Objective:To generate Epstein-Barr virus(EBV) Latent Membrane Protein 2A(LMP2A) recombinant adenovirus,and provide for further investigation on the therapy vaccine against EBV associated malignancies.Methods:Full length cDNA of encoding LMP2A of EBV had been amplified by reverse transcription-PCR and cloned into pGEM-T vector.The encoding cDNA of LMP2A was inserted into E1,E3-substituted adenovirus vector pAX1CW,then the LMP2A recombinant adenovirus vector was contransfected into 293 cells togetherwith EcoT221 digested Ad5-TPC.The LMP2A recombinant adenovirus was generated by homologous recombination,and primarily identificated by ClaI enzyme digestion.The expression of LMP2A on CV1 cells infected with recombinant adenovirus analyzed by fluorescence-activated cell sorting(FACS) and confocal microscope.Results:The replication-deficient LMP2A recombinant adenovirus was generated efficiently with the titers of 2.3?10 8 pfu/ml.The LMP2A could be seen on CV1 cells membrane with confocal microscope 48 h post infected with recombinant adenovirus and the percentage of CV1 cells expressing LMP2A was 94.4% by means of FACS analysis.Conclusion:These suggested that LMP2A could be expressed efficiently by recombinant adenovirus mediated transfer,and it was the foundation of further researching in its function and developing the suitable genetic engineering vaccine against EBV associated malignancies.

OBJECTIVETo explore early clinical effect of acetabular cup in total hip replacement for the treatment of Crowe II developmental dysplasia of hip.

METHODSEighteen patients (18 hips) with Crowe type II developmental dysplasia of hip were treated with total hip replacement from September 2001 to July 2013. Among them,including 13 males and 5 females aged from 42 to 60 years old with an average of 47.6 years old; the courses of diseases ranged from 9 to 22 years with an average of 13.5 years. All the patients had hip joint pain, limb shortening and limited hip function before operation. Harris score of hip joint were used to evaluate recovery of function at 1 day and 12 months after operation. Prosthetic coverage of acetabular cup at 1 week after operation was observed by using radiography.

RESULTSEighteen patients (18 hips) were followed up from 12 to 24 months with an average 17 months. All incisions were healed at stage I. No deep vein thrombosis, hip dislocation, periprosthetic joint infection and prosthesis loosening were occurred. No revision surgery during follow-up period. Prosthetic coverage of acetabular cup was more than 80% at 1 week after operation. Harris score were increased from 42.67 ± 5.06 before operation to 94.79 ± 3.27 at 12 months after operation (t = -45.269, P < 0.001).

CONCLUSIONFor type Crowe II developmental dysplasia of hip patients, 15° face-changing acetabular cups in THR could obtain higher actebular component coverage rate and satisfactory early clinical effects.

Acetabulum ; surgery ; Adult ; Arthroplasty, Replacement, Hip ; instrumentation ; Female ; Follow-Up Studies ; Hip Dislocation, Congenital ; surgery ; Hip Joint ; surgery ; Hip Prosthesis ; Humans ; Male ; Middle Aged

To probe into psycho-social factors related to relap se of heroin addict and to find main factor of effect on relapse． 186 cases of heroin addict had been devised into first and relapse group． Addict condition, family condition, motive of drug abuse and MMPI of addict had been studied． The results showed that relapse group were early and long time user, most of them with intravenous, the times per day of use, dosage and fee more then first drug abuse group． The motivation of relapse was strong dependent． Psychological dependent and release of depression and adolescence are high danger factor of relapse． Rehabilitation measure must be personal depend on cause of relapse of Individual．

BACKGROUND:Previous experiments have prepared shape memory polymer based on D,L-poly(lactic acid). According to domestic technical requirements for biological y evaluating biomaterials and medical equipments, tissue-engineered grafts must be subjected to preclinical experiment for biosecurity and cytocompatibility evaluation.
OBJECTIVE:To observe the biosecurity of the shape memory polymer based on D,L-poly(lactic acid).
METHODS:(1) Bacterial endotoxin test:polymer extract, endotoxin working standard solution and checking
standard water were added into limulus reagent, respectively. (2) Sensitization test:Polymer extract+Freund’s complete adjuvant+physiological saline and Freund’s complete adjuvant+physiological saline were injected into the scapula of Kunming mice. After induction by intradermal injection, local induction and excitation, stimulate skin erythema and edema degree were observed in animals. (3) Acute toxicity test:Kunming mice received intraperitoneal injection of 100%, 50%, 25%polymer leaching solution and physiological saline, respectively. (4) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cel proliferation:The direct method was that human umbilical cord vascular endothelial cel s were inoculated onto the polymer film, lactic acid and glass, respectively;the indirect method was that human umbilical cord vascular endothelial cel s were inoculated into the polymer leaching solution, acrylamide solution and 1640 culture solution.
RESULTS AND CONCLUSION:This shape memory polymer based on D,L-poly(lactic acid) is free of bacterial contamination in compliance with the biosecurity standards, and it has no al ergenic and toxicity but has good cytocompatibility.

10.3969/j.issn.2095-4344.2013.25.017

Objective It was reported that pretreatment with small dose of lipopolysaccharide (LPS) could protect the animal from lethal dose endotoxin. The purpose of this study was to investigate the effects of pretreatment with small dose of LPS on cardiac function in endotoxemic rats and the possible mechanism. Methods Sixty male Wistar rats weighing 200-280 g were randomly divided into 3 groups: group I normal saline(NS) ( n = 12); group Ⅱ LPS (n = 24) and group Ⅲ LPS-pretreatment ( n = 24). Group Ⅱ and group Ⅲ were further divided into 3 groups: 2 h,4 h and 6 h subgroups based the time when blood sample was taken and the animal was sacrificed. The table shows the LPS given in the 3 groups:groupⅠⅡ Ⅲ0hNSNSLPS 0.25 mg?kg-1ip24 hNSNSLPS0.5mg?kg-1ip96 hNSLPS 10 mg?kg-1 Ⅳ LPS 10 mg?kg-1 TVThe animals were anesthetized with 3 % pentobarbital 30 mg?kg ip and intubated. Right femoral artery and vein were cannulated for MAP monitoring and fluid infusion. Cardiac catheter was placed in the left ventricle for measurement of left ventricular systolic pressure (LVSP) and?dp/dt max. Blood samples were taken 2 h,4 h and 6 h after intravenous LPS (10 mg?kg-1) for determination of plasma levels of L-lactate-dehydrogenase (LDH) and creatine kinase (CK) . The animals were sacrificed right after blood sampling and the heart was removed for determination of myocardial HSP 70 expression using immuno-histochemical staining. Results LVSP and?dp/dt max gradually decreased 1h after intravenous administration of LPS in group Ⅱ (LPS group); while in group DI (pretreatment group) the cardiac contractility was maintained and LVSP,?dp/dt max did not decrease as compared with the baseline value. Plasma LDH concentration and CK activity increased significantly 4 h and 6 h after intravenous IPS in group Ⅱ . The plasma LDH and CK levels were significantly lower in groupⅢthan those in group Ⅱ ( P

BACKGROUNDTo study the effects of dendritic cells (DC) transfected with recombinant adenovirus encoding Epstein-Barr virus(EBV)latent membrane protein 2A(LMP2A)gene, and to provide evidence for further investigation on the therapeutic vaccine against EBV-associated malignancies.

METHODSDCs were transfected with EBV-LMP2A recombinant adenovirus (Ad5-LMP2A), which was generated by homologous recombination. The expression of LMP2A protein on mature DC transfected with Ad5-LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS), and the dead cells were counted by trypan blue staining. The alteration of surface markers on mature DC including CD1a, CD83, CD40, CD80, HLA?DR was detected by means of FACS before and after transfection. Meanwhile, the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H-thymidine uptake and fluorescent semi?quantitative polymerase chain reaction (PCR), respectively.

RESULTSAbout 80% mature DC expressed LMP2A protein and >92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection. Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL-12 P40 mRNA.

CONCLUSIONEBV-LMP2A gene,which was carried by adenovirus vectors, could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5-LMP2A.

Adenoviridae ; genetics ; Cells, Cultured ; Dendritic Cells ; physiology ; Gene Expression ; Genetic Vectors ; Humans ; Recombination, Genetic ; Transfection ; Viral Matrix Proteins ; genetics

Objective To investigate the correlation of infiltration of mast cells in kidney with renal interstitial fibrosis, expression of TGF-β1 and stem eel] factor (SCF) in rat models withprotein-overload nephropathy. Methods Sixty uninephrectomized SD rats were randomly divided into model group [intraperitoneal injections of bovine serum albumin (BSA)] and control group (intraperitoneal injections of equal volume of saline). Ten rats from both groups were sacrificed respectively at week 3, 7 and 11 after injection. 24 h urinary protein and serum biochemistry of these SD rats at the time of sacrifice were measured. The intensity of mast cell infiltration was examined by toluidine blue (TB) staining and immunohistochemistry using a monoclonal anti-MC chymase antibody. The expression of TGF-β1 and SCF was detected byimmunohistochemistry, using a monoclonal mouse anti-rat TGF-β1 antibody and a polyclonal rabbstanti-rat SCF antibody. Results Severe proteinuria was induced in the rats by BSA injectionpeaked at week 7 [(199.1±98.4) mg/d] after the BSA injection and gradually decreased until week11 [(133.7±67.8) mg/d]. Renal injury was accompanied with chymase-postitive and TB-postitive mast cell infiltration, in close proximity to areas of interstitial fibrosis. With aggravation oflesions degree, the number of mast cells increased,the difference between the modal rats and control rats was significant (P<0.05). Immunostainahle expression of SCIF and TGF-β1 was detected in tubular as well as interstitial cells, and increased with the BSA injection. The difference between the model rats and control rats was significant (P<0.05). Mast cells were positively correlated with interstitial fibrosis (r=0.772, P<0.01), expression of TGF-β1 (r=0.521, P<0.01) and SCF(r=0.916,P<0.01). Conclusions Increased infiltration of mast cells is involved in interstitial fibrosis of rats with protein-overload nephropathy. Proteinuria may attract mast cells to kidney by chemot actions of SCF,and mast cells may contribute to the development of renal fibrosis by secreting chymase and increasing expression of TGF-β1.

425 halophilic bacteria strains were isolated by using six different media from thirty salt-soil samples collected from Heijing ancient salt mine,Yunnan. By the growth on NaCl grandient concentration and other screening methods,79 strains of extremely halophilic microorganisms were chosen for further research. Based on colonies color,shape and size,15 stains were selected to be sequenced. The sequencing results revealed that 11 of them were haloarchaea. According to the phylogenetic analysis,they belong to four genera of the family Halobacteriaceae. The blast results showed that the similarities of six sequences were higher than 97 % with the validly described species of the following four genera: Halorubrum,Natronococcus,Natrialba,Halalkalicoccus. The similarities of other five sequences with any validly described species were less than 97 %,therefore,the taxonomic positions of the following five strains YIM-ARC 0032,YIM-ARC 0036,YIM-ARC 0037,and YIM-ARC 0050 could be determined according to further polyphasic taxonomy data.The results indicated that there was a considerable diversity of haloarcheaea in salt-mine environment of Heijing ancient salt mine,Yunnan and it was worth continuing to research on this area.

BACKGROUNDTo construct recombinant retroviral vector expressing HBcAg in eukaryotic cells.

METHODSThe HBV core gene fragment was amplified by using PCR from pADR which contains complement nucleotide sequence of HBV subtype adr and cloned into retroviral expression plasmid pLXSN, then transfected into packing cell (PT67) with lipofec AMINE. After 2-3 weeks selection with G418, large colonies were isolated and transferred to individual plates. Virus-containing medium was collected and used to infect NIH3T3, EL4 and mouse bone marrow derived dendritic cells(DC). DNA was extracted from infected cells and tested by PCR. Indirect immunofluorescence and FACS were used to detect the expression of HBcAg. Cell mediated immunity of immunized C57BL/6 mice with transduced DC was analyzed.

RESULTSThe insertion of HBV core gene fragment in the recombinant retroviral plasmid was confirmed by PCR as well as enzyme digestion with EcoR1 and BamH2. The viral titer reached 3 x 10(5) CFU/ml. The result of PCR showed that the HBV core gene had been integrated into the genome of infected NIH3T3 cells. Indirect immunofluorescence and FACS analysis showed that HBcAg had been expressed in these cells. HBcAg specific CTL responses could be generated in mice immunized with retrovirus transduced DC.

CONCLUSIONSHBV core gene had been integrated into eukaryotic cells with retroviral vector and target gene had been expressed efficiently. These results may have some impact on gene therapy of chronic hepatitis B.

3T3 Cells ; Animals ; Cloning, Molecular ; Dendritic Cells ; metabolism ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Mice ; Recombination, Genetic ; genetics ; Retroviridae ; genetics ; Transfection