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 optimize the technique of isolating and extracting oligosaccharide from Radix Rehmanniae.Methods The optimum conditions for the water extracting technique of Radix Rehmanniae were studied by orthogonal test design.Then the water extract was isolated by macroporous resins and active charcoal successively,meanwhile the pattern of macroporous resins,the volume of the eluents,and the decolorizing methods of active charcoal were studied.And then the oligosaccharide was obtained after being purified by Sephadex gel column chromatography.The content of stachyose was determined by HPLC.Results The optimal preparation process was as follows: 12 fold water,three times,0.5 h for each time.Then the water extract was isolated and purified by D-101 macroporous resins,0.1% active charcoal and Sephadex G15 column chromatography were successively used to obtain the oligosaccharide.The analysis of oligosaccharide indicated its main constituents are stachyose,rafinose,and manninotriose,and the content of stachyose is more than 60%.Conclusion This preparation technique of extracting oligosaccharide from Radix Rehmanniae is reasonable and feasible.And the yield of the oligosaccharide from Radix Rehmanniae achieves to 26%.

Objective To investigate the clinical efficacy of rhubarb acupoint sticking joint shuganjieyu diet in the treatment of chronic functional constipation patients with depressive anxiety.Methods 120 patients of chronic functional constipation with depression anxiety were selected,they were randomly divided into control group and obser-vation group,60 cases in each group.The patients in the control group were given promoting gastrointestinal motility drugs mosapride capsules,and orally given laxatives rhubarb with sodium bicarbonate.The patients in the observation group were treated with topical rhubarb acupoint,and orally given medicated shuganjieyu diet.7 days for a course of treatment,the patients were treated for four courses.The clinical efficacy of the two groups,defecation interval and fecal nature,SAS,SDS scale score changes and quality of life (PAC -QOL)scale score changes before and after treatment and adverse reactions during treatment were recorded.Results The total effective rate of the observation group was 90.00%,which of the control group was 91.67%,the difference between the two groups was not significant (χ2 =0.32,P >0.05).Before treatment,the bowel movement and stool ratings between the two groups had no statis-tically significant differences (t =0.23,0.33,all P >0.05).After treatment,the two points of the two groups were significantly decreased,but the differences between the two groups were not significant (t =0.18,0.41,all P >0.05).Before treatment,the SAS,SDS scale scores between the two groups had no statistically significant differences (t =0.78,0.69,all P >0.05).After treatment,the SAS,SDS scores of the two groups were significantly decreased, which of the observation group decreased more significantly than those of the control group (t =4.18,4.72,all P <0.05).Before treatment,the PAC -QOL scale scores for each dimension level had no statistically significant differ-ences (t =0.16,0.21,0.42,0.20,all P >0.05).After treatment,the dimensions of the two groups showed a decline trend,and those of the observation group decreased more significantly than the control group (t =2.10,3.56,2.76, 2.34,all P <0.05).In the control group during the treatment,2 cases of diarrhea,three cases of dry mouth,the patients were improved after given symptomatic treatment.No serious adverse event occurred during therapy in the observation group.Conclusion Rhubarb acupoint joint shuganjieyu diet in the treatment of chronic functional constipation patients with depression anxiety has significant clinical effect,which can significantly improve patients'depression and anxiety scores,quality of life,and without significant adverse reactions,it is worthy of clinical applica-tion.

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.

Objective To establish an inductively coupled plasma‐mass spectrometry (ICP‐MS) method for analysis of elements in peripheral blood of children .Methods A total of 474 healthy children in Hunan area were enrolled in this study ,and six ele‐ments ,including Ca ,Mg ,Fe ,Cu ,Zn and Pb ,in peripheral blood specimens were detected by using ICP‐MS method .Results The levels of elements ,including Ca ,Mg ,Fe ,Cu ,Zn and Pb ,in peripheral blood of healthy children showed skewed distributions ,and no significant differences were found in levels of these elements between male and female children(P>0 .05) .The reference intervals of Ca ,Mg ,Fe ,Cu ,Zn and Pb in peripheral blood of healthy children in this area were 57 .30 -81 .40 mg/L ,30 .40 -44 .80 mg/L , 361 .20-531 .40 mg/L ,848 .10-1 469 .20 μg/L ,2 .68 -6 .54 mg/L and 0 .00 -100 .00 μg/L respectively .Conclusion The ICP‐MS method for simultaneously detecting Ca ,Mg ,Fe ,Cu ,Zn and Pb in peripheral blood of children and reference interval of each ele‐ment are successfully established .

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.

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 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.