The clinical application of delayed double knotting over the entire microvascular ananstomotic for free tissues transform has the advantages of direct vision and it easy approachs,is useful to avoid the opposite wall biting and other mistakes.It is especially feasible in the management of the cases with endangium denudation.

Objective To obtain abundant human betacellulin(BTC) with biological activity. Methods The whole mature protein coding sequence of BTC gene was amplified by polymerase chain reaction(PCR) method applied to human pancreatic ?-cell tumors cDNA.The fragment was cloned into prokaryotic expression vector pET32a(+) plasmid.The recombinant plasmid was transformed into E.coli BL21 and the fusion protein was expressed under isopropyl-beta-D-thiogalactopyranoside(IPTG).The fusion protein was purified by Ni2+ affinity chromatography.SDS-PAGE and Western blot were employed to determine the expression and purification of the expected protein.BTC was added to culture NIH3T3 cells for 5 days,and cell proliferation was detected by MTT. Results Lots of fusion protein were produced,and the purified protein can stimulate the proliferation of NIH3T3 cells. Conclusion The human BTC can be successfully obtained from the pET32a(+) system with the biological activity of stimulating the proliferation of NIH3T3 cells.

Objective To construct eukaryotic expression vector of human pancreatic duodenal homeobox 1(PDX-1) gene,and to detect its expression in NIH3T3 cell lines. Methods The whole coding sequence of PDX-1 gene was amplified by polymerase chain reaction(PCR) from human pancreatic-cell tumors cDNA.The fragment was inserted into eukaryotic expression vector pcDNA3.1 plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.The expression of PDX-1 gene in NIH3T3 cells was assayed by Western blot. Results The length of specific fragment amplified by PCR was 852 bp,and the recombinant plasmid pcDNA3.1-PDX-1 showed two bands of 5.5 kb and 852 bp by digestion using respective restriction enzymes BamHⅠand EcoRⅠ.The sequence of PDX-1 gene was approved or confirmed by blasting to GenBank.It was suggested that PDX-1 gene had been cloned into pcDNA3.1 vector correctly.Western blot showed that PDX-1 gene was expressed,which was detected 24 h after pcDNA3.1-PDX-1 plasmid was transfected into NIH3T3 cells. Conclusion The recombinant eukaryotic expression vector pcDNA3.1-PDX-1 was successfully constructed and expressed in NIH3T3 cell lines.

Objective To investigate the CT,MRI features and differential diagnosis of central nervous system primitive neuroec-todermal tumors (cPNET).Methods The CT and MRI findings of 1 5 cases with cPNET proved by pathology were analyzed retro-spectively,and we summarized the imaging features and differential diagnosis.Results For this group,the average age was (8.82± 2.53)and the male to female ratio was 9 ︰6.All lesions located in supratentorial region,which had relatively large volume (average diameter of 6.3 cm),cystic necrosis (12/15),and no or mild peritumoral edema.cPNET showed isodensity,slight hyperdensity or slight hypodensity on CT plain scan,and demonstrated uniform or inhomogeneous enhancement.On MRI plain scan,solid part showed isointensity or slight hypointensity signal on T1 WI,isointensity or slight hyperintensity signal on T2 WI,hyperintensity sig-nal on DWI(12/15),isointensity(9/15)or slightly hyperintensity signal on FLAIR,and showed obvious uniform,honeycombed or irregular enhancement after enhanced scan,no enhancement in cystic necrosis.Conclusion cPNET have certain characteristics,inclu-ding the lower onset age,relatively large volume with well-defined edge and no or mild peritumoral edema,hyperintensity signal on DWI,isointensity signal on FLAIR.

Objective: To apply fuzzy clustering analysis to determination of 10 inorganic elements and Cu/Zn ratio in 13 kinds of traditional Chinese medicine cured for vascular disease. Methods: By FAAS (flame atomic absorption spectrometry) sodium, calcium, magnesium, potassium, iron, chromium, nickel, manganese, copper and zinc were determined. By range analysis, correlation coefficients and relative fuzzy matrix were calculated.Results: Correlation coeffieients ? were mostly in the range of 0.55～0.80, especially ?=0.95 between Buzhangnaoxintong Capsule and Xinbao Pill. Conclusion: On condition that trace and macro elements are relatively rich and clustering correlation coefficients are larger, therapentic effects among traditional Chinese medicines cured for vascular disease are also similar. This suggests that trace elements, macro elements and Cu/Zn ratio have much to do with therapeutic effect.

Objective To investigate the effect of acupuncture combined with human amniotic mesenchymal stem cells ( HAMCs ) transplantation on the osteoporosis in ovariectomized rats.Methods Forty-five healthy female non-pregnant Wistar rats were randomly divided into sham operated group, model group and experimental group ( acupuncture+HAMC transplantation), 15 rats in each group.At 7 days after the surgical wound healing, the rats received acupuncture every day and intravenous injection of HAMCs suspension ( except the sham operated group) once a week for consecutive 12 weeks.The body weight was measured every week.24 hours after the last intervention, the rats were killed and specimens were collected.Specimens of the mid-, proximal-and distal femur were taken to measure the bone mineral density using a dual energy X-ray absorptiometer.At 12 weeks after the intervention, changes of the expression of serum 25-hydroxy-vitamin D (25-HVD), C-terminal peptide of type I collagen (CTX-I), bone specific alkaline phosphatase ( BAP) and human tartrate-resistant acid phosphatase 5b ( TRACP 5b) were determined by ELISA, the biomechanical properties of the removed femur was measured, and the expression of transforming growth factor-β( TGF-β) in the vertebrae was assessed by RT-PCR assay.Results Body weight of rats in the sham operation group was increased gradually in accordance with the natural growth of animals, that of the model group was significantly higher than that of the sham operated group since two months after modeling, and the body weight of the experimental group was similar to that of the sham operated group.The bone mineral density and calcium content of the model group were significantly decreased compared with that of the sham operated group ( P<0.05) , and the bone mineral density and bone calcium content of the experimental group were significantly higher than that of the model group ( P <0.05 ) .The ELISA showed that the expressions of serum 25-HVD, CTX I, BAP, and TRAP5b in the model group were significantly lower than that of the sham operated group, and those of the experimental group were significantly higher than that of the model group ( P<0.05 ) . Measurement of biomechanical properties showed that the limit load, limit stress and elastic modulus of the femur of the sham operated group were significantly higher than that of the model group ( P<0.05 ) , and the limit load and elastic modulus of the experimental group were significantly higher than that of the sham operated group ( P <0.05 ) .T-PCR showed that the expression of TGF-β1 m-RNA in the experimental group was significantly up-regulated than that of the sham operated and model groups ( P<0.05) .Conclusions Acupuncture combined with human amniotic mesenchymal stem cell ( HAMCs) transplantation has a synergistic effect on the treatment of osteoporosis, and can improve the conditions in ovariectomized rats.

Objective To discuss the clinical efficacy of closed reduction and plaster splint fixation ( CRPSF) in the treatment of gerontal patients with distal radius fractures ( DRF) .Methods 76 elderly patients with DRF who treated by CRPSF were selected .According to AO classification of fractures ,the patients were divided into three groups,the A group had 27 cases;B group had 26 cases,C group had 23 cases.The treatment effect was evalua-ted by analyzing the follow-up data,the corresponding imaging measurement parameters and clinical scores .Results All patients had 12-month clinical and imaging follow-up.In the last follow-up, the arm-shoulder-hand dysfunction score and palmar angle ,ulnar deviation angle of A and B group were significantly better than those of C group , the differences were statistically significant(all P<0.05),the difference between A and B group was not statistically sig-nificant (P>0.05).In the last follow-up,the satisfaction score of C group was slightly lower than that of the A or B group,but had no statistically significant difference (P>0.05).Conclusion CRPSF in the treatment of gerontal pa-tients with DRF has good function and imaging effects ,and the improvement level has a certain relationship with the degree of fracture,but has no significant impact on the patients'satisfaction.

This paper reviewed the studies on the effect of electroacupuncture in improving learning and memory functions in the past 10 years. It showed an overview of the mechanisms that how acupuncture worksed: promoting synaptic plasticity, regulation of microvascular injury and oxidative stress; slowing down the progress of brain cell apoptosis, inhibition of inflammatory response;regulating neurotransmitter release and protecting the ultrastructure of neurons. In addition, this paper puts forth the problems which left in the current studies.

OBJECTIVE To investigate the ESBLs produced by clinical strains of Enterobacter cloacae.METHODS Production of ESBLs was identified with modification of the double-disk test(MDDT).Eight kinds of primers(CTXM,CTXM-1,CTX-M-2,CTX-M-9,SHV,TEM,VEB,and PER)were used for the PCR amplification.Gene clone and DNA sequencing were performed then.RESULTS The result of MDDT was positive,amplicons were gained by PCR amplification with CTXM-1.DNA sequencing of amplicons of this strain revealed ESBLs of CTX-M-3.CONCLUSIONS ESBLs are the important reason for E.cloacae resisting to the third-generation cephalosporins.

AIM:To study the application of ultrasonic extraction in process of extracting diosgenin from Dioscorea zingiberensis C.H.Wright,and to evaluate its merits.METHODS:A new process of ethanol extracting and hydrochloric acid hydrolysis was provided to extraction diosgenin,and five different extraction methods were studied and compared,and the reaction mechanism was discussed.RESULTS:The ultrasonic extraction method was more efficient.Compared with the traditional water-hydrolylis,extraction rate was 15 percent higher,dosage of hydrochloric acid was registered 90 percent decrease,and the production was purer.CONCLUSION:The grinding pretreatment of Dioscorea zingiberensis is help to enhance the effects of ultrasonic extraction,the method has some advantages in efficiency,energy-saving and low pollution.