Objective To construct the prokaryotic expression plasmid of human prostate stem cell antigen (PSCA),to induce the expression of GST-PSCA fusion protein in E. coli BL21,and to identify the purified recombinant fusion protein. Methods The fragment of PSCA gene was amplified by PCR,and then cloned into the pGEM-T Easy vector. The transitional plasmid Teasy-PSCA was identified by DNA sequencing. The PSCA gene was digested from the plasmid Teasy-PSCA by restrictive enzyme BamH I and Sal I,and then inserted into the pET42a vector which contains a glutathione s-transterase (GST) tag. Following the double restriction enzyme digestion,the recombinant plasmid pET42a-PSCA was obtained and transformed into E. coli BL21 (DE3). The expression of GST-PSCA fusion protein was induced with IPTG. The recombinant fusion protein was purified by passing over a Glutathione Sepharose 4B column,and was identified by SDS-PAGE and Western blotting. Results The length of amplified PSCA gene fragment was consistent with that expected,and the sequence was correct as exemplified by the PSCA gene reported in GenBank. The result of enzyme digestion indicated that the prokaryotic expression plasmid pET42a-PSCA was successfully constructed. After transformation with pET42a-PSCA and induction with IPTG,the recombinant target protein of about 43kD was obtained. The GST-PSCA fusion protein was correctly identified by SDS-PAGE and Western blotting. Conclusions The prokaryotic expression plasmid of human PSCA gene has been successfully constructed. The GST-PSCA fusion protein may express and be purified in E. coli BL21,and it lays a foundation for further study on the anti-prostate cancer gene vaccine.

Resident standardization training is an important means of clinical physician training in our country. Critical care medicine has important status in the training process. It is the important link to ensure the quality of resident standardization training. Residents should grasp the identification and early detection of critical ill patients. Residents should also get the ability of general basic management for critical condition and the doctor-patient communication ability. In practice, we have explored the training mode of standardized training of resident doctors in critical care medicine by developing detailed training outline, a variety of teaching methods and emphasizing the cultivation of clinical work ability.

Objective To expand the influence and promotion effect of the grassroots health demonstration base of appropriate technology at county level,explore the practice model for full coverage.Methods Four consortium and eight units in the county were engaged into the whole process,the whole cycle,synchronous implementation;the promotion practices were divided into different stages with different focuses based on priority setting;Stratified training,classified promotion strategies were involved to carry out the appropriate technology for all 11 items covered.Results The technical promotion training,technical promotion applications were completed with full coverage in the county,gained high satisfaction from both medical staff and public.Enhanced the technology radiation ability,also the base's annual development was increasing year by year.Conclusions The base construction full coverage promotion experiences can be shared and learned by other areas which aims for the promotion of fit health techniques.

OBJECTIVETo obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 (VEGFR2) fusion protein (mVEGFR2D1-4/GST) and identify its antiginicity and biological activity.

METHODSThe gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 (DE3) strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells (HUVECs) were evaluated in in vitro cell cultures.

RESULTSThe mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro.

CONCLUSIONThe mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.

Animals ; Cell Proliferation ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; isolation & purification

OBJECTIVETo amplify mouse prostate stem cell antigen (mPSCA) gene and construct a recombinant plasmid to obtain mPSCA protein and identify its antigenicity.

METHODSThe gene of mPSCA was amplified by RT-PCR from mouse prostate cancer cell line RM-1 with the signal peptide sequence removed. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mPSCA, which was transformed into BL21 (DE3) for mPSCA expression. The fusion protein was purified and identified by SDS-PAGE and Western blotting. The antigenicity of the purified protein was characterized by ELISA.

RESULTSThe mPSCA gene was obtained with an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mPSCA was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mPSCA protein.

CONCLUSIONThe purified mPSCA obtained possesses good antigenicity, which will facilitate further study of immunotherapy for prostate cancer targeting PSCA.

Animals ; Antigens, Neoplasm ; genetics ; immunology ; isolation & purification ; Cloning, Molecular ; Escherichia coli ; metabolism ; GPI-Linked Proteins ; genetics ; immunology ; isolation & purification ; Genetic Vectors ; Male ; Mice ; Neoplasm Proteins ; genetics ; immunology ; isolation & purification ; Plasmids ; Prostate ; cytology

OBJECTIVETo construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.

METHODSHuman IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.

RESULTSEnzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.

CONCLUSIONA novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.

Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; Electroporation ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Interleukin-12 ; genetics ; immunology ; Mice ; Plasmids ; Transfection

Objective: To understand the epidemic status and influencing factors of Mongolian children with ASD in central and eastern Inner Mongolia, so as to provide data support for formulating prevention and intervention strategies and improving the overall epidemiological investigation of ASD in Inner Mongolia. Methods: Sixteen kindergartens and primary schools were selected from Chifeng City, Ulanqab City, Tongliao City, Hulunbuir City and Xilingol League cities in Inner Mongolia by means of random cluster sampling. Firstly, 7 108 children aged 3-14 were initially screened with the Kirschner Autism Behavior Scale(CABS), and then the children with ASD positive were given the autism behavior test scale (ABC). According to the diagnostic criteria, the professionals, including chief physicians and associate chief physicians from the major of child psychiatry, diagnosed ASD with the total score of ABC scale ≥62. Univariate and Logistic regression multivariate analysis were carried out among Mongolian children to find out the influencing factors related to the occurrence of Mongolian ASD in Inner Mongolia. Results: The prevalence of Mongolian children was 0.37%. Mongolian ASD group and Mongolian normal children series in the household register, habitual twitch, hyperactivity, bite lips, families have extreme introverts, mothers age, father s cultural level, cultural degree of mother, father mother mild character, irritable, neonatal diseases, fetal gestational age distribution had statistical significance( χ 2/Z= 12.58 , 16.68, 14.93, 64.43, -3.76, -2.86, 4.57, 11.12, 12.33, 16.66, P <0.05). Conclusion Measures such as shaping a healthy growth environment, adjusting parental style, paying attention to the level of early childhood language development, and preventing neonatal diseases might lower the risk of ASD in children.

Objective: To understand the prevalence and influencing factors of children with ASD in central and eastern Inner Mongolia, and to provide theoretical basis for disease prevention and prevalence of ASD. Methods: Sixteen primary schools and kindergartens were selected from 5 cities in central and eastern Inner Mongolia through random cluster sampling. A total of 15 817 children aged 3-14 years were selected. Children who were positive using Clancy Autism Behavior Scale were further diagnosed according to the teacher s nomination form and the Autism Behavior Checklist, as well as the diagnostic criteria of the fifth edition of the American Diagnostic & Statistical Manual of Mental Disorders by 2 professionals. Results: The prevalence of ASD was 0.27% (42/15 817), with prevalence in urban areas (0.16%, 15/9 231) higher than that of rural areas (0.41%, 27/6 586) ( χ 2=8.89, P <0.01). Logistic regression analysis showed that maternal education and language development were negatively associated with ASD in urban children [ OR =0.29(95% CI =0.12-0.69) and 0.18(95% CI =0.05-0.60), P <0.05]. ASD in rural children were positively associated with enuresis and introverted family members [ OR =7.09(95% CI =1.60-32.27) and 8.63(95% CI =3.10- 24.01 ), P <0.05]. Conclusion High prevalence of ASD is found in urban area of central and eastern Inner Mongolia. Unhealthy habits, neonatal diseases, low parental education, delayed language development and poor exercise performance are primary factors associated with ASD in both urban and rural areas.

Objective To investigate the feasibility,safety and efficacy of transcatheter drug-loaded microsphere embolization(DLME)in treating patients with advanced bladder cancer with bleeding(ABCB).Methods A total of 26 ABCB patients who underwent DLME for tumor supply arteries were retrospectively selected.The postoperative efficacy and related complications were observed.The recurrence of hematuria and survival situation were followed up.Results All 26 surgeries achieved success with a technical success rate of 100.0％.There were 21 cases(80.8％)of bilateral bladder artery embolism and 5 cases(19.2％)of unilateral bladder artery embolism.Three days after the operation,24 patients(92.3％)had hematuria remission.And the other two patients(7.7％)had no hematuria remission,they were relieved after interventional embolization again.Compared with that before operation,the blood transfusion rate,blood transfusion volume,hematocrit and hemoglobin at one week after operation were significantly improved(P＜0.05).One month after the last intervention,there were 2 cases of complete response,19 cases of partial response,3 cases of stable disease,and 2 cases of progressive disease.The objective remission rate was 80.8％,and the disease control rate was 92.3％.Compared with that before operation,the T stage was significantly improved at one month after operation(P＜0.05).No patients had severe complications such as ectopic embolism.After follow-up for 3-36 months,5 cases(19.2％)had a recurrence of hematuria.Conclusion Transcatheter DLME is feasible,safe,and effective in the treatment of patients with ABCB.It is an optional,minimally invasive palliative measure.

Objective To explore the clinical effect of endovascular stent-graft exclusion in the treatment of acute Stanford B-type aortic dissection(AD)with distal left subclavian artery(LSA)rupture accompanied by arch intramural hematoma.Methods A total of 12 patients with acute AD treated by the endovascular stent-graft exclusion alone were retrospectively selected.All patients had primary rupture at the distal end of LSA with arch intramural hematoma and received endovascular treatment after 2 to 3 weeks of conservative treatment.The technique success rate and related complications were observed.Postoperative computed tomography angiography(CTA)of the aorta was reviewed to evaluate the remodeling of the true and false lumen,the absorption of the arch intramural hematoma,to observe whether there was a new rupture and endoleak,and to understand the position and shape of the stent and the blood supply of the branch arteries.Results All patients completed the operation with a technical success rate of 100％.Two patients partially blocked the opening of LSA,and one patient had type Ⅰ internal leakage after the operation,but the amount of internal leakage was small and was not treated.Other patients did not had serious complications such as aortic rupture,new rupture,paraplegia,stent displacement,stroke,upper limb ischemia or vertebral artery ischemia,internal leakage,and abdominal organ ischemia during and after the operation.The patients were followed up for 19 to 66 months,with an average follow-up of(36.7±13.9)months.During the follow-up period,no patient died.The aortic remodeling was satisfactory in all patients,the arch intramural hematoma was absorbed,and there were no new rupture,internal leakage,upper limb ischemia or vertebral artery ischemia,or other serious complications.One patient with type Ⅰ internal leakage showed no significant change in internal leakage after regular postoperative reexamination.Conclusion Endovascular stent-graft exclusion is safe and feasible in the treatment of acute AD patients with distal LSA rupture accompanied by arch intramural hematoma,and it is worth promoting and applying clinically.