Objective In order to study the role of APP SW mutation in the aetiology of Alzheimer′s disease(AD), the phenotypes of the PDAP SW transgenic mice were investigated. Methods Body weight, coat color and reproducibility of transgenic mice were observed;The pathological changes in the brain of the transgenic mice were examined using immunohistochemistry; Behavioral changes of transgenic mice were examined by Y-maze. Result At 3 month′s age, the mean body weight of the transgenic mice was (31.0?3.7) g, that of non-transgenic was (34.0?2.9) g;while the mean body weight of F1 transgenic mice was (32.0?3.3) g, of negative mice was (31.0?4.2) g. There were no significant difference between these two groups. There were three transgenic mice with abnormal coat and one male was infertile. The transgenic mice had amyloid deposit in their brains. In the Y-maze test, transgenic mice showed an increased number of arm entered (53?7 versus 37?4) and an impaired spontaneous alternation. The frequency of alternation was 48.2% in the transgenic mice and 76.4% in the non-transgenic mice,respectively.Conclusion The PDAP SW transgenic mice had typical pathological and behavioral changes similar to that of AD and could be used as an animal model for studying AD.

AIM: To clone the extracellular domain of N-cadherin cDNA, and to observe the antigenicity of the expressed protein. METHODS: Total RNA was extracted from CD34+ cells separated from fetal liver and bone marrow cells. The extracellular domain of N-cad cDNA was amplified with RT-PCR and inserted into a vector pOPE101-215. The recombinant pOPE-N-cad was expressed with IPTG induction. Then, mice were immunized with the protein. RESULTS: The extracellular domain of N-cad cDNA from CD34+ cells was identified by DNA sequencing. The recombinant pOPE-N-cad in host XL1-blue expressed a 70 kD protein after induced with IPTG, and anti-N-cad antibody was detected in serum of the immunized mice after 5 times injection of the recombinant N-cad protein. CONCLUSION: CD34+ cells bore N-cad gene and the recombinant protein of the extracellular domain of N-cad cDNA shows good immunogenicity.

ObjectiveTo explore the best time for replacing the long-term indwelling catheter in patients with silicone catheter.MethodsDomestic literature were retrieved and analyzed using Cochrane systematic review,and the meta-analysis of randomized controlled trials was conducted in accordance with inclusion criteria and exclusion criteria so as to look into the most suitable time for replacing the indwelling catheter for patients with silicone catheter at clinical trials.The replacement frequency for the catheters of different size together with the relevant infections in urinary tract infection and relative risk(RR)were used as values for effectiveness of interventions.ResultsA total of 11 literature were retrieved. Meta-analysis results suggested that the RR values of urinary tract infections when the catheters were replaced once every two weeks vs. every four weeks,and once every three weeks vs. every four weeks were 0.51[95% CI(0.40,0.66),P <0.001],0.79[95% CI(0.58,1.08),P = 0.14],respectively.The urinary infection rate of replacing a silicone cathether every 2 weeks was higher than that of every 4 weeks,but there was no difference between that of every 3 weeks and 4 weeks.Conclusion According to the nature of silicone catheter material as well as the clinical indexes, it is most reliable to replace a silicone catheter every four weeks to reach a best clinical outcome.

Objective To establish human embryonic stem cells (hESCs) feeder-independent and cell factor-free culture system. Methods Effect of high and low clone densities of hESCs culture was compared and impact of the clone densities to hESCs culture media was analyzed. Results HESCs could maintain their undifferentiated states at high clone density (34 clones/cm2) without cell factors. At the same time,the bone morphology protein (BMP)-like induction of N2 and B27 supplements (NB) medium could be modulated by the clone density,and high level of BMP-like induction was accompanied by high clone density. Conclusion High clone density of hESCs can change the environments by themselves to maintain the undifferentiated states,which provides a new clue to explore the mechanism of undifferentiated states of hESCs and simplify the culture medium.

Objective To study the relationship between umbilical cord leptin levels and fetal growth as well as neonatal birth weight. Methods One hundred and forty - two neonates selected from February 2009 to June 2013 in Shangqiu First People's Hospital according to the different gestational age and birth weight were divided into 3 groups. Group A included 44 cases(small for gestational age,birth weight below the average weight of the 10th percentile at the same gestational age),23 boy cases,21 girl cases;group B included 56 cases(appropriate for gestational age,birth weight at the average weight of the 10th to 90th percentile at the same gestational age),30 boy cases,26 girl cases;group C included 42 cases(large for gestational age,birth weight above the average weight of the 90th percentile at the same gestational age),22 boy cases,20 girl cases. Neonatal body mass index,birth weight,placenta weight and umbilical lep-tin levels of three groups were compared. Results Neonatal birth weight,neonatal body length,body mass index and the placenta weight leptin levels in group A were significantly lower than those of group B,having statistically significant difference(all P ﹤ 0. 001);Neonatal birth weight,neonatal body length,body mass index and the placenta weight leptin levels in group C were significantly higher than those in group B,with statistically significant difference( all P ﹤0. 001). Neonatal birth weight in the boy group was obviously higher than that of the girl group,and the difference was statistically significant(P ﹤ 0. 001). Neonatal leptin levels in the boy group were significantly lower than that of the girl group,and the difference was statistically significant(P ﹤ 0. 001). There were positive correlations between the umbili-cal cord leptin levels and the neonatal birth weight,neonatal length,neonatal weight index and the placenta weight(r =0. 382,0. 276,0. 358,0. 412,all P ﹤ 0. 01). Conclusions The umbilical cord leptin levels are closely associated with neonatal birth weight and intrauterine growth retardation,and it can be used as one of the important indicators for reflec-ting neonatal birth weight and fetal growth.

Objective To study the value of score for neonatal acute physiology Ⅱ(SNAP]Ⅱ) and its extension version Ⅱ (SNAPPE-Ⅱ) in predicting neonatal necrotizing enterocolitis (NEC) outcome.Methods We explored 73 NEC patients by statistics who were treated in our hospital from January 2002 to January 2012.The patients were divided into two groups:surgery group and non-surgery group,then they were divided into subgroups:alive group and death group.The general information including birth weight,age,clinical manifestations,treatment of patients were collected.Every patient was checked and scored by the methods SNAP-Ⅱ] and SNAPPE-Ⅱ in time.Results The scores (27.0 ± 2.3,26.5 ± 1.8) of surgery group including SNAP-Ⅱ and SNAPPE-Ⅱ were higher than those (14.0 ± 2.1,15.0 ± 2.5) in the non-surgery group(P ＜ 0.01).The scores(31.0 ± 3.2,31.0 ± 3.4) of the death group including SNAP-Ⅱ and SNAPPE-Ⅱ were higher than those(11.0 ± 2.5,10.0 ± 3.6) in the alive group(P ＜ 0.01).According to the area under the curve(AUC) analyzed by the receiver operating characteristic(ROC) curve for measuring the scores of surgery predicting,AUC was 0.726 for SNAP-Ⅱ and 0.732 for SNAPPE-Ⅱ.The value of predicting surgery risk was 20 and 24 respectively.According to the AUC analyzed by the ROC curve for measuring the scores for surgery predicting,AUC was 0.752 for SNAP-Ⅱ and 0.825 for SNAPPE-Ⅱ.The value of predicting mortality risk was 31 and 33 respectively.All P values were less than 0.01 and there were significant differences.Conclusion The two kinds of score for neonatal acute physiology have an important significance in predicting surgery and mortality risk of NEC.

Objective Increasing evidence suggest that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The study is to evaluate the effects of combined therapy of PI3K inhibitor (LY294002) and Ad-PTEN in athymic mice xenogeneic transplant model of human glioma and to reveal the possible mechanisms involved.Methods Twenty-four athymic mice were randomly divided into 4 groups (DMSO、Ad-vector plus DMSO、LY294002 alone and Ad-PTEN plus LY294002), and were treated, respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN、p-Akt、CyclinD1、Caspase-3、MMP-2、p-FAK in tumor tissues were analyzed with immunohistochemistry.Results The tumor-inhibiting rate of was significantly higher in Ad-PTEN plus LY294002 than in the LY294002 alone (92.46 vs 65.59%)( P <0.05).The protein expressions of PTEN and Caspase-3 were significantly higher, while PCNA、CyclinD1、bcl-2 and MMP-2、p-FAK was significantly lower in Ad-PTEN plus LY294002 group than in the other three groups ( P <0.05).Conclusions LY294002 plus Ad-PTEN achieve better outcome than either alone in treating glioma possibly through enhancement of the inhibitory action of PI3K/Akt pathway and Ad-PTEN pathway.

OBJECTIVETo investigate and establish the gene diagnosis methods for the frequent mutations of iduronate-2-sulphatase(IDS) gene in mucopolysaccharidosis type II patients.

METHODSpolymerase chain- reaction-single strand conformation polymorphism PCR-SSCP) analysis was applied to detect the mutations of exons 3, 8 and 9 which were hot spots in the iduronate-2-sulfatase gene; DNA sequencing was applied to analyze the mutations which had been detected by PCR-SSCP; PCR-restriction fragment length polymorphism (PCR-RFLP) was applied to detect the results of DNA sequencing.

RESULTSObvious and abnormal bands in exon 9 of the IDS gene were found by applying PCR-SSCP; the mutation(C1672T) of exon 9 was found in the patient through DNA sequencing, which led to amino acid replacement(R468W); the PCR-restriction enzyme digestion showed that only one band(554 bp) appeared in the patient, but there were two bands (257 bp and 297 bp) in his parents, and it verified the results of sequencing analysis.

CONCLUSIONPCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion are effective methods for MPS II diagnosis. Combined applications of these methods can verify and complement each other and improve the accuracy of diagnosis.

Amino Acid Substitution ; Base Sequence ; Child ; China ; Codon, Nonsense ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Humans ; Iduronate Sulfatase ; genetics ; Male ; Mucopolysaccharidosis II ; enzymology ; genetics ; Mutation ; Point Mutation ; Polymorphism, Single-Stranded Conformational

OBJECTIVETo prepare transgenic mice model of human Swedish mutation of amyloid precursor protein(APP(SWE)) gene.

METHODSBy pronuclear microinjection, human APP (SWE) genes were microinjected into pronuclei of zygotes of C57 and Kunming white mice. Integral zygotes were implanted into the oviducts of pseudopregnant female mice. The integration and expression of exogeneous genes in offsprings were detected by polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and reverse transcription-ploymerase chain reaction (RT-PCR) analysis.

RESULTSThe success rate of microinjection and the birth rate were 76.62% and 10.38%, respectively. There were 6 founders and the integration rate was 35.29%. The exogeneous gene was transferred to the third generation. Expression of human APP(SWE) gene was detected in the brain, heart and muscle but not detected in the liver and kidney of transgenic mice.

CONCLUSIONThe transgenic mice model of human APP(SWE) gene has been prepared successfully.

Amyloid beta-Protein Precursor ; genetics ; Animals ; Brain ; metabolism ; Cloning, Molecular ; Female ; Gene Expression ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Muscles ; metabolism ; Myocardium ; metabolism ; Plasmids ; genetics

To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.
AC133 Antigen ; Animals ; Antibodies ; genetics ; Antigens, CD ; immunology ; Antigens, CD34 ; immunology ; Base Sequence ; Cadherins ; immunology ; Female ; Glycoproteins ; immunology ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Library ; Peptides ; immunology