Objective:To investigate the impact of recombinant flagellin targeting TLR5 and NLRC4 simultaneously or respectively on innate immune cells in mice. Methods: Induction,expression,purification and identification of recombiant FliC,which were FliC(activating both TLR5 and NLRC4);FliCΔ90-97(unable to activate TLR5),FliC-L3A(unable to activate NLRC4),FliCΔ90-97:L3A(unable to activate both TLR5 and NLRC4). The mice were divided into five groups,namely group FliC,FliC-L3A,FliCΔ90-97,FliCΔ90-97:L3A and PBS,which were injected with 100μl PBS or 10μg recombinant flagellin intraperitoneally,three mice in each group. 12 h later,the mice were executed using dislocation of cervical vertebra and the splenic and peritoneal cells were isolated. The spleen was grinded into single-cell suspension. The proportion of neutrophils,NK cells,DCs and the expression level of CD80 and CD86 on DCs were evaluated with flow cytometry. Results:Group FliC,group FliC-L3A and group FliCΔ90-97 shared the similar proportion of neutrophils in peritoneal cavity ( P>0. 05 ) , and all of which were significantly higher than group PBS and group FliCΔ90-97 ( P<0. 01),and NK cells also showed the similar trend. Compared with group FliCΔ90-97 and FliCΔ90-97:L3A,the mean fluorescence intensities(MFIs) of CD80 and CD86 in group FliC and FliC-L3A increased significantly(P<0. 01). The proportion of Treg in spleen was highest among all groups. Conclusion:Activation of TLR5 and NLRC4 had similar chemotaxis of neutrophils and NK cells. The ex-pression of CD80 and CD86 on DCs were upregulated after stimulation by flagellin and TLR5-dependent. Activation of TLR5,but not NLRC4,increased the proportion of Treg in spleen.

Objective To evaluate the efficacy of pegylated interferon α-2a and entecavir (ETV) combination therapy for patients with HBeAg positive chronic hepatitis B (CHB).Methods Fifty eight HBeAg positive CHB patients were assigned to two groups:29 patients received ETV 0.5 mg daily for 72 weeks (ETV group) and 29 patients received ETV and pegylated interferon α-2a 180 μg weekly for 48 weeks followed by ETV alone for 24 weeks (combination group).Serum samples were collected from all patients every 12 weeks for assessment of biochemical,virological and serological responses to treatment.Results Fifty four patients completed the 72-week study,including 28 in ETV group and 26 in combination group.There were no significant differences in week 24,week 48 and week 72 of ALT normalization [72％ (21/29)vs.93％ (27/29),x2 =2.104;90％ (26/29) vs.97％ (28/29),x2 =0.269;90％ (26/29) vs.97％ (28/29),x2 =0.269],HBV DNA undetectable rate [31％ (8/26) vs.46％ (13/28),x2 =1.391;62％ (16/26) vs.57％ (16/28),x2 =0.108;77％ (20/26) vs.75％ (21/28),x2 =0.027],HBeAg loss rate[12％(3/26) vs.25％ (7/28),x2 =0.850;31％ (8/26) vs.32％ (9/28),x2 =0.012;46％ (12/26) vs.36％(10/28),x2 =0.609] and HBsAg levels (log10 IU/ml) (3.63 ± 0.45 vs.3.36 ± 1.18,t =-1.066;3.45 ±0.43 vs.3.23 ± 1.15,t =-0.915;3.36 ± 0.58 vs.2.88 ± 1.28,t =-1.762) between two regimens (all P ＞ 0.05).Among 58 patients,15 were HBeAg and anti-HBe double-positive (26％)and 43 were HBeAg mono-positive patients.The baseline HBV DNA level [(5.07 ± 1.50) vs.(6.40 ± 1.47) log10 IU/ml,t =2.858,P ＜ 0.05] and HBeAg titer [14 (4-45) vs.732 (296-1 012) S/CO,Z =-5.031,P =0.05] in double-positive patients were lower than those in mono-positive patients.The HBV DNA undetectable rate of double-positive patients was significantly higher than that of mono-positive patients in 24 weeks [10/15 vs.26％ (10/39),x2 =7.819,P ＜0.05] and 72 weeks [15/15 vs.69％ (27/39),x2 =4.287,P =0.05].The HBeAg loss rate of double-positive patients was higher than that of mono-positive patients in 12 weeks [6/15 vs.10％ (4/39),x2 =4.533,P =0.05] and 48 weeks [9/15 vs.26％ (10/39),x2 =5.608,P =0.018].This tendency was more significant in the combination therapy group,but the difference was not statistically significant.(5/6 vs.4/9,P =0.065).Conclusions Compared with Entecavir monotherapy,entecavir combined with interferon may not improve the therapeutic effect in HBeAg positive chronic hepatitis B patients.However,the therapeutic response of HBeAg/anti-HBe double-positive patients may better than that of HBeAg mono-positive patients.

Objective To study the effect of esomeprazole,clarithromycin,metronidazole triple therapy for helicobactor pylori(Hp) infection in children.Methods Ninty-eight cases of children,identified by ~ 13 C-urea breath test(~ 13 C-UBT) Hp infection,deparded into therapy group(66 cases) and control group(32 cases).Therapy group were given Esomeprazole[0.8 mg/(kg?d),1 time/d],clarithromycin[15 mg/(kg?d),2 times/d],metronidazole[30 mg/(kg?d),3 times/d]triple therapy.Control group were given the same treatment except ameprazole.The course was 1 week.They were followed up 4 weeks later after the course and re-tested by ~ 13 C-UBT.Results The recurrent abdominal pain of the two groups recovered in different degrees,and the efficacy rate was 100%.The eradication rate of Hp in therapy group and control group were separately 90.9%(60/66) and 87.5%(28/32).There was no significance difference of the eradication of Hp.Conclusions The trearment of esomeprazole,clarithromycin,metronidazole triple therapy on Hp infection in children is quite effective and safe.The side effect is moderate.

Objective To evaluate the effect of vacuum sealing drainage(VSD)ON the destroyed hand injuries.Methods From January 2006 to December 2008,forty pafients with destroyed hand injuries (surface of wound)were randomly divided into VSD group(20 cases)and control group(chlorhexidine wet compress)(20 cases)after debridement. The outcomes were retrospectively analysed by pain score,swelling score,bacterial positive rate and necrotic tissue.Results Three days after treatment,the pain score of VSD group was(2.62±1.54)degree,and that of control group was(3.39±1.64)degree.There wag significant difference between the two groups(P＜0.01).The swelling score of VSD group was(1.51±0.25)degree which was different from control group(2.29±1.26)degree(P＜0.05).The bacterial positive rate of control group was 20%(4/20),but that of VSD group was 5%(1/20).There was significant difference between the two groups(P＜0.05).The VSD group had no necrotic tissue occurrence,but the control group had 2 cases,one of which had serious necrosis and had escapology.Conclusion VSD Can inhibit bacterial infection,ease pain,exempt changing dressings and stimulate growing of granulation tissue also can maintain a good environment for tissue transplantation later.So VSD combining with tissue transplantation is an ideal therapy in destroyed hand injuries.

Objective To construct and screen effective shRNA expression vectors targeting human AQP1 gene ,and evaluate the interference efficiency of the AQP1 shRNA recombinant plasmids ,thus provide basis for further exploration on the effect and mechanism of AQP1 gene on human breast cancer cells .Methods Four pairs of shRNA sequences targeting human AQP1 gene were designed and synthesized ,and then inserted into the GV115 vector .AQP1 shRNA and control shRNA plasmids were trans‐fected into human breast cancer MCF‐7 cells .The expression of AQP1 mRNA and protein were detected by real time PCR(RT‐PCR) and Western blot to evaluate the interfering efficiency .Results RT‐PCR demonstrated that AQP1 was expressed in human breast cancer MCF‐7 cells .Sequencing showed that the shRNA vectors targeting AQP1 were successfully constructed .48 h after the AQP1 shRNA transfection ,AQP1 mRNA and protein expression levels in MCF‐7 cells were reduced to a significant degree ,and the AQP1 shRNA 4 plasmid vector could inhibit the AQP1 most efficiently .Conclusion The AQP1 shRNA recombinant plasmids vectors were successfully constructed and can significantly inhibit the expression of AQP1 in MCF‐7 human breast cancer cells .

Objective To isolate and identify exosomes from human serum , explore the feasibility of detecting exosomal miRNA in human serum.Methods Retrospective study.Serum samples from 10 healthy individuals in January 2013 were randomly selected.Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients ( n=20 ) and healthy controls ( n=20 ) were selected.Exosomes were isolated from these serum samples using ExoQuick , and then identified by using transmission electron microscopy , NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype.The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser.Then quantificational real-time polymerase chain reaction ( qRT-PCR) was carried out to detect miRNAs in different components of human serum ,and nonparametric tests were used for difference analysis.Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm.Moreover, they expressed HSP70 and four transmembrane protein CD 63.Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA.qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome , and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, U =2,P <0.000 1;let-7a, U=13,P=0.003 2) and exosome-depleted supernatant ( miR-21, U=15,P=0.006 5;miR-16, U=2,P<0.000 1;miR-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).miR-141, the molecular marker of prostate cancer ,were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients , 20 BPH patients and 20 healthy control people.The results showed that , in three groups , exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66,P=0.000 3; BPH group, U=83,P=0.001 6;PCa group, U=54,P<0.000 1).In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74,P=0.000 7 and 4.06 fold, U=70,P=0.000 5).Conclusion Exosome can be efficiently isolated from human serum.Compared with the whole serum , isolation of serum exosome may helpful to improve the detection of circulating miRNA.

Objective:To explore the impact of TLR5 and NLRC4 activation on the proliferation of different breast cancer cell lines,MCF-7 and MDA-MB-23 i.Methods:Induction,expression,purification and identification of recombiant flagellin,including FliC (activating both TLR5 and NLRC4),FliC△90-97 (unable to activate TLR5),FliC-L3A (unable to activate NLRC4),FliC△90-97:L3A (unable to activate both TLR5 and NLRC4).Using different concentration of recombinant flagellin to stimulate MCF-7 and MDA-MB-231 cell lines,72 h later,the proliferation of tumor cells were detected with CCK8.We also used soft AGAR forming experiments to detect the inhibition ratio of recombinant flagellin on breast cancer cell lines.Briefly,1 000 cells were plated in the 6-well plate,then stimulated with 1 μg/ml recombinant flagellin,14 days later,the number of cloning were counted after crystal violet staining.Results:After stimulation with four recombinant flagellins at the concentration of 0.1 μ,g/ml,the inhibition ratio on MCF-7 reached 30％,and FliC△90-97 were dose-dependent on the inhibition of MCF-7 proliferation.At the concentration of 1 μg/ml,FliC-L3A which only activated TLR5 showed stronger inhibition ratio than FliC.FliC△90-97:L3A which did not activate both TLR5 and NLRC4 also inhibited the proliferation of MCF-7.After adding transfection reagent,four recombinant flagellins showed inhibition effect on MDA-MB-231.Conclusion:Flagellin can inhibit the proliferation of MCF-7 and MDA-MB-231,and the mechanism of inhibition on the proliferation were not TLR5 and NLRC4 pathway dependent.There might exist new mechanisms to explain this phenomenon.

Objective To compare the efficacies and indications of locking compression plate (LCP) and external fixator plus Kirschner wires in treatment of complex intra-articular fracture of the dis-tal radius. Methods Ninety-eight patients with complex intra-articular fractures of the distal radius were treated with volar LCP or external fixator plus Kirschner wires, the efficacies of which were evaluated by comparing the grasping force and wrist function of the patients. Results All the patients were fol-lowed up for an average of 12.4 months, which showed fracture healing in all the patients. According to the wrist function assessment system of New York Orthopedic Hospital (1990), there was no statistical difference in the efficacy of LCP and external f'lxator plus Kirschner wires in treatment of types C1 or C2 fractures (P > 0.05), while the efficacy of external fixator plus Kirschner wires was significantly superior to that of LCP in treating type C3 fracture (P < 0.05). Conclusions For types C1 or C2 intra-articu-lar fractures of the distal radius, the efficacies of LCP and external fixator plus Kirschner wires are simi-lar, while the efficacy of external fixator plus Kirschner wires is superior to that of LCP in treating type C3 intra-articular fracture of the distal radius.

Objective To investigate the therapeutic effects and the possible mechanism of fecal transplantation on rats with Clostridium difficile-associated pseudomembranous colitis. Methods A total of 40 Sprague-Dawley rats were divided into four groups including the healthy control group, model group, fecal transplant treatment group and vancomycin treatment group. Rats in three experimental groups were subcuta-neously injected with clindamycin phosphate (10 mg), followed by treatment with toxin producing Clostridi-um difficile (ACTT43255) enema 24 hours later. The rats in fecal transplant treatment group and vancomy-cin treatment group were respectively treated with fecal suspension and vancomycin one day after modeling. The rats were fasted for one day after the last administration and then executed. The levels of potassium ion ( K) , sodium ion ( Na) , albumin ( ALB) , white blood cells ( WBC) , C-reaction protein ( CRP) , interleu-kin-1β ( IL-1β) , interleukin-10 ( IL-10 ) , interleukin-12 ( IL-12 ) and interleukin-17 ( IL-17 ) as well as the percentage of neutrophils ( N%) in serum samples were detected. The colon tissue samples were collect-ed for pathology examination. Results The rat model of pseudomembranous colitis was successfully estab-lished by subcutaneous injection of clindamycin in combination with toxin-producing Clostridium difficile (ACTT43255) enema. The signs of intestinal inflammation including serious weight loss, remarkably short-ened colon length and significantly increased colon wet weight index were observed in rats from the model group (P<0. 05). Compared with the rats from model group, the rats received fecal transplant showed sig-nificantly increased levels of K, ALB, IL-10 and IL-10/IL-12 in serum and decreased levels of WBC, N%, CRP, IL-1β and IL-17 (P<0. 05). Conclusion Fecal transplantation was proved to be an effective ap-proach for the treatment of pseudomembranous colitis. The therapeutic mechanism might due to its impacts on serum inflammatory factors.

Objective To investigate the radioprotective function and its mechanism of Sulforaphane (SF) in mice acute radiation-induced lung injury.Methods Totally 40 female C57BL/6J mice were equally divided into 5 groups randomly.Group A,treated by SF 3 mg/kg plus radiation;group B,treated by SF 5 mg/kg plus radiation;group C,treated by SF 10 mg/kg plus radiation;radiation group with a single dose of 12 Gy in 6 MV X-ray by a linear accelerator,and control group with sham radiation.The mice in drug group were administered intraperitoneally with different concentration of SF every other day from 7 d before irradiation to 7 d after irradiation,while the same volume of DMSO plus physiological saline solvent was given in the control and radiation groups.After being sacrificed at 14 d of SF administration,the pathomorphological changes of mice were observed in trauma lung tissue,the positioning and expression of NLRP3 was observed by immunohistochemical staining,the levels of IL-6,TNF-α and TGF-β1 in bronchoalveolar lavage fluid (BALF) were measured by ELISA,the expressions of NLRP3 and IL-1β mRNA in lung tissue were assayed by qRT-PCR,the expressions of NF-κB p65,NLRP3 and IL-1β proteins in lung tissue were assayed by Western blot,the activity of NF-κB was detected by EMSA.Results In comparison with radiation group,there was an obvious amelioration in pathological injury of lung tissue in the treatment groups:the expression of NLRP3 in lung tissue decreased;the concentration of NLRP3 in the drug intervention group (SF 10 mg / kg) markedly decreased (F =42.750,P ＜ 0.05).the IL-6,TNF-a and TGF-β1 levels in BALF decreased (tIL-6 =-62.65-21.00;tTNF-α =-32.18-16.57;tTGF-β1 =-58.22-46.11,P ＜ 0.05);the expressions of NLRP3 and IL-1β mRNA markedly decreased (tNLRP3 =-6.56-5.68;tIL-1β =-29.75--21.20,P ＜ 0.05),and the expressions of NF-κB p65,NLRP3 and IL-1β proteins decreased (tNF-κB p65 =-34.00--1.71,tNLRP3 =-25.01--16.91,tIL-1β =-73.70--55.14,P ＜ 0.05);the relative expressions of NF-κB p65 and NLRP3 were reduced in a dose-dependent manner (r =0.945,0.926);and the activity of NF-κB were obviously reduced (tNF-κB =-38.68,-614.82,-2 831.40,P ＜ 0.05).Conclusions Sulforaphane effectively alleviates the RILI in lung of mice by downregulating the expressions of inflammatory factor NLRP3.