Objective evaluate the mechanism that bacteria growing in biofilm always resist to antimicrobial agents,and to provide the theoretical basis for selecting antimicrobial agents in clinic.Methods Model of klebsiella pneumoniae bacterial biofilm were built up with the modified flat-board method and identified with the method staining with AgNO3 and confocal scanning laser microscopy.We used imipenem to induce the ESBLs production of BF bacteria.ESBLs production was performed by the standard disk diffusion method.Results The isolation rate of ESBLs producting strains in klebsiella planktonically planktonically were 20.0%(8/40).The isolation rate of ESBLs producting strains in klebsiella biofilm were 42.5%(17/40).The isolation rate of ESBLs producting strains in klebsiella biofilm being induced by imipenem were 65.0%(26/40).The isolation rate of group A and group B,groupB and group C was different from each other significantly with the statistic method of ?2-test.Conclusion One of the main reasons that klebsiella and escherichia coli resist to antibiotics is the synergetic effect of BF and ESBLs.

Based on link control concepts, the life cycle of science and technology projects was divided into five stages,, such as project application, selection, implementation, output and assessment.All the science misconducts that might occur among these five stages were listed.Finally, different precautions were made to prevent different misconducts during different stage..

Objective To study the early functional change of sinusoid endothelial cell after liver transplantation in rat, and to investigate the endothelia protective effect of prostaglandin E_1(PGE_1). Methods Rat orthotopic liver transplantation model was performed in "two-cuff method", grouped as follows: group A served as normal rat blank control, group B as operative control with normal donor, group C as experimental control with shock donor, and group D as experimental group with shock donor and PGE_1 administration ( n =8 in each group). Transplanted groups (referring to recipients without specific definition) were sacrificed 6 h after operation for blood taken to detect serum liver enzymes (ALT, LDH), malondialdehyde (MDA), nitric oxide (NO) and plasm endothelin (ET). Liver tissue was resected at the same time for standard pathologic examination. Comparison of the difference the results was made between groups. Results Cold preservation time and anhepatic phase were similar in each group, (2?0.5) h and (15?3) min respectively. All survived 6 h after transplantation (8/8) in group B and D with a survival rate of 100%, only 5 survived 6 h after transplantation in group C (5/8) with a survival rate of 62.5%. Comparing with group C, blood ALT, LDH, MDA, ET decreased and NO increased significantly in group D ( P

BACKGROUND: Increased expression of survivin in various tumor tissues can regulate cell proliferation, division, and plays an important role in protecting cells from apoptosis.OBJECTIVE: To construct the specific micro RNA (miRNA) expression vector that can block the C_(57)BL mice survivin gene by RNA interference (RNAi) technique.DESIGN, TIME AND SETTING: The single sample observation was performed at the experimental center of Department of Neurology, The First Afliliated Hospital of Chongqing Medical University from June to November 2008.MATERIALS: Ring-shaped pcDNA~(TM)6.2-GW/EmGFPmiR and BLOCK-iT~(TM) Pol II miR RNA interfered Expression Vector Kit with EmGFP was produced by Invitrogen Company. DH5a E. coli was preserved at the laboratory. Xho I and BamH I enzyme, spectinomycin were provided by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.METHODS: According to sequence of mRNA of C_(57)BL mice survivin provided by Genebank, four pairs of specific oligonucleotide sequences were designed and synthesized by using the software. The annealed oligonucleotide fragment was sub-cloned into pcDNA~(TM)6.2-GW/EmGFPmiR expression vector by gene clone technique and transformed into DH5a E. coli, subsequently, a single colony was incubated into liquid medium containing spectionmycin. Finally the plasmid was extracted.MAIN OUTCOME MEASURES: The recombinant vector was identified by sequencing and agarose gel electrophoresis.RESULTS: The sequencing revealed that insertion element was correctly cloned into the vector without nucleotide mutation, absence or insertion abnormality. The result of double enzyme digestion demonstrated that the fragment length was coincidence with expectation.CONCLUSION: The C_(57)BL mice survivin miRNA expression vector is successfully constructed.

Objective:To investigate the related factors and clinical significance of peritoneal micrometastasis in patients with gastric carcinoma,providing theoretical basis for resection range。Methods:CK19,CK20 immunohistochemistry were performed on 62 patients' tissues taken from anterior lobe of transverse mesocolon,posterior wall of omental bursa,pancreatic capsule and rectovesical pouch or Douglas pouch during the operations,compared with HE staining and peritoneal lavage cytology(PLC).Results:No metastasis was found by HE staining.Peritoneal micrometastasis were found in 27 cases out of 62 by immunohistochemistry,and its positive rate was 43.55%,obviously higher than PLC(14.52%).The peritoneal micrometastasis of gastric carcinoma had relations with diameter of tumor,depth of infiltration,clinical stage,lymph node metastasis(P0.05).Conclusion:Immunohistochemistric measure of CK 19 and CK20 can be effective to detect the micrometastasis of gastric carcinoma,which is helpful to guide clinical staging and useful to provide evidence for accurate selection of operation and postoperative treatment.Routine detection of peritoneal micrometastasis should be taken in patients of advanced gastric carcinoma,especially with a large size of serosa invasion.Multiple spots sampling is helpful to improve the detection rate.Anterior lobe of transverse mesocolon and pancreatic capsule should be peeled,and radical resection of omental bursa should be considered as routine operation in these patients.

Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B ( MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were ob-tained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plas-mid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results Three common MICB fragments contained the exons 2 and 3 were obtained. The recombinant proteins were expressed in E. coli BL21 DE3 strains carrying pET-28a-MICB and successfully purified by the Ni-NTA Spin column. Results of the Western blot assay confirmed that the obtained proteins were the target proteins. The ELISA method was successfully established and used for the detection of anti-MICB antibodies in 24 patients with kidney trans-plantation. The absorbance values indicated that the sensitivities of three recombinant MICB proteins were different. Conclusion The expression system for MICB gene was successfully constructed. The established ELISA for the detection of anti-MICB antibodies would pave the way for further investigation on the correla-tions between MICB protein and transplantation immunity.

Objective To investigate the effects of methylated CpG islands in the promoter region on the expression of killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1).Methods Three voluntary unpaid blood donors carrying high expression allele KIR 3DL1*01502 and three donors carrying low expres-sion allele KIR3DL1*005 were recruited in this study .The nucleotide sequences and the methylated CpG islands in the promoter regions of KIR 3DL1*01502 allele and KIR3DL1*005 allele were analyzed .The NK cells expressing KIR3DL1*01502 and KIR3DL1*005 were respectively treated with 5-aza for the dem-ethylation of CpG islands within the promoters .The expression of KIR3DL1 protein on the surface of NK cells was measured with flow cytometer .Results Two differences at nucleotide sites -65 and -269 were detected within the promoter regions of KIR3DL1*01502 and KIR3DL1*005, resulting in two distinct CpG islands.The CpG islands within the promoter of KIR 3DL1*01502 allele were highly methylated .The ex-pression of KIR3DL1 protein on NK cells which carried KIR 3DL1*01502 allele was significantly increased after the demethylation of CpG islands .However , the treatment of demethylation had no significant effects on the expression KIR3DL1 protein on NK cells harboring KIR3DL1*005 allele.Conclusion The methylated CpG islands within the promoter of KIR 3DL1*01502 allele affected the antigen expression on the surface of NK cells.Different KIR3DL1 alleles might show different mechanisms in regulating antigen expression .

Objective To analyze the molecular genetic basis of novel allele HLA-B * 9534 and establish the allele group specific primer PCR method. Methods Genomic DNA was extracted from whole blood by commercial DNA extraction kit. The HLA-B exons 1 to 8 coding sequences of the proband were am-plified by PCR and the amplification product was purified with double enzymes digestion and both strands of exons 2, 3 and 4 were sequenced. The exon 2-4 amplification of the HLA-B * 9534 was performed with al-lele group specific primers PCR and the PCR product was directly sequenced for exon 2 to 4. Results The proband has two HLA-B alleles. The result was assigned for HLA-B * 1518 and B * 4601 combination with a mismatch in 593A/G heterozygote by DNA sequencing of exon 2 to 4 with loci primers. After separating the two alleles of the proband with allele group specific primers polymerase chain reaction method, HLA-B * 4601 and HLA-B * 9534 alleles were identified after sequencing. The HLA-B * 9534 is identical to HLA-B * 1518 except for one nucleotide substitutions in exon 3 at position 593 A→G, this results in amino acid substitution at cedon 174 from Asn to Ser. The sequences of the novel allele have been submitted to GenBank (EU046491) and the allele has been officially nominated by the WHO Nomenclature Committee. Conclusion Identification of a novel HLA-B * 9534 allele and allele group specific primer PCR for HLA-B * 9534 was re-liable.

Objective To analyze the clinical characteristics of twin reversed arterial perfusion sequence (TRAP),and investigate its prenatal evaluation and clinical management.Methods Karyotype results and ultrasound data of 25 TRAP cases were retrospectively reviewed,including estimated weight and umbilical blood flow of acardiac twin,cardiac function and middle cerebral artery peak systolic velocity of pump twin.Various managements and the outcomes were analyzed.Results (1) Karyotype of amniotie fluid were tested in 16 pump twins.Mosaicism was found in 1 case (46,XX[36]/46,XY [14]).(2) According to the ultrasound evaluation,large acardia accounted for 87.0％ (20/23) cases.Abundant blood perfusion (inter-twin difference of umbilical resistance index ≤0.20) was indicated in 86.4％ (19/22) cases.Decompensation of cardiac function was suggested in 66.7％ (10/15) pump twins.Fetal anemia of pump twin indicated by middle cerebral artery peak systolic velocity＞1.5 multiples of the median was diagnosed in 75.0％ (12/16) cases.(3) The acardiac twin with abundant blood perfusion was more likely to be a large acardia than those without [94.7％(18/19) vs 1/3,Fisher exact test,P=0.04]; More pump twin with large acardia tended to have cardiac decompensation than non-large acardia pump twins [83.3 ％ (10/12) vs 0/3,Fisher exact test,P=0.02].(4) Eleven patients chose to terminate their pregnancies after being diagnosed.In 14 cases who continue the pregnancies,the survival rate of pump twin was 64.3％ (9/14).In 3 cases of non-large acardia without cardiac decompensation of pump twin,the patients selected conservative observation resulting in 2 term deliveries and 1 termination of pregnancy due to for exacerbation.Among 11 cases with large acardia,which the pump twins were complicated by cardiac decompensation or anemia,five cases selected conservative observation.One ended in spontaneous abortion; three exacerbated (one termination and two cesarean section before term with living births) ; one was stable until delivery.Another 6 cases received bipolar cord coagulation,and successful interruptions of acardiac blood flow were achieved in 5 cases among which 4 pump twins survived.Conclusions Prenatal diagnosis,cardiac function and fetal anemia of pump twin,together with the growth and blood supply of acardia are important indexes for prenatal evaluation of TRAP,on which our prompt management should be based.

Objective To investigate the intrauterine growth characteristics of twins and birthweight discordant twins (discordant twins ).MethodsTotal of 1010 twin pregnancies (2020 fetuses) with complete delivery records from the Department of Obstetrics and Gynecology,the First and Third Affiliated Hospital of SUN Yat-sen University between January 1,2000 and July 31,2010 were studied retrospectively.One handred and ninteen cases (238 fetuses) with intrapair birthweight difference ≥25％ were determined as the discordant twins group,and the other 891 cases (1782 fetuses) with intrapair birthweight difference ＜ 25％ were identified as the concordant twins group.The singleton control group included 4042 singleton pregnancies in the same period.Results ( 1 ) Comparison of clinical data between the twins groups:the birthweight of larger-twin,smaller-twin and intrapair birthweight difference in the discordant twins group and the concordant twins group were ( 2090± 827 ) g,( 1392 ± 592 ) g,( 33.9 ±9.3 ) ％,and ( 2408 ± 543 ) g,( 2191 ± 505 ) g,( 8.9 ± 6.5 ) ％,respectively,with significant differences (P＜0.01).The incidence of discordant twins was 11.78％ (119/1010).Compared with the concordant twins group,the discordant twins group bad higher proportion of monochorionic twins,and higher prevalence of pregnancy complications such as late miscarriage,abnormal umbilical insertion,twin-twin transfusion syndrome and hypertensive disorders in pregnancy ( P ＜ 0.05 ).( 2 ) The characteristics of twin birthweight distribution:① In all the 2020 twins,80.05％ (1617/2020) fetuses had birthweight below the 50th percentile of the singleton control group,while 23.71％ (479/2020) feeuses got birthweight below the 10th percentile of the singleton control group.② After 19th gestational week,the 50th and 90th percentile of all twins' birthweight were lower than those of singletons.After 38th gestational week,the birthweight of singletons kept increasing and reached its peak at 41 th week,while the birthweight of twins reached its peak at 38th week,followed by a decline at 39 weeks,which was even lower than the 10th percentile of the singleton control group.③ The distribution of birthweight of larger- and smaller-twin in the discordant twins group:65 (54.6％,65/119) larger-twins and one (0.8％,1/119) smaller-twin had birthweight above the 50th percentile of all twins,while 5 (4.2％,5/119) larger-twins and 97 ( 81.5％,97/119 ) smaller-twins got birthweight below the 10th percentile of all twins.Conclusions ( 1 ) The patterns of birthweight curves for each gestational week are different between twins and singletons.In order to evaluate the growth of twins, birthweight reference for twins shoull be employed.( 2 ) According to the reference of twins birthweight,the most discordant twins are complicated with fetal growth restriction at least in one twin.