Objective To explore the effect of the C gene truncated HBV mutant on HBV replication. Methods The C gene truncated HBV vector pHBV-?C was constructed through the molecular clone in vitro,and then transfected transiently into HepG2 cell.The expression of S protein was assayed by ELISA and SDS-PAGE Western blot.After co-transfection with pHBV-?C and wild HBV genome adwR9 into HepG2 cell the DNA was detected quantitatively by real-tine fluorescence quantitative PCR in the culture medium and the cell. Results There was no significant difference in expression of S protein assay by ELISA and Western blot.The DNA of the cotransfected group with pHBV-?C and adwR9 was lower than that of control group in the culture medium and the cell. Conclusions(C gene) truncated HBV mutant can cause the reduction of HBV replication.

Objective:To investigate the significance of T helper type 17 cells(Th17)/T regulatory cells (Treg) in the prognosis of chronic hepatitis B(CHB) by comparing the changes of Th17 cells ,Treg cells and their rate under different therapy in chronic hepatitis B patients.Methods: 40 patients infected with chronic hepatitis B(CHB) were divided into chronic hepatitis B without antiviral(CHBWA) group and chronic hepatitis B antiviral(CHBA) group depending on whether nucleoside antiviral was applied,with 20 cases each,10 cases in asymptomatic hepatitis b carriers(AsC) group.10 cases in health control (HC) group.Flow cytometer(FCM) was adopted to detect peripheral blood Th17 and Treg cell frequency in CHB patients at 0,1 month in CHBWA group ,at 0,1,3 month in CHBA group,AsC patients and HC cases ,then calculating Th17/Treg ratio and observing the dynamic changes at different disease states.Results: After the liver-protecting treatment in CHBWA patients,the Th17/Treg ratio at 1 month(0.39±0.11)decreased compared to 0 month(1.20±0.26)rapidly(P<0.01),and the ratio nearly reached the level of AsC and HC group (P>0.05).Ratio of Th17/Treg and ALT had good positive correlation (r=0.709,P=0).In CHBA patients group,after the liver-protecting and antiviral treatment,patients′ Th17/Treg ratio decreased rapidly in the first month(0.73±0.32) and third month(0.76±0.44),and there′s statistical difference compared with 0 month(1.18±0.27,P<0.01),but there was no significant change of Th17/Treg ratio after being treated for 3 months compared with 1 month,and the Th17/Treg ratios at 0,1,3 month were higher than that in AsC group and HC group(P<0.01).Ratio of Th17/Treg and ALT have good positive correlation (r=0.500,P=0.000),also the ratio of Th17/Treg and HBV DNA were positively correlated (r=0.345,P=0.007).Comparison of peripheral blood Th17,Treg cells frequency,Th17/Treg ratio between CHBWA and CHBA group at 0 month had no statistical significance(P>0.05),while the Th17/Treg ratio in CHBA group was higher than CHBWA group(t=4.471,P<0.01) after 1 month of treatment.Conclusion: In the CHB patients,antiviral treatment will influence the Th17,Tregs cells frequency,resulting the change of Th17/Treg ratio,but also the ratio may be associated with virus removal.

Laparoscopic pancreas- and spleen-preserving splenic hilar lymph nodes dissection is still difficult to accomplish,which restrains its application in total gastrectomy for advanced proximal gastric cancer.Based on our anatomical understanding of pre- and retropancreatic spaces,features of splenic vessels and distribution of perigastric lymph nodes,we combined the characteristics of laparoscopic surgery and developed a novel technique for laparoscopic pancreas- and spleen-preserving splenic hilar lymph nodes dissection through retropancreatic space.The key lies in mobilization of the splenic pedicle through retropancreatic space,dissection of peri-vascular lymph nodes in sequence of trunk-to-branch,in-sheath vascularization of the splenic vessels.From August 2009 to December 2010,this technique was performed on 6 patients in Nanfang hospital.The initial results suggested that this technique could be safe and feasible for skillful surgeons.Further studies on the application of this technique in total gastrectomy for advanced proximal gastric cancer would be needed.

Objective:To investigate the protective effect of intratracheal transplantation of different dose of human umbilical cord mesenchymal stem cells (MSCs) in rats with acute lung injury induced by severe burns.Methods:Seventy-five male Wistar rats were randomly divided into five groups:Sham(group A),Saline group(group B) and different doses of hUMSCs transplantation groups(C,D and E).The dosage ofhUMSCs was 1 × 105,5 × 105 and 1 × 106 respectively.Rats inflicted by 50 ％TBSA Ⅲ degree scalding employed as the model.After modeling,rats in group B and transplantation groups were immediately fluid resuscitated.Transplantation groups were intratracheally administered different dose hUCMSCs (0.2 mL),and group B were given normal saline in the same dose intratracheally.The lung tissue samples were collected on day 1,day 3 and day 7 after administration.HE staining was used to observe the pathological changes of lung tissue.MPO and CD68 immunohistochemical staining were used to observe the positive expression of neutrophils and macrophages in lung tissue.Results:Lung pathology showed that alveolar cavity was clear,alveolar structure integrity,occasionally a small amount of inflammatory cells of group A at each time point.At 1 day after scald,group B and the transplantation group (group C,D,E)the alveolar septum was thickened,and there was visible pulmonary capillary hyperemia,as well as a large amount of inflammatory cell infiltrations in the pulmonary capillaries and alveolar space.At 3 day,group B and the transplantation group alveolar structural damage,pulmonary hemorrhage and inflammatory cell infiltrations were better than those in 1 day.Compared with group B,the alveolar structure was clear and the septum was thinner,but there was no significant difference between the transplantation groups.On the 7 day after scald,the lung injury in the transplanted group was significantly less than group B,and the recovery of the injured lung tissue in E group was the most obvious.The number of the MPO positive cells increased significantly on the first day after scald (P ＜0.05) compared with group A,but there was no significant difference between the two groups.Compared with B group,the number of positive cells in transplantation group was significantly reduced at 3 and 7 day after scald,(P＜0.05),and the number of positive cells in group E was significantly lower than other groups (P＜0.05).CD68 staining showed a significant increase in positive cells in each group on day 1 (P＞ 0.05).The number of positive cells decreased in 3 day after transplantation (P＜0.05),but there was no significant difference between the transplantation groups.The number of positive cells in transplantation group was significantly lower than group B (P＜0.05) after 7 day.Compared with group C and D,there was significant difference in group E (P＜0.05).Conclusions:Intratracheal transplantation of different dose hUCMSCs have protective on severe burns induced acute lung injury models;the protection mechanisms may be that the hUCMSCs transplantation can inhibit the invasion of the inflammatory cells in lung tissues,and the optimal dosage is 1 × 106.

Correct implementation of laparoscopic total gastrectomy with D2 lymph node dissection depends on the understanding of anatomical features of peripancreatic space, the landmark of pancreas and blood vessels, and the diverse perigastric vascular anatomy and standardized surgical procedures designed according to the regional distribution of lymph nodes.From September 2006 to November 2009, laparoscopic total gastrectomy with D2 lymph node dissection surgery were completed in 12 cases in the Nanfang Hospital. A reasonable anatomy method and a simple, effective surgical procedure of laparoscopic D2 lymph node dissection were introduced.

Objective To evaluate the effect of dexmedetomidine administered locally on the median effective concentration ( EC50 ) of ropivacaine for paravertebral nerve block ( PVNB) . Methods Forty?eight ASA physical status Ⅰ or Ⅱ female patients, aged 20-64 yr, with body mass index<24 kg∕m2 , scheduled for elective unilateral segmental mastectomy under PVNB, were randomly divided into 2 groups ( n=24 each) using a random number table: ropivacaine group ( group R) and ropivacaine mixed with dexmedetomidine group ( group RD) . PVNB was performed at T4 on the operated side guided by ultrasound and nerve stimulator. Ropivacaine 20 ml and a mixture of ropivacaine and 20 μg dexmedetomidine 20 ml were injected locally in group R and group RD, respectively. The concentration of ropivacaine was determined by up?and?down sequential allocation. The initial ropivacaine concentration was set at 0. 35%, and the ratio between the two successive concentrations was 1. 2. The EC50 and 95%confidence interval of ropivacaine were calculated using Dixon?Massey method. Results The EC50 ( 95%confidence interval) of ropivacaine was 0.27% (0.23%-0.30%) and 0.22% (0.18%-0.25%) in group R and group RD, respectively. Compared with group R, the EC50 of ropivacaine was significantly decreased by 19% in group RD. Conclusion Small dose of dexmedetomidine administered locally can significantly enhance the efficacy of PVNB with ropivacaine.

Objective To investigate the anticancer effects and detailed mechanisms of Saikosaponin D (SSD) in human hepatoma HepG2 cells. Methods Cell proliferation and apoptosis were tested by MTT assay and Annexin-V/PI assay respectively. The expressions of CCAAT enhancer binding protein β(C/EBPβ) and p53 were detected by RT-PCR and Western blotting. Results SSD inhibited cell proliferation in a dose-dependent manner and induced apoptosis at the concentration of 5.0mg/L. SSD significantly increased the mRNA and protein levels of C/EBPβ and p53 in a dose-dependent manner. Conclusion SSD exerts its anticancer effect by inhibiting cell proliferation and inducing apoptosis partly through C/EBPβ-p53 signal pathway in HepG2 cells.

Objective To investigate the effects of osthole on neural stem cells ( NSCs) differentiation and explore the potential mechanism. Methods Brain-derived NSCs from newborn mice were isolated and cultured in vitro and determined by immunofluorescence. The P5 generations of NSCs were placed in culture solution with osthole at concentrations of (0,10,50, 100 μmol·L-1 ) . The neuron, astrocyte and oligodendroglia cell differentiation were determined by immunofluorescence. The mRNA expression of Notch 1 and its target genes Mash 1 and Neurogenin 2 were assessed by RT-PCR. Results The neurosphere displayed Nestin and Sox 2-postive by immunofluorescence, suggesting that the cultured cells were NSCs. Osthole promoted NSCs differentiating into more neuron(P<0. 01) and oligodendrocyte(P<0. 05), but not astrocyte. Meanwhile, osthole significantly reduced the mRNA expression of Notch 1(P<0. 01) and increased Ngn 2(P<0. 01)at the dose of 100 μmol·L-1. Conclusion Osthole enhances NSCs differentiating into more neuron and oligodendrocyte via probablly inhibiting Notch signal pathway.

To understand the Escherichia coli (E .coli) O157∶ H7 isolated from cow in Zhengzhou ,Henan Province ,a total of 146 samples of cow fecal and milk were collected in the different farms ,and E .coli O157∶ H7 was detected with mul-tiplex polymerase chain reaction (PCR) in our laboratory .Then the biochemical characteristics ,growth dynamic ,the biofilm formation ,and the toxin genes of the E .coli O157∶ H7 isolates were analyzed .The results showed that 2 strains of E .coli O157∶H7 were found ,with the detection rate of 1 .4% ,and the isolates were named as L1 and L2 in current study ,respec-tively .The E .coli O157∶H7 clinical isolates had the same biochemical characteristics with that of the typical E .coli .The L1 and L2 isolates presented similar growth curve ,which entered into the log phase earlier than that of the standard strain .L1 strain formed thick ,confluent ,complete biofilm after 48 hours post-inoculation ,and the biofilm of L2 strain was formed com-pletely in 36 hours .The two E .coli O157∶ H7 isolates were positive with eaeA and hlyA genes ,and the L1 strain also carried the Stx2 virulence gene .Our results reinforce the epidemiological data of E .coli O157∶H7 ,and underscore the need for more effective surveillance of animal-derived E .coli O157∶H7 isolates in Zhengzhou City ,China .

Objective To investigate the phenotype and the immunoregulatory function of CD8αα+TCRαβ+regulatory T cells in peripheral blood samples from mice.Methods The distribution profile and the phenotype of CD8αα+TCRαβ+regulatory T cells in C57BL/6 mice were detected by flow cytometry.The cytokines released by CD8αα+TCRαβ+regulatory T cells upon the stimulation with anti-CD3 antibody were analyzed by cytometric bead array.The in vitro immunosuppressive activity of CD8αα+TCRαβ+regulatory T cells on activated CD4+T cells was analyzed by using flow cytometry and carboxyfluorescein succinimidyl ester ( CFSE ) .An adoptive cell transfer assay was set up to evaluate the immunoprotective effects of CD8αα+TCRαβ+ regulatory T cells in a mouse model of experimental autoimmune encephalomyelitis ( EAE) .Results CD8αα+TCRαβ+regulatory T cells were detected in liver, spleen and peripheral blood samples collected from na?ve C57BL/6 mice.Compared with CD8αβ+TCRαβ+regulatory T cells, CD8αα+TCRαβ+regulatory T cells showed a memory-activated phenotype of CD25+CD122high CD44high CD62Llow CD69high NK1.1+DX5+.CD8αα+TCRαβ+regulatory T cells could produce IL-2 after 24 hours stimulation with anti-CD3 antibody, followed by producing IFN-γ, TNF-α, IL-4, IL-17A and traces of IL-6 and IL-10. In vitro, CD8αα+TCRαβ+regulatory T cells specifically suppressed the proliferation of activated CD4+T cells ( P<0.01 ).Moreover, they could delay the onset of EAE in mice and reduce clinical score (P<0.01).Conclusion CD8αα+TCRαβ+regulatory T cells were a unique population with immunoregula-tory function, which could be used as a potential therapeutic target in the treatment of autoimmune disease.