardiomyocytes (P<0.01). PGF₂α at concentration of 10-7 mol/L significantly promoted ANF and CaN mRNA expressions and the protein expressions of CaN/NFAT₃/GATA₄ compared with those in the vehicle control (P<0.05). Cyclosporin A, a CaN inhibitor, markedly inhibited the myocyte hypertrophy (P<0.01), reduced the increased ［Ca²⁺］_i(P<0.01) and decreased the expressions of CaN mRNA and CaN/NFAT₃/GATA₄ proteins (P<0.05) compared with those of only PGF₂α　10^-7 mol/L treatment. CONCLUSION: Cardiomyocyte hypertrophy induced by PGF2α may be, at least in part, mediated by CaN signal transduction pathway activated by increasing ［Ca²⁺］_i.     

Trichosanthin-induced apoptosis in melanoma B-16 cell line

AIM: To investigate the antitumor effect and mechanism of trichosanthin (TCS) on melanoma B-16 cells.METHODS: (1) The injury of B-16 cells by trichosanthin was observed with SCGE and hoechst33258 staining method.(2) LCSM and specificity fluorescent probe Fluo-3/AM,H₂DCF-DA,DAF-FM diacetate were applied to analyze the dynamic changes of Ca²⁺,ROS and NO in single cell cultured with TCS.Simultaneously,the relationship between ROS,NO and increase of Ca²⁺ was also revealed.RESULTS: (1)When treated with TCS (50 mg/L) for 3 h and 6 h,neither cytotoxicity assay nor SCGE showed the differences compared with control group.After 12 h incubation,specificity phenomena of DNA injury-comet tail appeared in SCGE and chromatin condensation even apoptotic body formation were seen by Hoechst33258 staining.（2） TCS (50 mg/L) evoked rapid enhancement of the production of Ca²⁺,ROS and NO in the cell and the differences between TCS and control group had statistical significance (P<0.01).Augment of ROS and NO was involved in the increase of Ca²⁺.CONCLUSION: Trichosanthin has relatively strong cytotoxicity on melanoma B-16 cells and contributes to apoptosis as well.Trichosanthin-induced apoptosis is related to the increase of Ca²⁺,NO and ROS in the cells.     

Effects of endothelin-1 on the induction of the transdifferentiation of human airway sub-epithelial fibroblasts

AIM: To explore the role of endothelin-1 (ET-1) in initiating transdifferentiation of sub-epithelial fibroblasts (SEFs) into myofibroblasts and its ionic and signal transduction mechanism.METHODS: Human SEFs or SEFs plated in collagen gels were co-cultured with human bronchial epithelial cells (16HBE) treated with lipopolysaccharide (LPS) plus mechanical scratch. ET receptor A inhibitor (BQ123) or the inhibitors specific for p38 MAPK, ERK1/2 were added, repectively. The expression of α-smooth muscle actin (α-SMA) in the SEFs and contractility of the collagen gels containing with SEFs as well as the effects of p38 MAPK or ERK1/2 on α-SMA expression were evaluated. Using Ca2+ sensitive Fluo-3/AM, dynamic changes of intracellular calcium concentration (［Ca²⁺］_i) were observed in the SEFs by laser confocal microscopy.RESULTS: Injured 16HBE induced the transdifferentiation of myofibroblasts, which expressd α-SMA and increased contractility. BQ123 blocked the induction to a certain extent. Injured 16HBE activated p38 MAPK and ERK1/2 pathways in SEFs, both inhibitors of p38 MAPK and ERK1/2 attenuated the induction of α-SMA by injured 16HBE. The addition of exogenous ET-1 enhanced α-SMA expression and activated p38 MAPK, ERK1/2 pathways in the SEFs. Additionally, ET-1 significantly facilitated Ca²⁺ inflow into the fibroblasts.CONCLUSION: Injured 16HBE induces the transdifferentiation of SEFs into myofibroblasts, which is involved in the activation of p38 MAPK and ERK1/2 pathways. The ET-induced influx of Ca²⁺ may be an early signal for initiating the myofibroblasts transdifferentiation.     

Mitogen-activated protein kinase is involved in the changes of intracellular free calcium concentration induced by wound exudate in cultured rat epidermal stem cells

AIM: To observe the effects of harvested wound exudate on intracellular free Ca²⁺ in epidermal stem cells (ESCs) in vitro, and to investigate the relationship between mitogen-activated protein kinases (MAPKs) signal pathways and Ca²⁺ mobilization in this condition.METHODS: Wound exudate was harvested from the 80 full-thickness wounds produced on both sides of the back in 40 adult Wistar rats. ESCs were isolated, purified from neonatal Wistar rats by referring to the formerly records and binding our ideas. When the cultured cells showed up clone growing, they were divided into five groups as follows: group A: control group (no-treatment); group B: only treatment with wound exudate; group C: treatment with wound exudate and PD98059; group D: treatment with wound exudate and SB203580; group E: treatment with wound exudate, PD 98059 and SB203580. Then, the cells were incubated with fluorescence Ca²⁺ dye fluo-3/AM at 37 ℃ for 30 min, and measured by using laser scanning confocal microscope.RESULTS: The results showed that the fluorescent intensity of group B was higher than that in group A. A phenomenon of calcium oscillation was found in group C and group D. Furthermore, a rapid decrease of fluorescent intensity was observed in the cells that were preincubated with PD98059 and SB203580 at the same time. CONCLUSION: Based on above results, we propose that wound exudate can directly induce an increase in intracellular free Ca²⁺concentrations of ESCs. MAPKs signaling pathway has an important function of feedback regulation for free Ca²⁺ mobilization of ESCs in this condition, and also is capable of affecting the biological behaviour of epidermal stem cells.     

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 pioglitazone on glucose metabolism and PPAR-γ expression in lipid-infused rats

AIM: To investigate the effect of pioglitazone (Pio) on glucose metabolism and peroxisome proliferators-activated receptor (PPAR)-γ expression in free fatty acid (FFA) -induced insulin resistance in rats. METHODS: A hyperinsulinaemic-euglycaemic clamp and ［3-3H］-glucose tracing technique were used in awake rats. Glucose metabolism in vivo and PPAR-γ in adipose tissue expression were assessed with elevation FFA by lipid infusion over 4 h in rats pretreated with or without Pio.RESULTS: During steady-state of clamp, there was a significant increase in plasma FFA in two lipid-infused groups, compared to control rats (P<0.01). The glucose infusion rates (GIR) in Pio-treated rats (P/L group), compared with controls, were significantly reduced ［(20.6±0.4) mg·kg^-1·min^-1 vs (33.6±0.6)mg·kg^-1· min^-1, P<0.01］, whereas the GIR was lower in the lipid group (L group) than that in the P/L group［(12.6±0.8) mg·kg^-1·min^-1 vs (20.6±0.4) mg·kg^-1·min^-1, P<0.01］. The hepatic glucose production (HGP) was significantly suppressed (85%) ［(18.3±2.1)mg· kg^-1·min^-1 (basal) vs (2.7±2.4)mg· kg^-1·min^-1, and (17.5±2.6) mg· kg^-1·min^-1 vs (2.6±1.0)mg· kg^-1·min^-1］, all P<0.01 during clamp in control and P/L groups. The suppressive effect of insulin on HGP was significantly blunted in L group［(17.3±2.1)mg· kg^-1·min^-1 vs (15.8±1.5)mg· kg^-1·min^-1］. The rate of glucose disappearance (GRd) was significantly reduced in two lipid-infused rats compared with controls［(26.6±1.6)mg· kg^-1·min^-1 and (23.2±0.9)mg· kg^-1·min^-1 vs (37.7±2.6)mg·kg^-1·min^-1,P<0.01］. The PPAR-γ expression of adipose tissue in P/L group was significantly upregulated. CONCLUSION: Lipid-infusion induces an acute insulin-resistance in vivo. Pio treatment upregulates the PPAR-γ of adipose tissue and suppresses HGP. Pio can protect partly against lipid-induced insulin resistance.     

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.