Objective To establish the method for simultaneous determination of epimedium glycoside and other 4 kinds of active ingredients in Xianling Gubao Capsules by three wavelength switching method. Methods An Waters Atlantis T3 C18 column (4.6 mm × 250 mm, 5 μm) was used with the mixture of acetonitrile-0.05％ formic acid solution as the mobile phase in gradient elution (0–5 min, 12％–20％ A; 5–15 min, 20％–55％ A; 15–35 min, 55％ A;35–55 min, 55％–76％ A; 55.1 min, 12％ A; 55.1–60 min, 12％ A). Detection wavelength was as follow: 0–30 min, 212 nm; 30–42 min, 246 nm; 42–60 min, 270 nm. The flow rate was 1.0 mL/min. The column temperature was 30 ℃. Results The calibration curves of asperosaponin Ⅵ, psoralen, angelicin, epimedin C, and icariin were in good linearity among the ranges of 101.6–3048 ng, 8.7–261 ng, 7.9–237 ng, 117.2–3516 ng, 78–2340 ng, respectively. In the instrument precision test, stability test, repeatability test, the RSD was less than 3％. The average recoveries were 99.83％, 100.35％, 100.59％, 100.60％, 99.72％, respectively, and all the RSD were less than 3％. Conclusion The method is sensitive, accurate, and separation effect is good, which can provide a basis for quality evaluation standard of Xianling Gubao Capsules.

Objective To investigate effect of serum apoptosis factor and long-term effect on medical abortion patients by sequential misoprostol. Methods 80 cases of early pregnancy patients who required termination pregnancy, were selected and randomly divided into 2 groups.40 cases in control group were treated with mifepristone 25mg oral, misoprostol 600μg, 4 times a day.40 cases in experiment group were treated with sequential use of misoprostol.Effect of medical abortion, survivin and caspase-3 were compared after 7d treatment.Gynecological inflammation, dysmenorrhea and infertility were compared for 2 years.Results Compared with control group, complete abortion rate of the experiment group was higher (P<0.05).The incidence of adverse drug reactions of the experiment group was lower than control group (P<0.05) after 2 years follow.up.Compared with the control group, HCG,E3 and P of experimental group were lower (P<0.05).After 7 days of medication,survivin decreased,caspase-3 increased,compared with control group, survivin of the experiment group was lower, caspase-3 was higher ( P<0.05 ) .Conclusion Misoprostol sequential can improve the success rate of medical abortion abortion, reduce the complications of abortion, presumably with the inhibition of survivin expression, is associated up regulation of caspase-3 levels.

Objective To explore the mixed infection of bacteria and viruses of community-acquired pneumonia (CAP) in children. Methods A total of 204 children with CAP were tested for sputum bacteria, viruses and atypical pathogen, and children with bronchoscope indications were performed with bronchoscope for alveolar lavage (BAL), and the BAL lfuid (BALF) was subjected to quantitative culture and intracellular bacteria detection. All the children were given antimicrobial sequential therapy. Results There were 153 strains of pathogenic bacteria isolated in 122 cases, the detection rate was 59.80%(122/204). Thirty cases were found with mixed bacterial and viral infections. BAL was performed on 70 cases, positive lavage germiculture were detected in 8 cases, of theses BALF specimen inducible co-stimulator (ICOS) positivity were found in 5 cases. Using BALF quantitative culture as control, the sensitivity of ICOS in the diagnosis of CAP was 37.50%and the speciifcity was 96.77%. In 30 cases of mixed infection with bacteria and viruses, 27 cases were younger than 5 years old, accounting for 90.00%. Duration of fever greater than 10 d in mixed infection group of children (43.33%, 13/30) was higher than that of the non-mixed infection group (23.12%, 40/173) (P??0.05). Average hospitalization time in children with mixed infection (13.5+1.5) d was higher than that with non-mixed infection (8.6+1.1) d (P?

Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombinant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hybridoma cell lines secreting McAbs against human perforin were established, and the three McAbs showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as lgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.

Objective To investigate the expression of Hippo pathway component in hepatic cancer tissues and investigate its effects on the tumor recurrence after Iiver transplantation.Methods The clinical data of 105 patients with liver cancer who were admitted to the Third Affiliated Hospital of Sun Yat-Sen University from July 2004 to September 2009 were retrospectively analyzed.The samples of liver cancer tissues were collected.The maximum diameter,number of foci,blood vessel involvement,preoperative level of alpha-fetoprotein (AFP),results of postoperative pathological examination were analyzed.All the patients were followed up via out-patient examination,mail and phone call.Patients were followed up once a week within the first month after operation,and once a month within the 6 months after operation,and then once every 3 months at 1 year later.The follow-up ended in December 2012 or tumor recurrence.The disease-free survival time began at the date of operation and ended at the time of tumor recurrence.The expressions of Yes-associated protein (YAP),phosphorylated YAP,Hippo pathway component (Lats1/2,pLats1/2,Mst1,pMst1/2) were detected by immunohistochemical staining.All data were analyzed using the chi-square test or Student t test.Factors might influence the postoperative tumorfree survival time after liver transplantation were analyzed using the Cox regression model.The survival curve was drawn by Kaplan-Meier method,and the disease-free survival was analyzed using Log-rank test.Results Positive expressions of YAP and phosphorylated YAP were detected in the nucleus and cytoplasm,and the positive expressions of Lats1/2,pLats1/2,Mst1 and pMst1/2 were detected in the cytoplasm.The positive expressions of YAP,phosphorylated YAP,Latsl/2,pLats1/2,Mst1 and pMst1/2 protein were 51.43％ (54/105),55.24％ (58/105),45.71％ (48/105),9.52％ (10/105),64.76％ (68/105) and 20.00％ (21/105),respectively.The positive expression of YAP was correlated with the tumor diameter,venous infiltration and AJCC stage (x2=4.173,9.611,7.233,P ＜ 0.05).The positive expression of Lats1/2 protein was correlated with tumor diameter and AJCC stages (x2=14.413,7.969,P ＜ 0.05).The positive expression of Mst1 protein was correlated with the tumor diameter (x2=4,129,P ＜0.05).The results of univariate analysis showed that the protein expressions of YAP,Lats1/2,pMst1/2,age,tumor diameter,tissue differentiation,preoperative level of AFP,venous infiltration and AJCC stages were risk factors influencing tumor recurrence after liver transplantation (HR =2.603,0.502,1.802,0.955,3.559,2.395,2.414,2.915,2.086,95％ CI:1.452-4.666,0.287-0.880,1.040-3.123,0.931-0.981,1.921-6.595,1.475-3.889,1.313-4.337,1.604-5.229,1.370-3.176,P ＜ 0.05).The results of multivariate analysis showed that the positive expression of YAP,tumor diameter ＞ 5 cm,low differentiation of tissue and AJCC stages Ⅲ were independent risk factors influencing tumor recurrence after liver transplantation (HR=2.011,2.176,2.390,1.574,95％CI:1.115-3.628,1.125-4.206,1.448-3.945,1.041-2.381,P ＜ 0.05).The median time of follow-up was 13.0 months (range,1.0-96.0 months).Eight patients missed follow-up.Fifty-four patients had tumor recurrence,and the mean time of tumor recurrence was 6.7 months (range,1.0-41.0 months).The disease-free survival time of patients with positive expression of YAP were significantly shorter than those with negative expression of YAP (Log-rank value =12.890,P ＜ 0.05).Conclusions Positive expressions of YAP and phosphorylated YAP were detected in the nucleus and cytoplasm,and the positive expressions of Lats1/2,pLats1/2,Mst1 and pMst1/2 were detected in the cytoplasm.The positive expression of YAP is the independent risk factor for tumor recurrence after liver transplantation.

Objective To investigate the correlation between the Yes-associated protein (YAP) expression and epithelial-mesenchymal transition (EMT) by transiently transfecting with YAP small-interfering RNA (siRNA) in MHCC97H and MHCC97L cells.Methods MHCC97H and MHCC97L cells were transiently transfected by YAP siRNA.Furthermore,protein expressions and mRNA levels of characteristic markers of EMT (E-cadherin,N-cadherin) were examined by Western blotting and real-time polymerase chain reaction.Transwell invasion assay was used to detect changes of invasiveness of MHCC97H and MHCC97L cells.Results The YAP siRNA transfected group in MHCC97H was examined after 72 hours by Western blotting.The result showed obviously higher expression of E-cadherin in transfected group compared with the control group (P ＜ 0.05),and lower expression of N-cadherin (P ＜ 0.05).In MHCC97L cells,the expression of E-cadherin was also significantly increased (P ＜ 0.05),however,N-cadherin expression did not significantly change (P ＞ 0.05).Moreover,compared with the control group,transwell invasion assay showed that the number of the transfected group cells significantly decreased in MHCC97H(66 ± 6.89 vs 117 ± 7.23,P ＜ 0.05),and compared with the control group,the number of the transfected group also significantly decreased in MHCC97L (40 ±2.65 vs 77 ±4.33,P ＜0.05).The result of real-time polymerase chain reaction indicated that mRNA levels of E-cadherin increased (P ＜ 0.05),but mRNA levels of N-cadherin did not significantly change (P ＞ 0.05).This is considered as post-transcriptional regulation after silencing YAP in MHCC97H and MHCC97L.Conclusions YAP silencing is able to inhibit EMT in MHCC97H and MHCC97L cells by modulating the characteristic markers of EMT.The inhibition of YAP expression can reduce the invasion ability of hepatocellular carcinoma cells.

Objective To express human B7.2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-α. The fusion protein consisted of GST and hB7.2(IgV+C) was identified by SDS-PAGE and Western blotting.T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated by anti-CD3 antibody. Results The fusion protein GST-hB7.2 (IgV+C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7.2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

Objective To explore the impact of sleep quality in early pregnancy on gestational diabetes. Methods A total of 657 pregnant women in the obstetrics clinic of our hospital were selected as the research subjects. The basic information of pregnant women, sleep time and sleep quality in early period of pregnancy were collected through questionnaires. Glucose tolerance screening tests were performed at 24-28 gestational weeks to screen out GDM pregnant women. Multivariate unconditional logistic regression analysis was used to analyze the effect of sleep status in early pregnancy on the incidence of gestational diabetes. Results 112 pregnant women were diagnosed with gestational diabetes, and 92 women in early pregnancy were indicated of abnormal sleep quality. Sleep disorders might lead to an increased risk of gestational diabetes (OR=1.031, 95％CI=1.027-1.115). Age, pre-pregnancy BMI, early pregnancy sleep quality and number of pregnancy and delivery were risk factors for gestational diabetes. Conclusion Poor sleep quality in early pregnancy is a high risk factor for gestational diabetes. Treatment of abnormal sleep during early pregnancy can reduce the incidence of gestational diabetes.

From April 2009 onward, a new strain of human H1N1 influenza virus has swept over the world. The genome of influenza virus consists of 8 segments, encoding 10 proteins, respectively. The reassortments among the 8 segments cause the variation of influenza virus. Therefore, phylogenetic analysis of the 8 genes is very important. In this paper, we choose neighboring word frequency as the genomic features, using VC++ programming to analyze evolution of the 8 segments of H1N1 virus. As a result, we found that PB2 genes and PA genes of these three isolated virus were originated from North American avian influenza virus, that PB1 genes were originated from the seasonal influenza virus of human, and that HA genes, NS genes and NP genes came from the North American classical swine influenza A virus. The NA segments and M segments were originated from the European swine influenza virus.
Cloning, Molecular ; Genes, Viral ; Genome, Viral ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza, Human ; epidemiology ; virology ; Mexico ; epidemiology ; Phylogeny ; Reassortant Viruses ; genetics ; United States ; epidemiology

OBJECTIVETo investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro.

METHODSThree fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation.

RESULTSThree recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.

CONCLUSIONSFunctional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

Antigens, CD ; pharmacology ; B7-2 Antigen ; Escherichia coli ; genetics ; Humans ; Immunoglobulin Constant Regions ; pharmacology ; Immunoglobulin Variable Region ; pharmacology ; Lymphocyte Activation ; Membrane Glycoproteins ; pharmacology ; Plasmids ; Recombinant Fusion Proteins ; pharmacology ; T-Lymphocytes ; immunology