Objective To construct prokaryotic expression vector carrying Japanese encephalitis virus (JEV) NS1 gene, and to express the vector in E. coli, so to lay a foundation for the further development of JEV early diagnosis kit. Methods The NS1 gene was amplified by RT-PCR, the target gene and prokaryotic expression plasmid pET28a(+) were digested by BamH I and Hind Ⅲ respectively. The target gene was then purified by DNA extraction kit. A 1∶3 molar ratio of vector: insert DNA ligated with T4 DNA ligase to construct the recombinant plasmid pET28a(+)-NS1. The ligated products were transformed into E. coli BL21 (DE3) and the colonies were selected on LB medium with karamycin. After cultivation, positive colonies were picked out and the recombinant plasmid were identified by endonuclease digestions, PCR rand sequencing. The target protein was expressed with induction of IPTG. The expressed proteins mentioned above were then identified and analyzed by SDS-PAGE and Western-blotting respectively. Results The sequencing results of amplified products showed that JEV-NS1 RNA fragments were about 1 300bp in length which were similar as respected. Compared with the published sequence of SA14-14-2 with Blast, the homology of the nucleotide sequence was 100%. The molecular weight of expressed protein was about 45kD, the result of Western blotting proved the specific antigenicity of the protein. Conclusion The specific JEV nonstructural protein 1 is expressed in E. coli successfully and shows high specificity to the antibody. The stable expression of the protein and the analysis of its antigenic specificity provide foundation to develop the early stage diagnosis kit.

BACKGROUND:In the process of cardiac stem cel culture in vitro, the growth microenvironment may have some effects on the cel proliferation.
OBJECTIVE:To investigate the possible mechanism of proliferation and migration of rat cardiac stem cel s cultured in vitro.
METHODS:Cardiac tissues from 10 Sprague-Dawley rats were obtained for primary culture and subculture. Passage 3 cel s were col ected for immunofluorescence staining, and stem cel growth factor receptor (c-kit) and CD45, CD90 were detected. Cultured tissues were col ected and randomly divided into two groups:in group 1, paraformaldehyde fixation, paraffin embedding, hematoxylin-eosin staining, Masson staining, and detecting apoptotic cel s using TUNEL method were conducted;in group 2, EdU labeling of proliferated cel s, immunofluorescent detection of c-kit positive expression, matrix metal oproteinases 2, 9 and transforming growth factorβ1 using immunofluorescent staining were done.
RESULTS AND CONCLUSION:After 7-10 days of myocardial tissue culture, bright and round cel s were visible, and after adhesion, fusiform cel s exhibited strong growth and proliferation ability. Immunofluorescence staining showed a large number of c-kit, CD45 positive cel s but CD90 negative cel s. After culture, a great number of newborn cel s were found, accompanied by apoptosis of myocardial cel s. After EdU staining, the positive cel s were distributed in the myocardial gap, and showed a smal amount of matrix metal oproteinases 2, 9 and transforming growth factorβ1, while in the surrounding myocardium there was a large number of matrix metal oproteinases 2, 9 and transforming growth factorβ1. Taken together, our findings show that cardiac stem cel s could be obtained through myocardial tissue culture in vitro, accompanied by cel proliferation and migration. The mechanism is related to the expression of matrix metal oproteinases 2, 9 and transforming growth factorβ1.

Objective To present the diagnosis and treatment of functional bladder outlet obstruction and to assess the results of transurethral bladder neck incision and alpha-blockers with regard to symptoms and urodynamic findings. Methods From October 1995 to October 2002,39 male patients (age range from 24 to 48 years,with a mean of 37 years) who had dysuria and underwent urodynamic examination,cystourethrography and urethral exploration were diagnosed with functional bladder outlet obstruction.The mean IPSS was 22.5.The mean maximum urinary flow rate was 10.2 ml/s and the mean residual volume was 124 ml.All the patients were treated with transurethral incision of bladder neck and alpha blockers. Results The mean operative duration was 15 min;mean blood loss was 50 ml;mean postoperative hospital stay was 3.5 d.During 1-year follow-up,most of the patients were satisfied with the treatment results.Subjective assessment showed a statistically significant reduction of the voiding complaints.The mean IPSS was 10.1 .The mean maximum urinary flow rate was 22.1 ml/s (range,12.7 to 42.1 ml/s) and the mean residual volume was 49 ml (range,0 to 84 ml). Conclusions Urodynamic examination,voiding cystourethrography and urethral exploration with dilator facilitate the diagnosis of functional bladder outlet obstruction.Treatments with transurethral incision of the bladder neck and alpha-blockers are effective and safe for functional bladder outlet obstruction.

Background The identification of mesenchymal stem cells(MSCs) have provided exciting prospects for cell-based regeneration after myocardic infraction.However cell therapy have inherent limitations such as low survival rate of transplanted cells and insufficient cell number.It is known that cell-matrix adhesion plays a key role in cell proliferation,differentiation and survival,and chemokine CXCL12 may involved in these prcesses.Transfected mesenchymalstem cells with CXCL12 for local secretion of CXCL12 and then explored CXCL12 triggered adhesion of mesenchymal stem cells to extracellular matrix proteins.Mesenchymal stem cells was transfected with CXCL12.?V and ?3 integrins content was evaluated by Western blot analysis.Cell adhesion to extracellular matrix was examined in vitro and cell prolife-ration after transplantation in vivo.Transfection of CXCL12 resulted increased CXCL12 in situ.Increased CXCL12 induced elevated adhesion to fibronectin in vitro and higher survival in vivo.CXCL12 mediated adhesion and proliferation was established by ?V and ?3 integrin subunits.Chemoattractive mechanisms are involved in adhesion processes of mesenchymal stem cells.Increased CXCL12 leads to enhanced expression of ?V and ?3 integrins,which may augment cell survival,proliferation and differrentiation.

Age Factors ; Cardiopulmonary Bypass ; Child ; Child, Preschool ; Humans ; Infant ; Infant, Newborn ; P-Selectin ; blood ; Platelet Glycoprotein GPIb-IX Complex ; analysis

Objective To investigate the effects of different duration of sevoflurane anesthesia in the neonatal period on the long?term cognitive function and hippocampal synaptic plasticity in rats. Methods Twenty?four pathogen?free healthy Sprague?Dawley rats of both sexes, aged 7 days, weighing 12-16 g, were divided into 3 groups ( n=8 each) using a random number table: control group ( group C) , sevoflu?rane anesthesia for 2 h group ( group S1 ) , and sevoflurane anesthesia for 6 h group ( group S2 ) . Group S1 and group S2 inhaled 2% sevoflurane for 2 and 6 h, respectively. Morris water maze test was performed at 30 days after the end of anesthesia ( postnatal day 37) to assess the cognitive function. After the end of the test, the rats were sacrificed, and hippocampi were isolated for determination of the expression of brain?de?rived neurotrophic factor ( BDNF) , postsynaptic density?95 ( PSD?95) and synapsin 1 in hippocampal tis?sues by Western blot. Results Compared with group C, the escape latency on 4th and 5th days of the test in group S1 and on 2nd-5th days of the test in group S2 was significantly prolonged, and the frequency of crossing the original platform was significantly decreased, and the time of staying at the platform quadrant was significantly shortened in S1 and S2 groups, the expression of BDNF, PSD?95 and synapsin 1 in hipp?ocampal tissues was significantly down?regulated in group S2 (P<0?05), and no significant change was found in the expression of BDNF, PSD?95 and synapsin 1 in hippocampal tissues in group S1 ( P>0?05) . Compared with group S1 , no significant change was found in the escape latency and frequency of crossing
the original platform (P>0?05), the time of staying at the platform quadrant was significantly shortened, and the expression of BDNF, PSD?95 and synapsin 1 in hippocampal tissues was significantly down?regula?ted in group S2 ( P<0?05) . Conclusion Short?time and long?time sevoflurane anesthesia both can induce long?term cognitive dysfunction in the neonatal period, and the severity is aggravated with prolonged anes?thesia; the partial mechanism is related to inhibition of the synaptic plasticity of hippocampal neurons of rats.

Objective To investigate the effect of imbedding chemotherapy of sustained release of 5-fluorouracil on the healing of colonic stoma in dog. Methods Twenty-eight adult hybrid dogs were randomly divided into chemotherapy group (n=22) and control group (n=6). The canine sigmoid colon were firstly detached and then anastomosed via median abdominal incision, 200 mg sustained release of 5-fluorouracil was imbedded in the mesentery 1.0-1.5 cm away from colonic stoma in chemotherapy group, whereas the control substance was injected into the dogs in control group. Tissue samples were collected from mesentery and stomas on 3, 5, 7, 10 and 15 days after operation, respectively, in order to observe the healing of stoma. The drug concentrations in the stoma and in the tissues that were 0, 1, 3, 5, 7, 10 and 15 cm away from the imbedding point were also measured by high performance liquid chromatography method at different phases. Results The tissues from colonic stoma only showed inflammatory reaction at early stage, with no necrosis and cellular degeneration. It was observed that the stoma healed basically on the tenth day after operation. The drug concentrations in the tissues gradually decreased at the range of 0-15 cm over time, but all of which were higher than the anti-tumor effective concentration (0.10 ?g/g). Conclusion The imbedding chemotherapy of sustained release of 5-fluorouracil in mesentery has little effect on the healing of stoma, and it could remain an effective anti-tumor concentration in a period of time.

Objective:To construct a recombination plasmid containing a kanamycin resistance gene,the upstream and downstream fragment of luxS of Streptococcus mutans so that luxS can be knock out by transforming the plasmid into S.mutans later.Methods:Kanamycin resistance gene,the upstream and downstream of luxS were cloned respectively by using plasmid pEGFP-N1 and DNA of Streptococcus mutans as template.Then the genes were ligated into Multiple Cloning Site(MCS) of vector pMD19-T in certain order and transformed into E.coli Competent Cells.Finally transformants were selected for resistance to kanamycin and ampicillin.Results:Kanamycin resistance gene and the upstream and downstream of luxS were successfully ligated into accurate enzyme digestion site of vector pMD19-T,and restriction digests analysis and sequencing result was correct.Conclusion:LuxS gene knock-out of Streptococcus Mutans recombinant plasmid is constructed and built a base of constructing Streptococcus Mutans luxS mutans in the future.

Objective:To study the effects of dexamethasone(DEX)on the cytotoxicity and apoptosis of NK-92MI cells and the mechanisms involved.Methods:NK-92MI cells were treated with different doses of DEX.The proliferative rate and cytotoxicity of the NK-92MI cells were detected by MTT colorimetry.The cell apoptotic rate was observed by flow cytometry with Annexin V and propidium iodide(PI)double staining.The expression of apoptosis-related gene,Bcl-2 and Bax was detected by RT-PCR.Results:After treated with 1?10-8mol/L to 1?10-3mol/L of DEX for 24 h,48 h and 72 h,the proliferation of NK-92MI cells was significant inhibited(P

Objective To study the effects of high albumin, high glucose and low bovine serum on the phenotypic transformation of renal tubular epithelial cells. Methods The normal human kidney proximal tubular eel] line (HKC) was cultured for 30 days in the presence of high albumin (1.5 g/L), high glucose(25 mmol/L) and low bovine serum(2% ) . Morphological changes were observed by electronic microscopy. Immunohistochemistry stain was used to examine the expression of cytokeratin, vimentin, a-SMA, collagen Ⅰ and TGF-pl protein. Western blot was applied to further detect the process of collagen I protein expression, and in situ hybridization was used to examined the expression of collagen Ⅰ gene. Results Renal tubular epithelial cells cultured in high albumin, high glucose and low bovine serum showed obvious morphologic changes, including elongated shape, decrease of microvilli and mitochondria, and increase of rough endoplasmic reticulum under electronic microscopy. Immunohistochemistry stain revealed the reduction of cytokeratin, and enhancement of vimentin, ?-SMA, TGF-?1 and collagen Ⅰ. Western blot demonstrated that the expression of collagen Ⅰincreased in a time-dependent manner, and in situ hybridization showed that collagen type Ⅰ mRNA increased as well. Conclusion High albumin, high glucose and low bovine serum induce phenotypic transformation of renal tubular epithelial cells into mesanchymal cells.