Objective The purpose of this study is to construct eukaryotic gene vector of herpes simplex virus type 1 thymidine kinase(HSV1-tk)and to observe the expression of HSV1-tk in lung adenocarcinoma AGZY cell line.Methods The full length HSV1-tk gene was amplified by PCR from plasmid pHSV 106 and was inserted into pMD18-T.The recombinant plasmid was recombined with eukaryotic vector plRES 2-EGFP u-sing gene recombinant technique .HSV1 -tk was transfected into adenocarcinoma AGZY cell line with Lipo-fectamineTM 2 000.Fluorescence microscopy was used to detect the transfection and expression of HSV 1-tk.RT-PCR was used to detect the mRNA levels of HSV 1-tk.The cell proliferation was measured by MTT assay .Re-sults A length of 1 130 bp gene sequence was obtained by PCR .The expressions of HSV 1-tk at mRNA and protein levels were displayed by RT -PCR and Western blot .MTT analysis showed that there were no significant changes cell survival on after transfection .Conclusion The eukaryotic expression vector of HSV 1 -tk report gene is successfully constructed and HSV 1-tk is effectively expressed in transfected AGZY cells .

AIM: To clone and express a human monoclonal anti-D Fab fragment in E. coli and make benefits for the expression of the whole immunoglobulin molecules of anti-D. METHODS: The gene of anti-D Fab fragment was cloned into the phagemid vector pComb3. After analyzing by PCR and restriction site analysis, the recombinant was expressed in E. coli and the expressed protein was analyzed by SDS-PAGE and ELISA. RESULTS: The result of SDS-PAGE confirmed that E.coli expressed a 48 kD protein. The ELISA result demonstrated that the cell culture supernatant reacted with Rh+ group O human erythrocytes, but was not recognized by Rh-group O human erythrocytes. CONCLUSION: Expressed Fab fragment has the antigenic specificity for human erythrocytes.

Objective To study the efficacy of hyperbaric oxygen(HO)for the delayed encephalopathy after acute carbon monoxide poisoning(DEACMP)Method One hundred and eleven patients who were diagnozed as the DEACMP from November 2000 to March 2007 in Tangdu Hospital the Fourth Military Medical University were randomly divided into two groups.Thirty-six cases were treated by onventional approach(group A),and 76 cases by HO besides conventional treatment(group B).The efficacy of HO was evaluated after 4courses of treatment. The curative effects were evaluated as(1)cured:clinical symptoms and signs fully disappeared,abnormal electroencephalogram recovered,patients were completely self-help and competent enough for routine work.(2)improved:chnical symptoms and signs partly disappeared,abnormal electroencephalogram partly recovered,patients were partial self-help and incompetent enough for routine work.(3)inefficacy:patient's condition didn't changed.Data were expressed as((x)±s)and analyzed with the chi-quare test and t-test.The statistical significance was established as P＜0.05.Results In group B,62(81.58%)were in good recover,9(11.84%)improved and 5(6.94%)were inefficacy;while in group A:21(58.33%)were in good recover,5(13.89%)were improved and 10(27.78%)were inefficacy.The effciency rate in group B was significantly higher(93.42%)than that(72.22%)in group A(P＜0.05),and the required time for the therapeutic effect noticed time in group B were significantly shorter(P＜0.05)Conclusions HO Can improve the therapeutic effects on DEACMP

OBJECTIVETo review the recent studies about human umbilical cord mesenchymal stem cells (hUCMSCs) and advances in the treatment of spinal cord injury. Data sources Published articles (1983 - 2007) about hUCMSCs and spinal cord injury were selected using Medline. Study selection Articles selected were relevant to development of mesenchymal stem cells (MSCs) for transplantation in spinal cord injury therapy. Of 258 originally identified articles 51 were selected that specifically addressed the stated purpose.

RESULTSRecent work has revealed that hUCMSCs share most of the characteristics with MSCs derived from bone marrow and are more appropriate to transplantation for cell based therapies.

CONCLUSIONSHuman umbilical cord could be regarded as a source of MSCs for experimental and clinical needs. In addition, as a peculiar source of stem cells, hUCMSCs may play an important role in the treatment of spinal cord injury.

Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Models, Biological ; Spinal Cord Injuries ; pathology ; therapy ; Stem Cell Transplantation ; Umbilical Cord ; cytology

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Humans ; Leptin ; chemistry ; genetics ; metabolism ; Molecular Weight ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism

Objective To clone and express the thioredoxin(Trx)from RH strain tachyzoites of Toxoplasma gondii,estab?lish the prokaryotic expression vector and purify the recombinant protein,then produce the polyclonal anti?Trx antibody in rab?bits. Methods Trx fragment was amplified by PCR and cloned into the pET?28a(+)vector,and the recombinant protein was in?duced with IPTG and purified by Ni?NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. Results The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx/pET?28a(+)was use?fully constructed,and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also ob?tained. The specific band of TRX was detected by Western blotting. Conclusion Western blotting can detect the specificity of polyclonal anti?Trx antibody,which will facilitate the biological functions of Trx.

Objective:To explore the clinical value of stereotactic wire-localized biopsy(SWLB)in digital mammography in the detection of microcalcification of the breast.Methods:A total of 45 patients with nonpalpable breast lesions which were positive for microcalcification by mammography but could not be detected clinically underwent SWLB.Their mammography fndings were analyzed in detail with pathology.Results:Among the 45 cases,13 cases(28.9%)had malignant lesions including ductal carcinoma in situ in 3 cases (20.1%),ductal carcinoma in situ with microinvasion in 4 cases(30.8%),invasive ductal carcinoma in 5 cases (38.5%)and intraductal papillary carcinoma in 1 case(7.7%).Thirty-two cases(71.1%)had benign lesions,2 cases(6.3%)of which were severe atypical hyperplasia.Conclusion:SWLB can accurately guide the surgical excision of nonpalpable breast microcalcification lesions and diagnose microcalcifications exactly,which is helpful for increasing the detection rate of eady-stage breast cancer.

Objective To evaluate the effect of various iterative reconstruction methods on phase analysis of gated myocardial perfusion imaging (MPI). Methods Thirty consecutive patients scanned by the Philips CardioMD system were recruited into this study. The gated SPECT (GSPECT) data were reconstructed with filtered backprojection (FBP),maximum likelihood expectation maximization (MLEM),three-di-mensional (3D) resolution recovery MLEM (AST),attenuation corrected (AC) MLEM,AC and 3D Monte Carlo scatter corrected (ACSC) MLEM methods. Parameters of left ventricular ( LV ) dyssynchrony ( phase standard deviation and histogram bandwidth) were measured using the software SyncTool. Paired t-test was used to compare the differences of the LV dyssynchrony indices between FBP and MLEM,AC MLEM,ACSC MLEM,AST respectively. Results The phase standard deviations of stress GSPECT MPI for FBP,MLEM,AC MLEM,ACSC MLEM,and AST were 11.6°,10.9°,11.2°,11.6°,11.4° respectively;while the histogram bandwidths were 35.7°,34.3°,35.1°,36.9°,35. 1 ° respectively. The phase standard deviations of rest GSPECT MPI for FBP,MLEM,AC MLEM,ACSC MLEM and AST were 15.2°,14. 5°,15.4° ,15. 4°,14.8° respectively; while the histogram bandwidths were 47.3°,46.4°,46.4° ,47.9°,46.1 ° respectively. No statistical significance was observed between the FBP and various iterative reconstruction methods for both the stress and rest GSPECT MPI study (t:-1. 179 to 1.554,P＞0.05 forall). Conclusion The standard FBP reconstruction method is accurate enough for the measurement of LV dyssynchrony indices using the widely used clinical software SyncTool.

Objective : To estimate the risk threshold of route index（RI）and mosq-ovitrap index（MOI）based on Breteau index（BI）,as supplements for dengue fever risk monitoring in specific habitats. Methods : Two towns and two streets were selected from nine towns（streets）in Jiashan County,and then one village（community）was selected from each of them as a Aedes albopictus monitoring site. The BI,RI and MOI were employed at the same time and area from April to October in the year 2018. Linear regression models were built with RI,MOI and BI to calculate the dengue risk threshold of RI and MOI according to BI. Results : The linear regression model of BI（X）and RI（Y）was Y=0.145+0.662X（P<0.05）,of BI（X）and MOI（Y）was Y=3.423+0.524X（P<0.05）. If BI=5（having risk of transmission of dengue fever）,then RI=3.455（95%CI：1.717-5.198）,MOI=6.043（95%CI：-0.327-12.417）. If BI=10（having risk of outbreak）,then RI=6.765（95%CI：5.018-8.518）,MOI=8.663（95%CI：2.260-15.071）. If BI=20（having risk of epidemic）,then RI=13.385（95%CI：11.326-15.453）,MOI=13.903（95%CI：6.352-21.461）. Conclusion The dengue fever risk threshold of RI estimated by BI had a narrow 95%CI and could be applied for dengue fever risk assessment,while the risk threshold of MOI had a wide 95%CI and the application value needed further study.

BACKGROUNDThe Ras/Raf/ERK1/2 signaling pathway controls many cellular responses such as cell proliferation, migration, differentiation, and death. In the nervous system, emerging evidence also points to a death-promoting role for ERK1/2 in both in vitro and in vivo models of neuronal death. To further investigate how Ras/Raf/ERK1/2 up-regulation may lead to the development of spinal cord injury, we developed a cellular model of Raf/ERK up-regulation by overexpressing c-Raf in cultured spinal cord neurons (SCNs) and dorsal root ganglions (DRGs).

METHODSDRGs and SCNs were prepared from C57BL/6J mouse pups. DRGs or SCNs were infected with Ad-Raf-1 or Ad-Null adenovirus alone. Cell adhesion assay and cell migration assay were investigated, DiI labeling was employed to examine the effect of the up-regulation of Ras/Raf/ERK1/2 signaling on the dendritic formation of spinal neurons. We used the TO-PRO-3 staining to examine the apoptotic effect of c-Raf on DRGs or SCNs. The effect on the synapse formation of neurons was measured by using immunofluorescence.

RESULTSWe found that Raf/ERK up-regulation stimulates the migration of both SCNs and DRGs, and impairs the formation of excitatory synapses in SCNs. In addition, we found that Raf/ERK up-regulation inhibits the development of mature dendritic spines in SCNs. Investigating the possible mechanisms through which Raf/ERK up-regulation affects the excitatory synapse formation and dendritic spine development, we discovered that Raf/ERK up-regulation suppresses the development and maturation of SCNs.

CONCLUSIONThe up-regulation of the Raf/ERK signaling pathway may contribute to the pathogenesis of spinal cord injury through both its impairment of the SCN development and causing neural circuit imbalances.

Animals ; Cell Movement ; physiology ; Dendritic Spines ; metabolism ; physiology ; Female ; Ganglia, Spinal ; cytology ; MAP Kinase Signaling System ; physiology ; Mice ; Neurogenesis ; genetics ; physiology ; Neurons ; cytology ; Pregnancy ; Signal Transduction ; genetics ; physiology ; Spinal Cord ; cytology ; Synapses ; metabolism ; physiology ; Up-Regulation ; raf Kinases ; genetics ; metabolism ; ras Proteins ; genetics ; metabolism