Objective To evaluate the potential of S100 calcium binding proteins S100A2 and S100A6 levels in serum as diagnostic markers for non-small cell lung cancer (NSCLC). Methods Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of S100A2 and S100A6 in 141 NSCLC patients and 150 healthy subjects. Results The average level of serum S100A2 in NSCLC group was (15.02±0.79) ng/ml, that in healthy control group was (11.18±0.64) ng/ml, and the difference was statistically significant (P=0.002). The average level of serum S100A6 in NSCLC group was (12 760±651.8) pg/ml, that in healthy control group was (8 434±408.2) pg/ml, and the difference was statistically significant (P<0.001). The serum levels of S100A2 and S100A6 in stageⅠ/ⅡNSCLC patients were higher than those in healthy controls (P<0.05), respectively. Receiver operating characteristic (ROC) curve analysis showed that S100A2 and S100A6 could distinguish NSCLC patients from healthy controls [area under the curve (AUC) were 0.646 and 0.668, respectively]. Meanwhile, these two proteins showed notable capabilities for distinguishing stage Ⅰ/ⅡNSCLC from healthy controls (AUC were 0.708 and 0.702, respectively). Conclusion Serum levels of S100A2 and S100A6 are significantly elevated in the early stage for NSCLC patients, which can be the potential biomarkers for the diagnosis of NSCLC.

Objective To summarize the clinical characteristics and causes of ischemic cerebrovascular disease(ICD)in children.Methods A retrospective analysis was conducted on the clinical data of 53 cases with ICD from Feb.2002 to Jun.2008 at the department of neurology in Wuhan Children's Hospital.The self-designed questionnaire of children with ICD was used,whose items included patients' age,gender,personal history,clinical features,cerebrospinal fluid examination,neurological imaging,immunologic examination,metabolic examination,and so on.Results Of 53 children with ICD,30 cases(56.6%)were male,and 23 cases(43.4%)were female.Patients' age varied from 9 months to 12 years old,in which 45 cases(84.9%)were less than 6 years old.Patients from rural area(60.4%)were more than those from city(39.6%).Ratio of limb paralysis was 75.5%(40 cases)in first clinical symptomatology of children with ICD,including hemiplegia in 32 cases(60.4%),alternate hemiplegia in 5 cases(9.4%)and monoplegia in 3 cases(5.7%).Skull CT/MRI scan was performed to reveal 27 cases(50.9%)with basal ganglia region infarction and secondly 15 cases(28.3%)with multi-lobar infarction.Forty cases were found in abnormal cerebrovascular image by means of magnetic resonance angiography/digital subtraction angiography,in which middle cerebral artery and its branches were involved in 21 cases(52.5%).There were 41 cases(77.4%)of patients to be found with clear causes,of which 13 cases(24.5%)were of infections,8 cases(15.1%)of moyamoya disease,5 cases(9.4%)of cerebral vascular malformations,4 cases(7.5%)of head trauma.However,another 12 cases(22.6%)of patients had unknown etiology.Conclusions Children with ICD had characteristics themselves.The limb paralysis was mostly the first symptoms,and the middle cerebral artery and its branches lesions were the most common locations in children with ICD,and next the internal carotid artery involvement,anterior cerebral artery involvement,posterior cerebral artery involvement,cerebral vascular malformations,and so on.Their major cause was infection,followed by Moyamoya disease,cerebrovascular malformations and head trauma,and there were still some unknown causes.

Objective To study the chemical constituents of Clematis manshurica. MethodsThe extract from the roots and rhizomes of C. manshurica was subjected to chromatography on silica gel and Sephadex LH-20 column. The compounds obtained were identified on the basis of their physicochemical and spectroscopic evidences. ResultsEleven compounds were isolated and their structures were elucidated as squalene (Ⅰ), friedelin (Ⅱ), hexacosoic acid (Ⅲ), ?-sitosterol (Ⅳ), stigmasterol (Ⅴ), oleanolic acid (Ⅵ), ?-daucosterol (Ⅶ), 5-hydroxymethyl-2-furaldehyde (Ⅷ), methyl 3, 4-dihydroxy-phenyl lactate (Ⅸ), 5R-dihydro-5-hydroxymethyl-2(3H)-furanone (Ⅹ), 5R-5-hydroxymethyl-2(5H)-furanone (Ⅺ). ConclusionAll the compounds except for ?-sitosterol are isolated from the plant for the first time.

Objective To evaluate the inhibition effects of shuanghuanglian injection powder and its active components on the activities of CYP1A,CYP 2D and CYP 3A in rats liver microsomes. Methods Three probe substrates including phenacetin for CYP1A,dextromethorphan for CYP2D and midazolam for CYP3A were incubated with shuanghuanglian injection powder and the active components (baicalin,phillyrin,forsythiaside A,lutin,chlorogenic acid,coffeic acid and lutiolin) in rat liver microsomes. Contents of three probe substrates were simultaneously determined by HPLC to evaluate the metabolic rates. Results Shuanghuanglian injection powder and baicalin inhibited the activities of CYP2D and CYP 3A, but didn 't affect CYP1A. The other active components showed no effect on CYP1A,CYP2D and CYP3A. Conclusion Drug-drug interactions may occur when combining shuanghuanglian powder injection with CYP2D and CYP 3A substrates and baicalin may be the effector substance responsible for the interactions.

Objective To observe the effect of polydatin on nerve cell apoptosis and the influence of the caspase-8 and FLIP protein expression after ischemia reperfusion injury. Methods Thirty rats were randomly divided into sham operation group, model group and polydatin group. The middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia was established by the method of thread embolism. Rats were subjected to adaptive feeding for the first 7 days and then recieved the treatment for another 10 days. The model of ischemia reperfusion injury was established at the 14th day. The polydatin group received 15 mg/kg of polydatin, and the sham operation group and the model group with the same volume of saline once a day for 15 days. The expression of caspase-8 and FLIP protein in the hippocampus of rats were observed by immunohistochemical staining. The expression of caspase-8 and FLIP protein in the hippocampus of rats were observed by TUNEL method at 72 h after cerebral ischemia-reperfusion. Results Compared with the model group, the number of apoptotic cells of polydatin group significantly decreased in hippocampus CA1 region (P<0.01); The expression of caspase-8 (148.78 ± 6.82 vs. 89.61 ± 7.76) in the polydatin group significantly decreased and the expression of FLIP (127.60 ± 8.52 vs. 150.22 ± 8.53) in the polydatin group was increased significantly in hippocampus CA1 region(P<0.01). Conclusions Polydatin have a protection effect on ischemia reperfusion injury in rats and its mechanism may be inhibition of caspase-8 protein expression, promote the FLIP protein expression.

Objective To estimate the effect of different doses of ultraviolet (UV) radiation on the proliferation of and apoptosis in kertatinocytes,as well as on the expression of p53,matrix metalloproteinase-2 (MMP2) and-9 (MMP9) in actinic keratosis (AK) lesions and normal human skin.Methods Tissue specimens were obtained from the lesions of 20 patients with AK and sun-exposed normal skin of 20 healthy human subjects,and subjected to an air-exposed culture.Each of the specimens was divided into 4 areas to remain untreated (control area) or be irradiated with UV of 5,10 and 20 J/cm2 (irradiated areas) for 4 consecutive days.After another 24-hour culture,the tissue cultures were collected followed by the evaluation of apoptosis in and proliferation of keratinocytes by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Ki-67 staining,and determination of mRNA and protein expressions of p53,MMP2 and MMP9 by using real time PCR and immunohistochemistry respectively.Results A statistical increase was observed in the percentage of apoptotic cells in the normal skin irradiated with UV of 10 and 20 J/cm2 (46.8％ ± 2.1％ and 56.7％± 2.4％,both P ＜ 0.05) and in the AK lesions irradiated with UV of 20 J/cm2 (43.5％ ± 1.5％,P ＜ 0.05)compared with the corresponding unirradiated tissues.The normal skin showed a higher percentage of apoptotic cells than the lesional skin after irradiation with UV of 10 and 20 J/cm2 (both P ＜ 0.05).The percentage of Ki67-positive cells was significantly decreased in the normal skin after irradiation with UV of 20 J/cm2 (3.34％ ±0.76％,P ＜ 0.05),but experienced no statistical changes in the lesional skin after different doses of UV irradiation (all P ＞ 0.05).There was a statistical elevation in the expression of p53 mRNA (5 J/cm2:1.106 ± 0.025,10 J/cm2: 1.259 ± 0.045,20 J/cm2:1.425 ± 0.053,all P ＜ 0.05) and protein(10 J/cm2:0.1169 ± 0.0032,20 J/cm2:0.1454 ± 0.0047,both P＜ 0.05) in the normal skin,but a statistical reduction in the expression of p53 mRNA(10 J/cm2.0.611 ± 0.050,20 J/cm2:0.578 ± 0.070,both P ＜ 0.05) and protein (20 J/cm2:0.0404 ± 0.0027,P＜ 0.05) in the lesional skin after irradiation compared with the corresponding unirradiated skin tissues.Further more,a statistical increment was observed in MMP2 mRNA and protein expression in normal skin irradiated with UV of 10 J/cm2 (1.086 ± 0.013,0.0843 ± 0.0024,respectively,both P ＜ 0.05) and 20 J/cm2 (1.417 ± 0.036,0.1236 ±0.0042,respectively,both P ＜ 0.05) and in lesional skin irradiated with UV of 20 J/cm2 (1.296 ± 0.028,0.0744± 0.0032,respectively,both P ＜ 0.05),as well as in MMP9 mRNA and protein expression in normal skin irradiated with UV of 20 J/cm2 (1.395 ± 0.026,0.3065 ± 0.0162,respectively,both P ＜ 0.05) and in lesional skin irradiated with UV of 10 J/cm2 (1.298 ± 0.035,0.0992 ± 0.0053,respectively,both P ＜ 0.05) and 20 J/cm2(1.286 ± 0.032,0.1010 ± 0.0063,respectively,both P ＜ 0.05) compared with the corresponding unirradiated tissues.Conclusion Ultraviolet may accelerate the progression of AK by down-regulating p53 expression but up-regulating MMP2 and MMP9 expression.

Objective To establish a time resolved fluoroimmunoassay (TRFIA) method for detecting Glypican 3 (GPC3) and to explore the diagnostic value of serum GPC3 for hepatic carcinoma (HCC). Methods Microplate coated with anti-GPC3 monoclonal antibody 7C8 and GP9 labeled with Eu3+ were used to establish TRFIA kit. The serum concentrations of GPC3 in 41 HCC patients and 44 chronic hepatitis (CH) patients were quantitatively analyzed. AFP was detected by with lowest limit of 2.06 μg/L. The CV of inter and intra assay were 12.25% and 12.91%, respectively. The average serum concentration of GPC3 in HCC patients was (86.68±110.39) μg/L (median: 56.98 μg/L). But in CH patients it was only (14.77±29.48) μg/L, which was significantly lower than that in HCC (Wilcoxon W=1335.00, Z=-4.99, P<0.001). With diagnostic cut-off value set at 42.94 μg/L, the diagnostic sensitivity and specificity of TRFIA GPC3 for HCC were 58.5% (24/41) and 95.5%(42/44) respectively. The diagnostic sensitivity of AFP was 46.3% (19/41) in 41 HCC patients, and was raised to 78.0% (32/41) when combined with GPC3. Conclusions Serum GPC3 assay by TRFIA is established and it could increase the diagnostic sensitivity for HCC when combined with AFP.

Objective:To explore the mechanisms of the inhalation of atomized 1.0% anti-IL-1βand TNF-αimmunoglobulin yolk ( IgY) on treating guinea pigs with allergic asthma induced by the inhalation of aerosolized ovalbumin ( OVA).Methods:Healthy guinea pigs were randomly divided into the normal controls ( group C ) , the allergic asthma model group ( group M )-treated by the inhalation of atomized ovalbumin ( OVA ) , the inhalation of atomized 1.0% anti-IL-1βand TNF-αimmunoglobulin yolk ( IgY ) treatment group (group Z1)-treated asthma model guinea pigs by the inhalation of atomized 1.0% anti-IL-1βand TNF-αIgY,and positive control the inhalation of atomized budesonide treatment group (group Z2)-treated asthma model guinea pigs by the inhalation of atomized budesonide.The blood was gotten by cardiac puncture and the bronchoalveolar lavage fluid ( BALF ) was collected by bron-choalveolar lavage at 2 h,4 h,8 h and 24 h after the last time atomization.The inflammatory cells in the peripheral blood ( PB) were counted by methylene blue and eosin staining.Cytokine concentrations of IL-1β,IL-4,IL-6,IL-8,IL-13,IL-16,TNF-α,TGF-β1 and IgE in the plasma and bronchoalveolar lavage fluid (BALF) were measured using an enzyme-linked immunosorbent assay (ELISA).Results:In PB,eosinophils was decreased from 2 h to 8 h in group Z1 compared to group M.In plasma,the levels of IL-1βat 4 h and 24 h,IL-16 at 2 h,4 h and 24 h,TGF-β1 from 4 h to 24 h and IgE at 24 h,as well as the levels of IL-1βand TNF-αfrom 2 h to 8 h,IL-4,IL-6,IL-8 and IL-13 from 4 h to 24 h,IL-16 at 8 h,and TGF-β1 and IgE from 4 h to 8 h,especially the level of IL-1βand TNF-αstarting at 2 h,in BALF were significantly reduced in group Z 1 compared to group M ( P<0.05 ).The levels of IL-1βand TNF-αwere positively cor-related with that of IL-4,IL-6,IL-8,IL-13,IL-16,TGF-β1 and IgE (P<0.05).Conclusion: The inhalation of aerosolized anti-IL-1βand TNF-αIgY effectively alleviates inflammatory responses in guinea pigs with allergic asthma induced by aerosolized OVA inhalation may be due to the significant decrease in the levels of various allergic inflammatory cytokines .

[Objective] To estimate the effects of ginsenoside Rb1 on melanogenesis in human melanocytes and underlying mechanisms.[Methods] Epidermal melanocytes were obtained from circumcision specimens of children,and subjected to primary culture.After 2 to 5 passages,the melanocytes were treated with different concentrations of ginsenoside Rb1,dimethyl sulfoxide (DMSO,vehicle control),forskolin at 10 μmol/L(positive control) or remained untreated (blank control).After additional culture for 72 hours,methyl thiazolyl tetrazolium (MTT) assay and NaOH lysis method were used to evaluate cell viability and melanin content in melanocytes respectively,spectrophotometer to determine dopa oxidase activity of tyrosinase,Western blot to quantify the protein level of tyrosinase,microphthalmia-associated transcription factor (MITF),phosphorylated and total cAMP response element binding protein (p-CREB and t-CREB) in melanocytes.[Results] After treatment with ginsenoside Rbl of 25,50 and 100 μmol/L for 72 hours,the melanocytes experienced no significant changes in viability (P ＞ 0.05 ),but a significant dose-dependent increase in melanin content (112.4％± 5.7％,155.7％ + 6.3％,217.2％ ± 11.7％ vs.100％,P＜ 0.05 or 0.01) and tyrosinase activity(117.9％ ± 5.7％,158.2％ ± 9.6％,182.6％ ± 10.0％ vs.100％,P＜ 0.05 or 0.01 ) compared with the vehicle control melanocytes.The protein expressions of tyrosinase,MITF and p-CREB were statistically higher in melanocytes treated with ginsenoside Rb1 of 100 μmol/L for 72 hours than in the vehicle control melanocytes (225.4％ ± 12.8％ vs.100％ ± 7.9％,313.5％ ± 16.7％ vs.100％ ± 9.8％,322.5％ ± 21.1％ vs.100％ ± 9.1％,all P＜ 0.01).The increase in MITF protein expression was inapparent in melanocytes at 8 hours after the treatment with ginsenoside Rb1 of 100 μmol/L,but statistically significant at 24 hours compared with the melanocytes at baseline (P＜ 0.01).The pretreatment with H-89 (a 8elective inhibitor of PKA) at 10 μmol/L,significantly suppressed the ginsenoside Rb1 (100 μmol/L for 72 hours) -induced phosphorylation of CREB,increase in MITF,tyrosinase expression,as well as tyrosinase activity and melanin content in melanocytes (all P ＜ 0.01 ).[Conclusion]s Ginsenoside Rb1could enhance the melanogenesis and tyrosinase activity in normal human melanocytes.The PKA/CREB/MITF/ tyrosinase signaling pathway may contribute to the pro-melanogenic effect of ginsenoside Rb1.

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK).The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay.GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells,no GCRV-specific siRNA could be detected.Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene.These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway,unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.