Objective:To investigate the immunogenicity of SARS-CoV N DNA vaccine and the feasibility of live-attenuated Salmonella typhimurium as the carrier to deliver the N DNA vaccine.Method:The recombinant attenuated salmonella strain CS022 harboring the pcDNA-N DNA vaccine was constructed.And mouse was immunized with the recombinant strain via intranasal and oral routes.Cellular and humoral immune responses were assessed by ELISA,lymphocyte proliferation assays,ELISPOT and FACS.Result:The oral immunization with the transformed salmonellae elicited strong immune responses mainly including high level of N-specific antibody,a dramatic activation of IFN-?-secreting cells,a high level of lymphocyte proliferation,and a high level of activated CD8+ T cells.Conclusion:Live-attenuated Salmonella typhimurium could effectively deliver the SARS-CoV N DNA vaccine in vivo.These encouraging pre-clinical data provide a rational basis for undertaking a new immune style to investigate SARS vaccine.

OBJECTIVE:To compare genetic diversity of Spatolobi caulis from different areas of Guangxi by random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR). METHODS:Through using POPGENE 32 software,Ntsys software and SPSS 17.0 software,RAPD and ISSR methods were used to study genetic diversity of 9 samples of S. caulis from dif-ferent areas of Guangxi. RESULTS:After amplification of screened 3 RAPD primers and 4 ISSR primers,and there were 198 and 315 locus,and 37 and 80 polymorphism locus. Rates of polymorphism locus were 18.7% and 25.4%;the number of effective al-leles were 1.416 8 and 1.584 0;genetic diversity index were 0.269 4 and 0.351 3;Shannon diversity index were 0.431 6 and 0.529 9. All the values of ISSR marker were higher than RAPD marker. The average genetic similarity coefficient of ISSR and RAPD were 0.757 64 and 0.683 80,indicating ISSR was more sensitive for the detection of genetic diversity. The clustering result of them was close to each other. The correlation coefficient of them were 0.847,indicating very significant positive correlation at the level of 0.001. CONCLUSIONS:ISSR could reflect more information of genetic diversity than RAPD,and is more suitable for research of genetic diversity of S. caulis from different areas of Guangxi.

Objective To investigate the association between extramural vascular invasion (EMVI) detected by multi-detectors computed tomography (MDCT) with contrast enhanced (ceMDCT) and clinicopathologic characteristics in patients with colon cancer.Methods Between February 2009 and December 2013,patients with histologically proven primary colon cancer and undergoing curative resection were included in this retrospective study.According to American Joint Committee on Cancer TNM staging system,patients of stage Ⅱ and Ⅲ were included in this study.EMVI status detected by MDCT (ctEMVI) was defined according to the EMVI scores.Chi-square test was used to analyze the association between clinicopathologic characteristics and ctEMVI.Results 165 stage Ⅱ and stage Ⅲ patients were included in this study as confirmed by pathology based on AJCC.Positive ctEMVI was demonstrated in 51 patients (34.5％,51/165).There were significant association between positive ctEMVI and age ＜ 65 years (x2 =4.810,P =0.031),ceMDCT defined tumor stage (x2 =17.911,P =0.000),ceMDCT defined metastatic lymph node (x2 =5.436,P =0.022),tumor size≥5 cm (x2 =3.799,P =0.036) and pathological T stage (x2 =13.346,P =0.001).Conclusions EMVI,detected by ceMDCT,is significantly associated with age,tumor size and T staging in colon cancer.

This paper describes a new technique in the repair of the hand defect with digital extensor tendon injury. The anterolateral thigh flap with the thick femoral fascia has been used in the reconstruction of the composite defect of the dorsal hand, especially the defect of tendon. This technique requires short period of treatment and hence causes less damage to the donor site but shows a better recovery of the hand function. A favorable curative effect has been obtained in this patient.
Adult ; Contusions ; surgery ; Fingers ; surgery ; Free Tissue Flaps ; Hand Injuries ; surgery ; Humans ; Male ; Reconstructive Surgical Procedures ; methods ; Tendons ; surgery ; Thigh

Lymphocyte function associated antigen-1 (LFA-1) is a member of integrin family, that plays an important role in the adhesion of lymphocytes with other cells and matrix. To investigate the role of LFA-1 in collagen-induced arthritis (CIA), the incidence of CIA, histological and radiological assessments in the LFA-1 deficient (LFA-1~ -/- ) mice and control mice were examined. LFA-1~ -/- mice and control mice were immunized with 100?g collagen type II(CII) emulsified with an equal volume of Freund’s complete adjuvant (CFA), followed by the booster injection of the same amount of CII in CFA on day 21. Then, clinical, histological and radiological assessments were done. It showed that 57% control mice developed arthritis and apparently changed in the histological and radiological assessment, whereas the all of LFA-1~ -/- mice had the normal histological and radiographic response and none developed arthritis. These results suggeste that LFA-1 is indispensable for the onset of CIA.

Objective To explore the clinical characteristics of pancreatic gland damage in children with Henoch-Sch(o)nlein purpura(HSP).Methods The serum and urine analysis of 95 examples were detected by the automatic biochemical analyzer,which were diagnosed as HSP from Aug.2009 to Jun.2010 in Department of Nephrology,Hunan Children's Hospital,and the clinical characteristics of them were analyzed.All children were treated with anti-infection and anti-allergic drugs.The pancreatic morphology of patients was observed by B ultrasonic,and the clinical features of skin,joints,digestive tract,and kidney damage were observed.And the relationship between damage of pancreas and damage of other systems was analyzed.Results There were 64 cases suffering from pancreatic gland damage in 95 HSP children:34 cases were male(53.1％),30 cases were female(46.9％),and there was no significant difference (x2 =0.56,P ＞ 0.05).The incidence of pancreatic gland damage of the patients with allergic purpura combined with abdominalgia was 82.8％ (53 cases),which was obviously higher than that in the patients without abdominalgia (11 cases,17.2％)(x2 =14.24,P ＜0.05).The incidence of pancreatic gland damage in mixed type of allergic purpura(61 cases,95.3％) was obviously higher than that in the patients which only possess the skin rash(3 cases,4.7％) (x2 =18.18,P ＜0.05).The pancreatic glands of the total 64 patients were detected by type B ultrasonic.None of them had been detected with pancreatic gland edema and deformation of structure.The mean hospital stay of the HSP patients with pancreatic gland damage was (10.80 ± 6.39) days (5-39 days),while mean hospital stay without pancreatic gland damage was (8.42 ± 3.51) days (4-13 days),and there was no significant difference between them (t =5.68,P ＞ 0.05).Conclusions HSP children usually were accompanied with pancreatic gland damage,and if they get abdominalgia and multi-system damage they are more likely to be accompanied with pancreatic gland damage,which should be paid attention to.

Objective To analyze the clinical and pathological characteristics of children in different stages and to investigate the clinical significance based on clinical diagnosis and staging criteria of acute kidney injury(AKI).Methods Based on the clinical diagnosis and staging criteria of AKI,165 AKI children admitted to the Department of Nephrology of Hunan Children's Hospital,between Oct.2004 and Oct.2011 were divided into 3 groups:stage 1,stage 2 and stage 3.Clinical characteristics,age,etiology,pathology and prognosis were compared among 3 groups of children with AKI.Results (1) A total of 165 patients(109 males and 56 females) were included in this study,average age of (6.26 ± 4.43) years,including 69 patients in stage 1,19 patients in stage 2,and 77 patients in stage 3.(2) The 3 groups of patients had an average age of(9.09 ± 3.69) years,(4.34 ± 3.90) years,and (4.22 ± 3.78) years,respectively,which showed significant differences (P ＜ 0.01).(3)Three most frequent causes of AKI were drugs (24.8％),acute glomerulonephritis (AGN) (22.4％) and septicemia (15.2 ％),which showed significant differences (P ＜ 0.01).(4) Renal histopathological examination was performed on 140 AKI children,3 most main types of pathology were acute tubular interstitial nephritis 56 cases (40.0％),endocapillary proliferative glomerulonephritis 33 cases (23.6％) and mesangial proliferative glomerulonephritis 18 cases(12.9％).In the stage 1 patients,glomerular disease was predominant(84.4％).In the stage 2 patients,glomerular disease(38.5％) and tubulointerstitial lesions(38.5％) were the major pathological types.In the stage 3 patients,tubulointerstitial damage (73.0％) was the major pathological type (P ＜0.01).The patients in 3 different groups showed significant differences in the ratio of tubulointerstitial disease and glomerular disease(P ＜0.01).(5)The median recovery time of serum creatinine to the baseline was 9 days(3-41 days) for stage 1 patients,11 days(3-25 days) for stage 2 patients,and 16 days(3-∝ days) for stage 3 patients,which showed a significant difference (P ＜ 0.05).(6)Of the 165 A KI patients,124 cases had hematuria,126 cases had varying degrees of proteinuria.There were significant differences in the hematuria incidence and the duration among the 3 groups(P ＜ 0.01).There was no difference in the proteinuria incidence among the 3 groups (P ＞ 0.05),while the stage 1 patients showed significant differences from the other 2 groups (P ＜ 0.01).Conclusions The patients are mostly seen in stage 1 and stage 3.The stage 1 AKI children are largely school-age children and acute glomerulone phritis is the main etiology.The stage 3 AKI children are mainly infants and the etiology of AKI is mainly drugs and septicemia,the pathological type is mainly acute tubulointerstitial nephritis,and the renal functional recovery is slow.

OBJECTIVETo investigate the expression of PTEN (phosphatase and tension homology deletion on chromosome 10, PTEN) and its pseudogene PTENP1 in acute leukemia (AL) and correlation between them, and to explore the role of PTENP1 on the PTEN expression in AL cells.

METHODSPTEN and PTENP1 mRNA expression were evaluated in bone marrow (BM) samples from 138 newly diagnosed AL patients and 15 healthy controls by quantitative real-time RT-PCR (qRT-PCR). pCDH1-PTENP1 3'UTR-GFP lentivirus vectors were constructed. 293T cells were transfected by calcium phosphate precipitation to produce retrovirus. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP and pCDH1-PTENP1 3'UTR-GFP respectively. The flow cell sorter was used to sort the HL-60 with GFP positively expressed. The mRNA expression of PTEN and PTENP1 was detected by qRT-PCR, the expression of PTEN protein by western blot, and the impact of PTENP13'UTR on the proliferation of HL-60 cells by MTT assay.

RESULTSAML patients showed significantly lower PTEN and PTENP1 mRNA expression in BM compared to healthy controls. Correlation analysis showed that the expression of PTEN and PTENP1 mRNA were positively correlated (P < 0.05). The 108 cases of PTENP1(+) AML were classified according to the prognostic classification of 2011 NCCN Clinical Practice Guidelines in AML, there was no difference among different subgroups. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP (control group) and pCDH1-PTENP1 3'UTR-GFP respectively. Compared with the control group, PTENP1 mRNA level of HL-60 infected with the retroviral vectors expressing pCDH1-PTENP1 3'UTR-GFP increased significantly, and PTEN mRNA level also increased. While the PTEN protein level and the cell growth rate of the PTENP1 3'UTR group didn't change significantly.

CONCLUSIONPTEN and PTENP1 mRNA expression level of BM cells from AL patients is significantly lower. There is a positive correlation between expression of PTEN and PTENP1 mRNA. PTENP1 may regulate the expression of PTEN in mRNA level.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; Female ; Gene Expression ; HL-60 Cells ; Humans ; Leukemia ; genetics ; Male ; Middle Aged ; PTEN Phosphohydrolase ; genetics ; Pseudogenes ; genetics ; RNA, Messenger ; genetics ; Transfection ; Young Adult

To explore the relationship between the expression of CD133 and pathogenesis of leukemia and MDS, immunocytochemistry method was used to examine the expression of CD133 in bone marrow cells of patients with leukemia and MDS. The results showed that the positive rate of CD133 in 41 acute leukemia patients was 51.2%. The expression of CD133 in AML patients (16/29, 55.2%) was significantly higher than that in control group (2/15, 13.3%). There was no significant difference in CD133 expression between CML and control group. The positive rate of CD133 in 9 patients with MDS was 55.56% (5/9). There was no significant difference between MDS and normal control. The expression of CD133 in all leukemia cells with CD34(+) was higher than that in leukemia cells with CD34(-), and there was significant difference in expression of CD133 between them (P < 0.05). The expression of CD133 had no relationship with the clinical prognostic factors such as sex, age, the percentage of leukemic cells in peripheral blood and in bone marrow, WBC counts, hemoglobin concentration, platelet counts and LDH level. It is concluded that the expression of CD133 in bone marrow cells of patients with AML is higher than that in control group. The expression of CD133 is significantly correlated with the expression of CD34. The high expression of CD133 may be an adverse prognostic factor in acute leukemia.
AC133 Antigen ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; metabolism ; Antigens, CD34 ; immunology ; metabolism ; Bone Marrow Cells ; immunology ; metabolism ; Child ; Female ; Glycoproteins ; metabolism ; Humans ; Immunohistochemistry ; Leukemia, Myeloid, Acute ; immunology ; metabolism ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; metabolism ; Peptides ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; metabolism ; Prognosis ; Young Adult

OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods