Objective To analyse the encoding gene sequence of extended spectrum ? lactamase produced by Klebsiella pneumoniae E95 strain in Zhejiang Province and identify its ESBLs subtype. Methods The gene of ESBLs produced by E95 strain was amplified by PCR. The purified PCR product was cloned into pGEM Teasy vector and then sequenced by Sanger's dideoxy chain termination composition method. Results The encoded gene of ESBLs was identified as SHV by PCR. Its PCR product had 812 nucleotides. It had the same gene sequence as the gene encoding SHV 12 discovered in Swiss. Conclusions The ESBLs produced by Klebsiella pneumoniae E95 strain isolated from a patient in Zhejiang Province is SHV 12.

Objective To investigate the effect of resistance nodulation division (RND) efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii.Methods Totally 631 isolates of Acinetobacter baumanii were collected from 16 hospitals in 7 provinces in 2010.Genes oxa-51 and oxa-23 were detected by PCR method,and the ST profiles were determined by multilocus sequence typing (MLST).The disk susceptibility assay was used to determine the inhibition zone diameters of β-lactams,aminoglycosides,macrolides,tetracyclines,carbapenems,tigecycline and polymyxin.The minimum inhibitory concentration (MIC) of tigecycline was determined by E-test in strains with inhibition zone diameters ≤ 12 mm on tigecycline.The expression of operon genes adeB,adeG and adeJ was determined with efflux pump inhibitor NMP (N-methyl-2-pyrrolidone) for detection of efflux pump inhibitor phenotype.The isolates of Acinetobacter baumanii which were resistant both to tigecycline and carbapenems and with the inhibited phenotype of efflux pump inhibitor were collected as the experiment group,the isolates which were susceptible to tigecycline but resistant to carbapenems were collected as the control group,and ATCC 19606 was used as the reference strain.The expressions of adeABC,adeFGH and adeIJK were quantified by q-PCR at the transcriptional level.Genes adeR,adeS and adeL were amplified and sequenced using PCR method to find polymorphic locus and insertion sequences.Results There were 32 isolates of Acinetobacter baumanii with reduced susceptibility to tigecycline and carbapenem-resistant.Eight isolates were with the inhibited phenotype by efflux pump inhibitor.And 4 strains which were susceptible to tigecycline but resistant to carbapenems were selected as the control.The expressions of adeABC in A518,Z1219 and A527 of experiment group were 13-fold,5-fold and 7-fold higher than reference strain ATCC19606,respectively.The expressions of adeFGH and adeIJK were up-regulated slightly in some isolates.Transcript of adeABC was not found in control group strains A207 and A1731,and the expressions of adeABC,adeFGH and adeIJK were not up-regulated in other isolates.The single nucleotide polymorphism (SNP) was detected in adeR (E220K) and adeS (A130D),respectively.ISAab1 insertion sequence was identified in adeS of adeABC-over expressed isolates.No mutation was found in adeL.Conclusion High expression of adeABC pump may play an important role in tigecycline resistance in carbapenem-resistant Acinetobacter baumannii,mainly due to the insertion of ISAba1 sequence in its regulator gene adeS,but other mechanism of tigecycline resistance may not be excluded.

Objective:To construct and evaluate a novel tissue-engineered bone composed of murine stromal cell-derived factor 1(mSDF-1), simvastatin (SIM) and collagen scaffold (Bio-Oss?), serving as a cell-homing approach for bone formation .Methods: In the study , 32 ICR mice were randomly divided into 4 groups,each group including 8 mice.The drug-loaded collagen scaffolds were implanted subcutaneously onto the cranium of each mouse according to the groups: ( 1 ) 1 ∶50 ( volume ratio ) dimethyl sulfoxide ( DMSO ) /phosphate-buffered saline ( PBS ) solution +collagen scaffold ( blank control group ); ( 2 ) 10 -3 mol/L SIM solution +collagen scaffold ( SIM group ); ( 3 ) 200 mg/L mSDF-1solution +collagen scaffold (mSDF-1 group); and (4) 10 -3mol/L SIM +200 mg/L mSDF-1 solution +collagen scaffold ( SIM +mSDF-1 group) .One week after implantation , the mice were trea-ted by injecting the same drug solution mentioned above around the scaffold once a day for two days .The specimens were harvested 6 weeks after implantation and the bone formation was evaluated by soft X-ray analysis , HE staining and immunohistochemical staining .Angiogenesis of each group was checked by calculation of vessels in each tissue section .Results:Six weeks after implantation , the collagen scaffolds were retrieved.The value of gray scale for the SIM +mSDF-1 group[(421 836.5 ±65 425.7) pixels] was significantly higher than that of the blank control group [(153 345.6 ±45 222.2)pixels, P<0.01], the SIM group [(158 119.2 ±100 284.2) pixels, P<0.01], and the mSDF-1 group[(255 529.5 ± 152 142.4) pixels, P <0.05 ]; HE staining analysis revealed that significant bone formation was achieved in the SIM +mSDF-1 group; The immunohistochemical staining showed the existence of os-teopontin and osteocalcin in the SIM +mSDF-1 group; There were more vessels in the SIM +mSDF-1 group[(46 ±8)vessels/mm2] than in the blank control group [(23 ±7) vessels/mm2, P<0.01], and the SIM group[(24 ±6) vessels/mm2 , P<0.01].Conclusion:The novel tissue-engineered bone com-posed of mSDF-1, SIM and collagen scaffolds has the potential to form bone subcutaneously in vivo.It re-presents a novel method of in vivo bone re-generation without seed cell delivery .

Objective:Human adipose-derived stem cells (hASCs)are a highly attractive source in bone tissue engineering.To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs ) in vitro,and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo.Methods:The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct.Then,the OCpro-Luc-Puro construct together with three assistant vectors:pM-DLg/pRRE,pRSV-REV,and pVSVG,were transiently transfected into HEK293T cells followed by viral supernatants collection,filtration and concentration.Next,the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection.The OCpro-Luc-hASCs were then cultured in the absence or presence of osteo-genic differentiation medium.On the 7th and 1 4th days,after osteogenic induction,cellular extracts were collected and analyzed by luciferase reporter assay.Meanwhile,alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR)analysis of osteogenic associated genes osteo-calcin (OC),runt-related transcription factor 2 (Runx2)and alkaline phosphatase (ALP)were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs.Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated.On the 7th and 1 4th days after osteogenic induction,the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased.Consistently, the extracellular matrix mineralization,as shown by Alizarin red S (ARS)staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC,Runx2 and ALP, although to variable extent,was observed upon osteogenic differentiation.These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs.Conclusion:We established a luciferase reporter system that could be used to rapidly,quantitatively and specifically determine osteo-genic differentiation ability of hASCs.Comparing with the traditional time-consuming methods,the system we generated here was highly effective.This system not only can be used to examine ostogenic differentia-tion of hASCs in a high throughput manner,but also provides a way to monitor ostogenic differentiation of cells in vivo.

Objective To investigate the expressions of metastasis suppressor 1 (MTSS1 )and calcium activated protein 43 (Cap43)in esophageal squamous cell carcinoma (ESCC)tissue,and to clarify the relationship between the expressions of MTSS1,Cap43 and the clinicopathological features of ESCC.Methods 80 cases of ESCC tissue and 30 cases of normal adjacent-cancer tissue were collected,and the protein and mRNA expressions of MTSS1 and Cap43 in ESCC tissue and normal tissue were detected by streptavidin-perosidase (SP)immunohistochemistry and RT-PCR;their relationships with the clinicopathological features of ESCC were analyzed.Results The positive expression rate of MTSS1 in normal esophagus tissue was significantly higher than that in ESCC tissue detected by SP (83.3%vs 21.3%,P<0.01)and RT-PCR (0.703±0.085 vs 0.295±0.065,P<0.01),However,the positive expression rate of Cap43 in normal esophagus tissue was significantly lower than that in ESCC tissue by SP method (16.7%vs 76.3%,P<0.01)and by RT-PCR (0.236±0.052 vs 0.693±0.078,P<0.01).The mRNA expression levels of MTSS1 and Cap43 in ESCC tissue were significantly related with the invasive extent, histological differentiation,TNM stage,and lymphatic metastases (P<0.05)of ESCC,but not related with the age,sex,tumor size and pathological type (P>0.05). The mRNA expression of MTSS1 was negatively correlated with the expression of Cap43 (r=-0.457,P<0.05).Conclusion The low-expression of MTSS1 and over-expression of Cap43 in ESCC tissue may contribute to tumor invasion and metastasis;the imbalance of MTSS1 and Cap43 may be one of the mechanisms of tumor invasion and metastases.

OBJECTIVE To identify the antibiotic resistance,homology and the carbapenemases determinants of imipenem-resistant strains of Acinetobacter baumannii isolated from elderly in the Zhejiang Hospital.METHODS All 142 strains of A.baumannii were isolated from Zhejiang Hospital through Jan 2005 to Jan 2007.K-B method was used to screen imipenem-resistant strains.The MICs of imipenem-resistant strains to 14 antimicrobial agents were determined by agar dilution method.The homology of these isolates was analyzed by pulse-field gel electrophoresis(PFGE).The coding genes of carbapenamases and the gene environments were investigated by PCR,clone,and sequencing.RESULTS Ninety-seven strains of imipenem-resistant A.baumannii were isolated from 142 strains.All of the strains of carbapenem resistant A.baumannii belonged to 4 epidemic PFGE-clones.Ninety carbapenem resistant strains contained OXA-23-like carbapenemase gene and 91 isolates were positive for OXA-51-like gene. OXA-23-like gene of 86 strains was just on the down-stream of insert sequence ISAba1.OXA-51-like gene of 6 strains had an ISAba1 sequence just on the up-stream.CONCLUSIONS All imipenem-resistant strains of A.baumannii are pan-resistant isolates.Clone dissemination is the most important style of strains spread.No OXA-24-like,OXA-58like,IMP-like,and VIM-like gene are detected.OXA-23-like and OXA-51-like gene are the most popular carbapenemases coding genes of these strains in the Zhejiang Hospital.ISAba1 has close relationship with OXA type carbapenemases genes in Zhejiang Hospital.

Objective To investigate the effects on anti-expression of Osteopontin (OPN) by an antisense oligodeoxynucleotide (ASODN) targeting to low free energy region of OPN mRNA and its effects on the proliferation and apoptosis of human breast carcinoma cell line MCF-7. Methods Designing and synthesizing an ASODN based on minimum free energy algorithm in vitro,which targets to the low free energy region of OPN mRNA. Transfecting it into breast carcinoma cell line MCF-7 which expresses OPN in high level. The cell proliferation-inhibitory rate was determined by MTT method;The morphologic change was observed through transmission electron microscope (TEM);The OPN mRNA expression level was checked by RT-PCR method;Cell cycle and apoptosis rate were detected by flow cytometry (FCM) after transfection respectively. Results OPN ASODN inhibited the proliferation of the cells by a time and concentration dependent manner (P

Objective To investigate the prevalence of the genotypes of extended spectrum ? lactamses (ESBLs) in klebsiella pneumoniae and eschrichia coli, which were isolated in Hangzhou City from 2000 to 2001. Methods Total of 198 isolates of E. coil and K. pneumoniae were isolated from five hospitals in Hangzhou. ESBLs-producing isolates were detected by inhibitor-potentiated disc diffusion test, Agar dilution method was used to determine the minimum inhibitory concentrations (MIC) of cefotaxime and ceftazidime.ESBLs genes were analysed by isoelectric focusing (IEF) assay and PCR initially,genotypes were determined by sequencing. Results 51 isolates (25.76%) from 198 E. coil and K. pneumoniae strains were obtained. In ESBLs producers, the ESBLs types of SHV, TEM, CTX-M were 17.65%,84.31% and 80.39% respectively. genotypes were: SHV 11,12;TEM 1;CTX M 3and CTX M 3 like, CTX M 13,14. respectively. Conclusions CTX M type were the major genotypes of ESBLs in klebsiella pneumoniae and eschrichia coli isolated in Hangzhou city.

Objectives To investigate the properties and distributions of ampC gene among different drug-resistant strains of Serratia marcescens,and the relationship of control gene ampR with AmpC enzymes′ expressions.Methods According to the results of inducting experiment with 1/2 MIC of beta-lactam antibiotics (CTX),three-dimensional testing and isoelectric focusing electrophoresis testing,143 strains of S.marcescens were classified into three groups:including induction group, continuous low-production group and hyperproduction group. In each group, the sequences of ampC and ampR genes were amplified using the method of PCR. The products of PCR were analyzed. The plasmid-mediated beta-lactamases were detected using the method of conjugation experiment.Results Among 143 strains of S.marcescens, the continuous low -production strains, induction strains and hyperproduction strains were 14,103,and 18, respectively.125 and 99 strains were ampC and ampR gene positive, respectively.The detection rate of ampR in hyperproduction group was lower than other groups.5 sites of ampC genes and 4 sites in the Open Reading Frame (ORF) of ampR gene were easily mutated in 5 induction strains and 2 hyperproduction strains.Conclusions The production of inducing drug-resistance of some S.marcescens might be related to mutation of ampC gene encoding AmpC beta-lactamases and the ORF mutation in ampR. The continuous hyperproduction drug-resistance had something to do with deletion mutation in ampR in segmental hyperproduction strains.The plasmid-mediated AmpC enzymes hadn′t been found in S.marcescens.

Early detection and treatment of high-risk adenomas and colorectal cancer (CRC) can reduce mortality of this disease.CRC screening is aimed at minimizing its harm and colonoscopy is presently the gold standard for it.However,colonoscopy needs bowel preparation and is invasive with high risk of intestinal perforation,causing a bad compliance,which is unfavorable to its popularization and application.Recently,non-invasive detection methods for CRC have gone through a rapid development.Tests based on CRC-related biomarkers in fecal and blood samples provide new options for non-invasive CRC screening.However,detection methods for these biomarkers still need further research and improvement because of the complex composition of feces and blood.In the two aspects of fecal tests and blood tests,the progress of recent studies on non-invasive screening methods for CRC was reviewed in this article.