Objective To explore the method to develop a gene vaccine of human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) against its infection. Methods HCMV strain AD169 was propagated in WI-38 cell and viral DNA was extracted as a template for polymerase chain reaction (PCR) amplification of UL83 (pp65), the resulting of PCR was subcloned into pUC118HincII/BAP plasmid and DNA sequence analysis conformed the fidelity of the PCR. The vector pcDNA 5.0 was designed to correctly place CMV promoter sequence, pp65 sequence and secret signal sequence (mouse immunoglobulin kappachain for efficient secretion of recombination protein) into its genomic DNA. Exchanged primers of pp65 sequence, CMV promoter sequence and secret signal sequence to confirm the result by PCR screening. The vector pcDNA 5.0 was transfected into CHO cell, supernatants of transfected cells were extracted and purified. Recombination protein from supernatants was detected by gel electrophoresis and dot blot hybridization of Western- ECL system. Results Compared the sequence of pp65 gene with the standard sequence of pp65 from Medline,it was found that the concordant rate between them was 99.99%,only a nonsense mutation occurrences at 1 455 base.A pcDNA 5.0 Eukaryotic expression vector was established, which including CMV promoter sequence,secretion signal sequence and pp65 sequence. PCR screening and the pp65 protein expressed in CHO cell confirmed it. Extraction from supernatants in transfected CHO cells was recombination protein of pp65, which was detected by gel electrophoresis and dot blot hybridization of Western- ECL system and western blotting.Conclusion Subunit vaccine of HCMV is gained,which is a transfer eukaryotic expression vector pcDNA 5.0 constructed by CMV promoter sequence,secretion signal sequence and pp65 gene sequence.

Objective To explore the migration and differentiation of the human neural stem cells (hNSC) after being transplanted to the neonatal rat lateral ventricle,to provide some data on therapy for neonatal cerebropathy by using of neural stem cells.Methods N2 medium containing EGF+FGF2+LIF was used to culture the NSC spheres from the forebrain tissues of aborted human fetus.The hNSC was identified by detecting the NSC marker nestin antigen and showing the potency to differentiate into neural cells( including astrocytes,oligodendrocytes and neurons)by using indirect immuno-fluorescence assay(IFA).The part of the hNSC in-vitro cultured for 14 d was digested to suspensions of cell.Cultured for 14 d, the hNSC in-vitro and the suspension were transplanted into the lateral ventricles of the neonatal rat brains.The rats were respectively killed at 24,48 and 72 h respectively post-transplant,the whole brain was sectioned,and the special immuno-response detection was performed by using anti-human nuclei(anti-hNuc)and anti-human neurofilament(anti-hNF).Results In vitro culture,the typical NSC spheres were obtained from the forebrain of the human fetus.The suspensions of cells were obtained from the neurosphere.In neurosphere group, the results of anti-hNuc detecting tracing at 72 h post-injection showed that the grafts had migrated into the cortex grand layers of olfactory bulbus,medial precentral area of lobus frontalis,hippocampal,and lobus occipitalis.The label-positive cells lined along the Cerebellar Purkinje cell layers and appeared in most parts of mesencephalons.The immuno-respons results of anti-hNF showed that the positive cells scattered in the grand layer of cortex,the connection among positive cells was watched.In suspensions group,the results of anti-hNuc detecting tracing at 24 h post-injection show a great quantity of positive cells in the ventricles and injection track.At 72 h, a small quantity of positive cells remained in the ventricle and nearby brain tissue.Conclusions Whole neurospheres migrated intensely and differentiated into neurons and gliocytes.At the same time,transplants of cells from suspension transplants showed limited or no migration because of internal environment of the brain and construction of neurospheres.

Objective To investigate the efficacy of spectral entropy measurement in reflection of depth of anesthesia and noxious stimulation. Methods Forty-five patients of ASAⅠorⅡ were randomly divided into three groups(n=15).Group A,B and C received fentanyl 1,3 and 5 ?g/kg,respectively,3 min before target controlled infusion(TCI) of propofol.Intubation was performed when the effect-site concentration(CE)reached 3.5 ?g/mL,which was maintained until 5 min after incision.Response entropy(RE),State entropy(SE) as well as heart rate(HR),mean arterial pressure(MAP) were measured at the time points of before fentanyl and 2,3 min after fentanyl,every CE of propofol steps,before intubation,immediately and 1,3,5 min after intubation,before skin incision,and 0.5,1,3,5 min after skin incision,respectively. Results Three minutes after receiving fentanyl,the values of RE and SE in the three groups decreased significantly in a dose-dependent manner,and increased obviously at the same degree during intubation and after skin incision.The values recovered to the level before stress stimulation 1 min after intubation and 5 min after skin incision.There were no differences in the fluctuation of RE and SE among the three groups when the CE of propofol reached 1.0 ?g/mL.Conclusion Spectral entropy may effectively reflect the depth of anesthesia,but not analgesia during anesthesia.

OBJECTIVETo investigate the genetic polymorphism of 9 short tandem repeats (STR) gene loci, namely CSFIPO, TPOX, TH01, D16S539, D7S820, D13S317, F13A01, FESFPS and vWA in Chinese Korean population in Mudajiang area.

METHODSAmplified fragment length polymorphism (Amp-FLP) method was used to get the allele frequency distribution.

RESULTSThe genotype distributions of the 9 STR loci are conformed to Hardy-Weinberg equilibrium by chi(2) test analysis. The total accord frequency, the accumulated total discrimination power and the the accumulative excluding probability of paternity were calculated.

CONCLUSIONThe result suggested that all 9 gene loci have high power of excluding probability of paternity and individual identification. They can be used in paternity testing and individual identification for forensic medicine. The gene frequencies of CSFIPO, TPOX and TP01 gene loci have significant differences between the Korean population in Mudanjiang area and those in Yanji area, but there is no difference in gene loci of D7S820, D17S317 and vWA.

Amplified Fragment Length Polymorphism Analysis ; Asian Continental Ancestry Group ; Humans ; Korea ; Microsatellite Repeats ; genetics ; Polymorphism, Genetic ; genetics

Objective To explore the effectiveness of impulse oscillometry(IOS)in airway responsiveness measurement and to find out the positive threshold of IOS for asthma diagnosis. Methods Seventy-nine children aged 6-14 years who had suspicious asthma,were recruited into the study. The positive criteria of the methacholine bron-chial provocation test was a 20％ reduction in forced expiratory volume in the first second (FEV 1 )compared to base-line. Simultaneously measured changes in various parameters of IOS,including resonant frequency(Fres),impedance at 5 Hz(Zrs),resistances at 5 and 20 Hz(R5,R20),reactance at 5 Hz(X5),and area of reactance(AX). The results of the challenge test were divided into positive and negative groups according to the pulmonary ventilation function me-thod. The differences between the 2 groups of IOS parameters before and after the challenge test,and the correlation be-tween the change rate of FEV1 and the change rate of IOS parameters were compared,and the positive judgment criteria of IOS parameters in the determination of respiratory responsiveness were determined. Results The positive group of bronchial provocation test had 37 patients and negative group had 42 patients. There was no significant difference in the basic values of parameters between the positive group and the negative group (all P > 0. 05). Changes in Zrs,R5,X5 of IOS were correlated with changes in FEV1 (r = 0. 374,0. 310,0. 449,all P < 0. 05). By single factor analysis,the area under the receiver operating characteristic(ROC)carve (AUC)showed:basic value of Zrs increased by 45. 85％,R5 increased by 45. 72％,X5 increased by 80. 74％ respectively compared to the baseline showed the optimal combination of sensitivity and specificity. In multivariate Logistic regression models,when Zrs and R5 were combined to measure the airway responsiveness,the sensitivity and specificity were 73. 0％ and 81. 0％,respectively. Conclusions IOS and spirometry can be used to determine airway responsiveness in children during methacholine bronchial challenge. Zrs≥45. 85％,or R5≥45. 72％,or X5≥80. 74％,or Zrs and R5 of multiple regression formula can be used as the positive criteria for young children with airway heperresponsiveness,the combination of Zrs and R5 has higher sensitivity and specificity.

OBJECTIVEWe investigated the in vivo effects of recombinant adenovirus-associated virus type-2 (AAV-2) mediated interleukin-10 (IL-10) gene transfer on the expression of matrix metalloproteinase (MMP)-2, 9, tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I and type III in a rat acute myocardial infarction model.

METHODMale Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 6): sham operation group, MI/AAV2 group, and MI/AAV2-IL-10 group (10(10) vg/ml x 0.1 ml injection at peri-infarct regions immediately post MI). Five days later, the expressions of MMP-2 and MMP-9 were measured by RT-PCR, Western blot and zymography. The expression of TIMP-1 was measured by RT-PCR and Western blot. Collagen type I and type III were assessed by RT-PCR and immunohistochemical stain.

RESULTSThe myocardial expressions of MMP-2, MMP-9 and collagen contents in MI/AAV2 group were significantly increased than those in sham operation group. Myocardial expressions of MMP-2, MMP-9 were significantly decreased and the expression of TIMP-1 significantly increased in the MI/AAV2-IL-10 group than those in MI/AAV2 group. Moreover, the expressions of collagen type I, collagen type III and the ratio of I/III collagen in border zones of infarcted myocardium were decreased by 47.6% (P < 0.01), 23.6% (P < 0.05), and 17.9% (P < 0.05) respectively, while the expression of TIMP-1 increased by 73.1%(P < 0.05) in MI/AAV2-IL-10 group compared to MI/AAV2 group.

CONCLUSIONIn vivo myocardial IL-10 transfer reduced myocardial MMP and collagen expression and increasing the TIMP expression.

Animals ; Extracellular Matrix ; metabolism ; Gene Expression ; Genetic Therapy ; Interleukin-10 ; genetics ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Myocardial Infarction ; genetics ; metabolism ; physiopathology ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transfection ; Ventricular Remodeling

OBJECTIVETo investigate the effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib.

METHODSTumor suppressor gene p14(ARF) was transduced into K562 (K562-p14(ARF)) and 4 blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector with cells transduced by empty vector as control. Fluorescence microscopy and flow cytometry were applied to measure transduction efficiency, and Western blotting assay was used to detect p14(ARF) protein of K562 cells. WST-8 method was used to determine cell growth inhibition rate of K562 cells transduced by the target gene under different concentrations of imatinib (0, 0.015, 0.062, 0.125, 0.25, 0.5, 1.0, 2.0 μmol/L). Cell apoptosis and leukemic cellular colony-forming ability were detected by Annexin V-FITC/PI dyeing using flow cytometry (FCM) and semi-solid culture method respectively.

RESULTSFluorescence microscopy and FCM showed that transduction efficiency (GFP positive cells) of K562-p14(ARF), K562-VSV and CML-BC1 cells were close to 100%, and CML-BC 2-4 cells were 80% to 90% on average. Results of Western blotting showed that the levels of ARF protein expression of K562 cells transduced by p14(ARF) were significantly higher than of untransduced cells; the apoptosis rate of K562-p14(ARF) was 20%; the mean apoptosis rate of 4 primary leukemic cells transduced by the p14(ARF) [(71.1±22.4)%] was significantly higher than of control group [(12.4±6.2)%] (P<0.05). Imatinib significantly inhibited the proliferation of K562-p14(ARF) cells in a dose-dependent manner. The mean leukemic cellular colony-forming unit of 4 primary leukemic cells transduced by the p14(ARF) (41.5±13.2) was significantly lower than of the control group (88.5±7.9) (P<0.05).

CONCLUSIONIncreased p14(ARF) gene expression could induce apoptosis of CML cells; Moreover, it could enhance inhibitory effect on cell proliferation when combined with imatinib.

Apoptosis ; Gene Expression Regulation, Leukemic ; Genetic Vectors ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Tumor Suppressor Protein p14ARF ; metabolism ; Up-Regulation

OBJECTIVESTo evaluate the relationship between the operative methods and the therapeutic results of the patients with hypospadias.

METHODSNine operative types and different tissue materials taken in hypospadias operations in the past 12 years were retrospectively analyzed.

RESULTSThe operative types and tissue materials had significantly effects on the therapeutic results besides the clinical experience of the operators. The cure rates of Tunneltron Urethroplasty, Preputial island flap urethroplasty and Bladder mucosa graft urethroplasty were 86.4%, 83.3% and 83.0% respectively.

CONCLUSIONSAll the nine types and different tissue materials of Hypospadias operations have its own advantages and disadvantages. They are worth further study and improvement.

Adolescent ; Adult ; Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Infant ; Male ; Mucous Membrane ; transplantation ; Penis ; surgery ; Surgical Flaps ; Treatment Outcome ; Urethral Stricture ; surgery ; Urinary Bladder ; transplantation ; Urinary Fistula ; surgery ; Urologic Surgical Procedures, Male ; methods

OBJECTIVETo conduct a molecular epidemiological survey on the mitochondrial DNA C1494T mutation in non-syndromic hearing loss patients in Chinese population.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to screen the mitochondrial DNA 12S rRNA C1494T mutation in 20 patients with aminoglycoside antibiotic induced hearing loss, 136 sporadic non-syndromic hearing loss patients and 50 probands of pedigrees with non-syndromic hearing loss.

RESULTSThe C1494T mutation did not appear in all cases except for the positive control.

CONCLUSIONIncidence of mitochondrial DNA C1494T mutation is much lower than that of mitochondrial DNA A1555G mutation in non-syndromic hearing loss of Chinese population. Mitochondrial DNA C1494T mutation may be a rare variation in non-syndromic hearing loss and is not the main cause of aminoglycoside antibiotic induced-deafness.

Adolescent ; Aminoglycosides ; adverse effects ; Anti-Bacterial Agents ; adverse effects ; Asian Continental Ancestry Group ; genetics ; Child ; China ; DNA, Mitochondrial ; genetics ; Female ; Hearing Loss ; chemically induced ; ethnology ; genetics ; Humans ; Male ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal ; genetics

Drainage ; Humans ; Pancreas ; surgery ; Pancreaticoduodenectomy ; Stents