ObjectiveTo explore the effects and underlying mechanisms of acid sensing ion channels (ASICs) on pain behavior in a rat model of post-incision pain.MethodsFifty-eight adult male Sprague Dawley rats were used in this study,four rats were used for immunofluorescence test,thirty rats were employed for pain behavior test,and twenty-four rats were used for Western blot.Rats used for pain behavior test and Western blot were randomly divided into 3 groups:control group ( C group),incision pain model group ( I group) and amiloride group (A group).Plantar skin of rats in A group were infiltrated with 20 μl(200 μg)amiloride solution.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) of all rats in pain behavior test was tested at 24 h preoperative,2 h,4 h,8 h,12 h,24 h postoperative.Western blot was tested at 4 h postoperative.ResultsImmunofluorescence test displayed ASIC3 was expressed in plantar skin of all rats.The basal level of PWMT and PWTL of all rats in three groups was C group( (23.15 ± 5.10) g,( 11.32 ± 1.21 ) s),I group ( (23.26 ± 5.69) g,( 11.75 ± 2.01 ) s),A group ( (23.63 ± 4.96 ) g,( 11.47 ± 1.96) s) respectively,which was no significantly difference (P ＞ 0.05 ).PWMT and PWTL of I group and A group was significantly lower than that of C group at all time points postoperative (P ＜ 0.05) ; PWMT and PWTL of A group was at 2 h( ( 13.75 ±3.25)g,(9.96±1.32)s),4h((14.05±3.75)g,(9.17±2.11)s),8 h((9.75 ±2.74)g,(8.11 ±1.22)s)postoperative,which was significantly higher than that of I group (P ＜ 0.05 ).Compared with that of C group,the level of pERK1/2 expression was significantly increased in I group at 4 h postoperative (P ＜ 0.05 ),which could be inhibited by amiloride local infiltration (P ＜ 0.05 ).ConclusionASIC3 can mediate incision pain in a rat model of post-incision pain,through pERK1/2 signaling pathway,which can be inhibited by amiloride.

Objective To observe the effect of bone morphogenetic protein-7 (BMP-7) on proliferation,activation and TGF? signaling in TGF?1 inducing hepatic stellate cells (HSC),and its anti-fibrosis mechanism.Methods Human HSC LX-2 cell line was treated with BMP-7 at different concentrations (80,40,20 ng/ml) and TGF?1(5 ng/ml).Proliferation of HSC LX-2 cells was detected with cell counting kit-8 (CCK8).Expressions of ?-smooth muscle actin (?-SMA) and collagen Ⅰ,as well as TGF receptors Ⅰ and Ⅱ (TGF?RⅠ,TGF?RⅡ) mRNA,Smad 3,7 mRNAs,were detected by immunocytochemical assay and RT-PCR,respectively.Results No significant difference was found in proliferation of LX-2 cells before and after treatment with TGF?1.BMP-7 used at different concentrations (80,40,20 ng/ml) inhibited the proliferation of LX-2 with an inhibition rate of 28.9%,19.6% and 10.5%,respectively (P

Growth hormone (GHRH) and pitutary adenylate cyclase activating peptide (PACAP) are the members of the PACAP/Glucagon superfamily,who are related in both sequence and function.Their stimulation of GH secretion and animal growth is concerned.A series of expression plasmid,pIRES1-GHRH-PACAP (P-G-P),plRESI-GHRH (P-G) and plRESI-PACAP(P-P),were constructed,extracted and purified,then transfected into CHO cell line with Lipofectamine.The expression was examined by RT-PCR,dot-ELISA and Western blotting.The biological activity of expression products was detected in rats.At 8 h after injection of transfection supematant,serum IGF-I concentrations in P-G-P group were significantly higher than that in other groups(P ＜ 0.05).PLGA encapsulating plasmid microspheres were prepared and injected intramuscularly into rabbit legs.Growth behavior and IGF-1 level were measured at day 0,15,30 and 45 after injection.Greater body weights gain and higher serum 1GF- [ levels were observed in three plasmid microsphere injection groups,compared with control group.At day 30,the body weight gain in P-G-P group was greater than saline group (81%,P＜ 0.01),P-G mierosphere group (15%,P＜ 0.05) and P-P microsphere group (7%,P＞ 0.05),serum IGF-I concentration in P-G-P microsphere group showed a 16.68% increase to P-G microsphere (P ＞ 0.05),a 17.14% increase to P-P microsphere(P ＞ 0.05) and a 50.46% increase to control (P ＜ 0.05).These results suggest that co-expression of GHRH and PACAP in one expression plasmid might exert an additive stimulation of GH secretion and growth when delivered into rabbit skeletal muscle with PLGA mierosphere.The results may provide a new approach to regulate animal growth.

AIM: To investigate the apoptosis of a vascular smooth muscle cell line A10 caused by mild K+ depolarization. METHODS: Apoptosis was evaluated by nuclear staining, DNA fragmentation gel electrophoresis and propidium iodide-stained flow cytometry. Mitochondrial transmembrane potential (Δψm) was measured by flow cytometry. RESULTS: K+ depolarization caused dose correlated A10 cells apoptosis; nifedipine, BAPTA/AM, ryanodine inhibited the cytotoxic effect of K+ completely.The combination use of nifedipine and cyclosporin A made it clear that mitochondria was involved in the apoptosis of A10 cells,and Δψm measurement further confirmed this speculation; A10 apoptosis caused by K+ depolarization was not influenced by heparin or Zn2+,a effective capacitative calcium entry(CCE) blocker. CONCLUSION: Ca2+ entry through voltage-dependent ca channels increases intracytoplasm Ca2+, then triggers further Ca2+ release from endoplasmic reticulum via ryanodine receptor, and the microdomains of elevated intracytoplasm Ca2+ are sensed by adjacent mitochondria, which ultimately lead to cell apoptosis.

OBJECTIVE To investigate the distribution of pathogenic bacteria and the status of drug resistance in intensive care unit(ICU) of Anhui Provinceical Hospital,to provide reference for rational use of the antibiotics in clinical practice and effective control of hospital infection.METHODS From Jan 2007 to Dec 2008,a total of 873 clinical isolates were collected from different samples of infective patients in ICU,drug sensitivity test was conducted based on disk diffusion testing(K-B),to analyze the drug resistance of the pathogenic bacteria.RESULTS The highest isolating rate came from the sample of respiratory tract,which was 68.96%.Among 873 clinical isolates,Gram-negative bacilli and Gram-positive cocci accounted for 65.95% and 19.22%,respectively.Gram positive cocci rised obviously in 2008 compared to 2007.The rate of bacterial drug resistance was relatively high,meticillin-resistant Staphylococcus aureus(MRSA) detection rate was 56.58%;The detection rates Escherichia coli and Klebsiella ESBLs were 75.00% and 57.41%.CONCLUSIONS There is a higher rate of infection for ICU patients,and the condition of drug resistance is serious.To strengthen the pathogen distribution and drug resistance monitoring is of great guiding significance for rational clinical use of drug,reducing multi-drug resistance and nosocomial infection control.

AIM: To explore method of stimulating murine bone marrow-derived dendritic cells by lipopolysaccharide(LPS), transforming growth factor-β1 (TGF-β1)and to study their biological character. METHODS: Murine bone marrow-derived dendritic cells were cultivated with cytokine GM-CSF and IL-4 for 6 days, BMDC was stimulated by control, LPS, TGF-β1, LPS +TGF-β1 for 48 hours respectively. Morphological characters of BMDC were observed by a inversed microscope, surface molecules such as CD_(11C), CD_(80), CD_(86)and MHC Ⅱ were detected by flowcytometry, Interleukin-6 and interleukin-12 p70 in co-culture medium was quantified by ELISA. RESULTS: In LPS group it presented the most typical DC morphology with the highest expression of CD_(80), CD_(86) and MHC Ⅱ, the strongest ability in mixed lymphocyte reaction, higher level of IL-6 and IL-12 p70 compared with control, TGF-β1, LPS + TGF-β1 ( P < 0. 05). While in TGF-β1 group it presented the less typical DC morphology with the lower expression of CD_(80), CD_(86), MHC Ⅱ, weaker ability in mixed lymphocyte reaction, and lower levels of IL-6 and IL-12 p70 compared with control and LPS (P < 0.05). CONCLUSION: LPS can stimulate maturation of BMDC in its late differentiation which makes it presents a more significant biological characteristics. TGF-β1 can inhibit maturation but not differentiation of BMDC thereby can prevent its biological characteristic presentation.

Objective To establish a real-time quantitative polymerase chain reaction (qPCR) assay for B-cell lymphoblastic leukemia according to individualized and specific immunoglobulin gene rearrangements in leukemia cells, and to use it for the monitoring of minimal residual disease (MRD) of B-cell lymphocytic leukemia. Methods The immunoglobulin gene rearrangements of bone marrow samples from 15 cases of B-cell lymphoblastic leukemia were analyzed with a validated European BIOMED-2 system, and the individualized and specific qPCR-based quantification of leukemic immunoglobulin gene rearrangements was established. Results Unique and specific gene rearrangements of immunoglobulin light and heavy chains were identified in 14 cases and Ig-qPCR based on these gene rearrangements had a sensitivity of 10-5 and high specificity which met the international criteria in 10 patients. Leukemia MRD quantification with immunoglobulin gene rearrangement-based qPCR was similar as compared with other MRD detection methods. Conclusion Immunoglobulin gene rearrangement-based leukemia MRD quantification is feasible, sensitive, specific, precise and much valuable for clinical decision of treatments in B-cell lymphoblastic leukemia.

Objective To test the protective effect of a new Ringer's malate solution on intestine's apoptosis caused by hemorrhagic shock in rats.Methods Forty-eight Sprague-Dawley male rats, weighing 280-320 g, were randomly assigned into four groups: sham shock group (group SS), normal saline group (group NS), Ringer's lactate group (group RL) and Ringer's malate (group RM), n=12 each.The group SS was served as control group, the other groups were subjected to 60 min of hemorrhagic shock followed by crystalloid resuscitation.Those rats were sacrificed 3 h after resuscitation.Intestinal tissue was harvested to detect Bcl-2/Bax protein level, the bioactivity of superoxide dismutase (SOD) and malondialdehyde (MDA) level.The level of intestinal cell apotosis was measured using TUNEL method and apoptosis index was calculated.The intestinal histopathology was examined.Results Compared with group SS, the expression of Bcl-2 and the bioactivity of SOD were lower, the level of Bax protein, MDA and apoptotic index were higher in groups NS, RL and RM (P<0.05).Compared with groups NS and RL, the expression of Bcl-2 and the bioactivity of SOD was higher, the level of Bax protein, MDA and apoptotic index were lower in group RM (P<0.05).Histopathological examination showed that group RM was better than group NS and group RL.Conclusion Ringer's malate alleviated intestinal apoptosis caused by hemorrhagic shock in rats.The study suggests that Ringer's malate solution could be a potential new therapeutic agent for fluid resuscitation.

Objective To analyze the phenotype and genotype of a Chinese family with inherited hypofibrinogenemia,and to investigate its molecular mechanism.Methods Peripheral blood was collected from seven people of this family and then plasma was separated.Activated partial thromboplastin time ( APTT),prothrombin time ( PT),thrombin time ( TT),reptilase time ( RT),the activities of antithrombin( AT∶ A ),protein C ( PC ∶ A ) and protein S ( PS ∶ A ) were tested.The activity and antigen of plasma fibrinogen were analyzed by Clauss method and immunoturbidimetry method,respectively.The fibrinogen peptide chain of the proband was semiquantitatively assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thrombin generation test was performed by calibrated automated thromhogram.The dynamic process of blood coagulation was evaluated by the thrombelastography (TEG).Genomic DNA was extracted from the peripheral blood.The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes FGA,FGB and FGG were amplified by polymerase chain reaction ( PCR ) and analyzed by direct sequen(c)ing.Results The activity and the antigen levels of the proband' s plasma fibrinogen were reduced to 0.48 g/L and 0.68 g/L,respectively.TT prolonged to 29.2 s and RT prolonged to 75.8 s.The assays of SDS-PAGE showed no abnormal molecular weight of fibrinogen.Peak height of thrombin generation was reduced to 249.93 nmol/L and endogenous thrombin potential was reduced to 1007.0 nmol · L-1 · min.Hypocoagulability state of the whole blood was found by TEG test.The coagulation index was - 8.6.The proband was diagnosed as inherited hypofibrinogenemia by phenotype analysis.Two mutations (Gln143Pro and g.4642delC) were found in the proband's fibrinogen Aa-chain gene,Gln143Pro came from her mother and g.4642delC came form her father.Conclusion Compound Heterozygous Mutations (Gln143Pro and g.4642delC ) of fibrinogen Aa-chain causes the proband congenital hypofibrinogenemia.

Objective To analyze the phenotype and genotype of three patients with yon Willebrand disease (vWD),and to explore its molecular pathogenesis.Methods Bleeding time (BT),APTT,ristocetin induced platelet aggregation (RIPA),von Willebrand factor (vWF):ristocetin cofactor (Rco)(vWF∶ Rco),vWF antigen (vWF∶ Ag),vWF activity (vWF∶ A) test,vWF collagen binding assay (vWF∶ CB) and multimer analysis were detected for phenotype diagnosis.The dynamic process of blood coagulation was evaluated by using the thrombelastography.Genomic DNA was extracted from the peripheral blood.The vWF gene mutation was detected by sequencing.Results APTT,BT were prolonged in the three probands.Plasma vWF∶ Rco,vWF∶ Ag,vWF∶ A and vWF∶ CB were decreased in different degrees.RIPA was reduced in probands B and C.vWF multimer analysis found the lost of the large molecular weight multimers in proband B,while basically normal in probands A and C.The dynamic process of blood coagulation of proband C presented obvious hypocoagulability by using the thrombelastography.Heterozygous missense mutation g.106782G ＞ T resulting in Cys1130Phe in exon 26,g.110988G ＞ A resulting in Gly1579Arg in exon 28 and g.110373C ＞T resulting in Arg1374Cys in exon 28 were found in the probands A,B and C,respectively.Conclusion Three probands were diagnosed as type 1,type 2A or type 2MvWD by phenotype detection.Heterozygous missense mutation Cys1130Phe,Gly1579Arg and Arg1374Cys induced vWD of three probands,respectively.