AIM:The homologous recombination takes place in E.coli BJ5183 between shuttle vector and adenoviral backbone vector.Recombinants are selected for kanamycin resistance,and the linearized recombinant plasmid is transfected into 293 cells.In this study,AdEasy system was used to generate recombinant adenovirus vector carrying rat interleukin-10(IL-10) gene.METHODS:The experiment was conducted in Department of Microbiology,Qingdao University Medical College from August 2006 to May 2007.①The materials included five clean-grade SD rat,a shuttle vector pAdTrack-CMV,an adenoviral backbone plasmid pAdEasy-1,E.coli.BJ5183 and DH10B,and human embryo kidney 293 cells,which were given as a present by professor Luo.All animal treatment was accorded with the ethical standards.②Total RNA was extracted from spleen cells of rat stimulating by lipopolysaccharide.IL-10 cDNA was amplified by using RT-PCR.The gene of interest was firstly cloned into a shuttle vector pAdTrack-CMV.The resultant plasmid was linearized by digesting with restriction endonuclease Pme Ⅰ,and subsequently transformed into E.coli.BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1.Finally,the linearized recombinant plasmid was transfected into adenovirus packaging cell lines 293 cells.Recombinant adenovirus vAd-IL-10 was obtained.The expression of green fluorescent protein(GFP) was observed under fluorescent microscope.293 cells were cultured in 96-well culture plate with 1 ?104 cells per well.Condensed virus stock solution was added into the plate at different ratios.Recombinant adenoviruses titer was determined.Three days later,total RNA was extracted from 293 cells by TRIzol one-step method,and contaminant DNA was digested by DNaseⅠ.Electrophoresis detected the expression of IL-10 mRNA.RESULTS:①GFP expression was observed 16 hours after packing of the linearized recombinant adenoviruses in 293 cells.The amplification product of replicationdeficient Ad-IL-10 virus was transfected into adenovirus packaging cell lines 293 cells.When the fourth and fifth generations were seeded in virus for 24-48 hours,fluorescence was found in almost cells,and 5.5?108 pfu/mL titer of Ad-IL-10 was obtained.Titer of vAd-GFP was 9.0?108 pfu/mL.②Total RNA was extracted from transfected 293 cells.Electrophoresis showed that 580bp amplification product was expressed obviously and recombinant adenovirus was duplicated in 293 cells.CONCLUSION:The recombinant adenoviral vector carrying IL-10 gene is successfully constructed using AdEasy system.Moreover,vAd-IL-10 recombinant adenovirus with high titer is prepared and transcribed in 293 cells.

Objective To study the expressions and clinical significance of E cadherin(E CD) and CD44v6 in bladder transitional cell cancinoma. Methods The expressions of E CD and CD44v6 of 96 cases of bladder transitional cell cancer were detected by streptavidin peroxidase immunohistochemical method. Results The positive expression rates of E CD and CD44v6 were as follows:Grade Ⅰ,86.7%(26/30),80.0%(24/30);Grade Ⅱ,52.8%(19/36),69.4%(25/36);Grade Ⅲ,26.7%(8/30),33.3%(10/30), P

Objective To explore surgical strategy for patients with hilar cholangiocarcinoma (HCC) and study prognostic factors after curative treatment. Methods We retrospectively reviewed medical records of 41 patients with HCC surgically treated in our department during the 9-year period from January 1999 to February 2007. Clinicopathological factors were evaluated for their association with post-operational survival by univariate and multivariate analysis using Cox proportional hazard model. Results All the 41 patients underwent laparotomy following preoperative assessment of extent of disease and 21 patients (resectability rate 51.2%) ultimately underwent resection with curative in-tent. In the resection group, R0 radical resection was possible in 11 patients, while R1 resection in 6and R2 in 4. Different types of hepatectomy were combined to accomplish resection. Meanwhile, por-tal vein wedge resection or reconstruction was needed in two patients. The 1-, 3-and 5-year survival rates were 41.5%, 14.6% and 4.9% in the overall group and 71.3%, 28.6%, 9.5% in the resection group, respectively. In R0-resection, Rl-resection and R2 resection group, the 1-,3-and 5-year sur-vival rates were 81.8% ,45.5% ,18.2% ;66.7% ,16.7% ,0 and 50% ,0,0, respectively. Survival rates after resection were significantly higher than those after palliative drainage and exploratory laparotomy (P<0. 001). Higher survival rates were seen in R0-resected patients when compared with Rl-or R2-resected patients (P<0.001). Multivariate analysis revealed that tumor-free margins, pTNM stage and combined hepatectomy were independent prognostic factors affecting survival. Conclusion Only surgery can provide chance to achieve the possibility of cure and long-term survival. Tumor-free margins, pTNM stage and combined hepatectomy are the most important prognostic factors affecting the survival.

Objective To discuss the main high-risk factors,clinical features and prognosis of global developmental delay(GDD),so as to provide effective basis for reducing incidence of children with GDD,early diagnosis,early intervention and improving prognosis.Methods One hundred and eighty-five cases of children with GDD,who were first diagnosed and treated in the Pediatric Neurology Rehabilitation Center,the First Affiliated Hospital of Anhui Medical University from October 2011 to September 2013,were included and high-risk factors,clinical features,and prognosis were analyzed.At the same time,the patients were followed up for 2 years and the children with abnormal development received continuous intervention and treatment during the follow-up.x2 test was used to compare high-risk factors and prognosis of different clinical features and Logistic regression models were selected to analyze high-risk factors influencing prognosis.Results In 185 cases with GDD,there were 119 children (64.3％) with motor and language developmental delay,which were the most common features,and followed by types of motor combined cognitive and language developmental delay which make up 30 cases (16.2％) and cognitive merged language developmental delay which make up 22 cases (11.9％) and the rarest type of 14 cases (7.6％) was motor and cognitive developmental delay.The main high-risk factors included neonatal asphyxia,premature birth,pathologic jaundice,intrauterine growth retardation,intrauterine hypoxia,neonatal hypoxic-ischemic encephalopathy (HIE),neonatal infection and pregnancy-induced hypertension syndrome and the differences of various clinical features with premature birth,intrauterine growth retardation,pathologic jaundice were statistically significant.Up to 2 years of follow-up,40 cases (21.6％) turned normal,but 145 children (78.4％) were still abnormal,including 97 children (52.5％) having significantly improved after intervention,30 cases(16.2％)of intellectual developmental disorder and 18 cases (9.7％) of cerebral palsy.The differences in various clinical features showed statistically significance (x2=60.960,P=0.017).The main high-risk factors affecting prognosis were intrauterine growth retardation [β=0.777,odds ratio (OR)=2.174],intrauterine hypoxia (β=0.706,OR=2.026),HIE(β=0.547,OR=1.729) and neonatal asphyxia (β=0.070,OR =1.073).Conclusion Causes of GDD are complex and prognosis is poor and the etiology and prognosis of children with different clinical features are also different.It is important to enhance perinatal care,early diagnosis and intervention for reducing the incidence of GDD and improving prognosis.

Objective To investigate the effects of Atorvastatin Combined with valsartan on the degree of coronary artery lesion and the level of serum lipoprotein and C reactive protein in patients with coronary heart disea.Methods 95 cases of patients with coronary heart disease from September 2015 to September 2016 in our hospital were randomly divided into observation group and control group,the control group were treated with atorvastatin,observation group of patients in the control group patients on the basis of the combination of valsartan treatment,severity of coronary artery disease,serum lipid and protein levels in patients with C reaction protein levels before and after treatment were compared between two groups.Results After treatment,the patients in the observation group were fibrous plaque,calcified plaque,lipid plaque,mixed plaque were significantly decreased,and lower than that of control group,and control group before and after treatment of fibrous plaque,calcified plaque had no obvious change after treatment,the observation group were HDL,LDL,TG,TC were(2.12±1.01),3.27±0.94),(1.53±0.98),(3.35±1.78)was significantly higher than the control group,the difference was significant,2 months after treatment,3 months to observe the levels in patients with C reactive protein were(10.27±1.78)and(7.26±2.63)was significantly lower than the control group with significant difference,with statistical significance(P<0.05).Conclusion Atorvastatin Combined with valsartan can help to reduce the coronary plaque,regulate lipid metabolism,reduce the level of C reactive protein.

ObjectiveTo investigate the effect of NF-?B on the differentiation and maturation of mu rine bone marrow-derived dendritic cells (DC) in vitro.MethodsThe activity of NF-?B in dendritic cells was blocked by oligodeoxyribonucleot ide Decoys (ODN Decoys) containing two special binding sites for NF-?B. Morpho logical changes of DS were observed. The expression of assistant molecules on th e cell surface was detected by using flow cytometer, and the stimulating activit y of allogeneic T lymphocyte proliferative response was determined in culture mu rine DC. The effect of NF-?B on the maturation and immunobiological activity o f murine DC was studied.ResultsNF-?B ODN Decoys were efficiently incorporated by DC, markedly suppressed the maturation of DC, the expression of assistant costimulatory molecules (CD80, CD8 6 and CD40) on the surface of DC and the secretion of IL-12, blocked the develo pment of DC and this blocked function was not reversed by lipopolysaccharide (LP S). In mixed lymphocyte reactions, DC treated with NF-?B ODN Decoys could indu ce allogeneic T cells hyphoresponsiveness, and this was associated with the inhi bition of Th1-type cytokines (IL-2 and IFN-?) production.ConclusionsNF-? B is a key gene to the development and maturation of DC. Specific interference w ith NF-?B in DC using ODN Decoys approaches could offer a novel strategy for i nducing and generating tolerogeneic immature DC and provide a promising means to induce transplant immune tolerance.

Objective To investigate the effect of murine dentritic cells vaccine modified with IFN-? inducible protein-10 gene on CTL induction. Methods DC was propagated from bone marrow (BM) of mice and pulsed with mouse prostate cancer cell line RM-1's whole lysate (Tuly-DC).IP-10 DNA fragments were inserted into pcDNA3.1(+) vector to construct recombinant plasmid IP-10/pcDNA3.1.Tuly-DC was transfected with IP-10/pcDNA3.1 by DOTAP liposome.Reversal transcript-polymerase chain reaction (RT-PCR) was used to evaluate gene transfer efficiency and chemotaxis assay was used to estimate the tansfected DC's chemotactic activity on T cells.Antitumor activity of the DC vaccine was studied in vitro by using Mixed leukocyte reaction (MLR) and cytotoxic assay (MTT assay). Results The IP-10 plasmid vector was successfully transfected into DC,which was confirmed by RT-PCR.The DC tranfected with IP-10 gene was capable of synthesizing and secreting IP-10 chemokine,which could increase the preferential chemotaxis of DC to T cells.MLR showed that the DC pulsed with whole tumor lysate and modified with IP-10 gene (IP-10/ Tuly-DC) could induce T cell proliferation significantly compared with other groups (P

OBJECTIVE:To establish the method for determining the contents of neferine and liensinine in plumula nelumbini.METHODS:Neferine and liensinine in plumula nelumbini were extracted by ultrasound method and determined by TLCs.RESULTS:The average recovery and relative standard deviation were(97.91?2.49)% and (99.28?3.22)%,respectively.The correlation coefficient were 0.996 and 0.999,respectively.CONCLUSION:The method,operated simply and attained the reliable results with good reproducibility,can be used to determine the contents of bioactive alkaloid in plumula nelumbini or other compound preparations.

Yin-Yang1 (YY1),as a ubiquitous nuclear transcription factor with double transcriptional activity in eukaryotic cells,regulates many genes transcription and plays an important role in the cellule biological processes.Overexpression of YY1 is found in several tumor types recently,and may play a role in tumor development and progression by regulating oncogenes,tumor suppressor genes,angiogenesis related factors,as well as apoptosis.YY1 may become a new kind of tumor markers,is conducive to estimate the prognosis of patients with tumor and gain important breakthrough in cancer targeted therapy.

Objective To explore Fms-like tyrosine kinase 3(FLT3)gene internal-tandem duplications(ITD)mutation in hematologic malignancy.Methods FLT3/ITD gene mutation was analyzed by polymerase chain reaction(PCR)amplification on the DNA samples from 332 patients with hematologic malignancies at the Nanfang Hospital from 2001 to 2005.Results The mutation of FLT3/ITD gene was detected in 22.3% acute myeloid leukemia(AML)cases,in 6.5% chronic myeloid leukemia(CML)-blast crisis(BC)cases,in 5.6% myelodysplastic syndromes(MDS)cases and in 2.6% acute lymphoblastic leukemia(ALL)cases;FLT3/ITD gene mutation was not detected in CML-chronic phase(CP),multiple myeloma(MM),non-Hodgkin lymphoma(NHL)and chronic lymphocytic leukemia(CLL)cases.FLT3/ITD+ AML indicate high white blood cell count and high percentage of bone marrow blast cells and had a unfavourable cumulative relapse rates.Conclusion AML patients with FLT3/ITD have a poor prognosis.Detection of FLT3/ITD gene mutation may be valuable in hematologic malignancy.