Objective:LAPTM4B is a novel gene which is over expressed in HCC.To evaluate LAPTM4B gene promoter on gene transcription,we construct luciferase reporter plasmid pGL3-LAPTM4B-Luc.Methods:The LAPTM4B cDNA was used as a template in the polymerase chain reaction to amplify its upstream regions from the translation initiation codon.The PCR product was directly cloned into the luciferase reporter vector pGL3-Basic.Results:Restriction enzymes digestion and nucleotide sequencing confirmed that the recombinant plasmids were correct without base mutation and deletion.Conclusion:A known gene promoter sequence could be freely obtained from Genbank database.This is very useful for the gene promoter cloning and promoter vector constructing.The construction of LAPTM4B recombinant plasmid lay a foundation of the analysis of promoter activities.

Objective To establish the assay method of hydroxyproline(HYP) level in rat lung tissue for evaluating the lung fi-brosis degree .Methods The impact of different acid hydrolyzable time ,oxidative time ,developing time on the assay results of the HYP level in rat lung tissue was studied .On this basis ,the assay method for determining the HYP level was established and prelim-inarily applied in the detection of the HYP level in rat lung tissue .Results The optimal condition for the HYP level detection in rat lung tissue was as follows :7 .50 mol HCL was hydrolyzed for 16 h at 110 ℃ ,oxidized for 10 min at room temperature and devel-oped for 25min at 60℃ .The sensitivity of this method was 0 .067μg/mL .The recovery rate and the average CV of this method were 88 .85% -110 .88% and 4 .70% -6 .60% ,respectively .In the study of bleomycin induced rat lung fibrosis ,the HYP level of the model group was obviously higher than that of the control group .Conclusion This method has high sensitivity ,high recovery rate and good reproducibility ,and may be used as a reliable quantitative method to judge the lung fibrosis level in clinic .

Objective To clarify the helical CT features and anatomic distribution of recurrence and metastasis of postoperative esophageal cancer.Methods Fifty patients ( 43 men , 7 women , ages 42~76 , mean 58 years ) with postoperative esophageal cancer underwent helical CT examination.On the basis of axial CT imaging , CT features were evaluated , including types , anatomic distribution and time of recurrence in patients with postoperative esophageal cancer .Results Among 50 patients , 31 patients developed recurrence and metastasis. Of 31 patients , 12 patients had recurrence of upper gastrointestinal tract , including esophagogastric anastomoses (n = 8), esophageal stump (n =1) , and intrathoracic stomach (n=3). Lymph node metastasis were investigated in 28 cases , which included right upper paratracheal (2R, n=11), and subcarinal right(7+8, n=11) ; 13 patients possessed lymph node metastasis of two and over-two regions ; Five patients had abdominal para-aortic lymph node metastasis. Metastasis of lung, liver and pancreas were discovered in 8, 7 and 1, respectively. There were six with pleural fluid . All of recurrence and metastasis occurred in the periods from 3 months to 1 year after surgery (P

Objective:Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro .Methods:PBMC were isolated from healthy EBV carriers and NPC patients, and then cocultured with autologous mature Ad5 LMP2A transfected DC at the ratios of 20∶1. Cytotoxicity of LMP2A specific CTL was determined with LDH release assay, the populations of CTL were performed by FACS,the IFN ? secretion and FasL mRNA expression of the CTL were detected by biological activity assays and RT PCR, respectively.Results:The results showed that high cytotoxicity of LMP2A specific CTL could be elicited by autologous transfected DC. The cytotoxicity boosted with the increase of transfected DC stimulation times, but there were no significant changes between two and three stimulations.The phenotypic analysis demonstrated that the LMP2A specific CTL induced at day 14 consisted of a majority of CD4 + and CD8 +T cells, and only a small percentage of CD56 + cells. The IFN ? secreted in the supernatants of cell culture also increased with the stimulation times. In addition, the specific CTL at day 14 from EBV healthy carriers could express FasL mRNA.Conclusion:Strong and functional EBV specific CTL could be generated by autologous mature DC transfected with adenovirus encoding LMP2A, which could provide a rationale for the immunotherapy of EBV associated NPC. [

Objective: To express the protein of HPV18E6 based on pET-32a(+) at high level and study the expression and significance of HPV18E6 proteins in laryngeal carcinoma. Methods: The HPV18E6 gene was amplified by PCR and cloned into pET-32a(+). The amplified fragment was inserted into the plasmid pET32a (+) that was digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET32/E6 was transformed into E.coli JM109 which was selected with ampicillin. The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the confusion protein. Finally, the optimization of expression conditions, such as temperature, concentration of IPTG, was studied. Results: The recombinant plasmids were identified and confirmed with enzyme digestion and sequencing. The BL21(DE3) transformed recombinant plasmid pET32/E6 had expressed HPV18E6 recombinant protein effectively. The optimum conditions of expression were 37 ℃, 1 mmol/L IPTG. Conclusion:Prokaryotic expression vector pET-32a(+)-HPV18E6 was successfully constructed. The high-level expression of HPV18E6 was achieved in E.coli BL21(DE3).

Objective To synthesize and identify artificial antigens of lung elastin degradation peptide and for the purpose of preparation of COPD test.Methods The artificial antigens were synthesized by Sulfo-SMCC and KLH.The complete antigens were identified by ultraviolet spectrum and SDS-PAGE.Immunize Balb/c mice was used to prepare antibody.The antiserum activity was evaluated by indirect competitive ELISA.Results The artificial antigens were identified by ultraviolet spectrum and SDS-PAGE. The protein concentration was 1.181 mg/mL.The titer of antiserum was 1∶64 000,and IC50 was 13.7 ng/mL.The antiserum had no cross-reaction with nonsense peptide.Conclusion The artificial antigens were acquired successfully,which had good immunoge-nicity.The results have laid basis for COPD test.

AIM:To evaluate the effects of treatment with HP 1188-immunoglobulin yolk ( HP1188-IgY) on Helicobacter pylori ( H.pylori)-infected gastritis in BALB/c mice.METHODS:BALB/c mice were used to establish an animal model of H.pylori-infected gastritis, and the mice were divided into 8 groups (10 mice per group).Oral antibiotics were used in group 1, 1 mg HP1188-IgY in group 2, 1 mg HP1188-IgY plus 30%sucralfate in group 3, 5 mg HP1188-IgY in group 4, 5 mg HP1188-IgY plus 30%sucralfate in group 5, PBS in group 6, and 30% sucralfate in group 7 with the treatment once per day for 10 d;and 2.5 mg HP1188-IgY was injected hypodermically twice with a 48-h interval in group 8.Another 10 mice were used as normal control in group 9.The planting of bacteria in the stomach was assayed by bacteri-al culture, rapid urease test, PCR and pathological sectioning .RESULTS:Intragastric administration with 1 mg HP1188-IgY plus 30%sucralfate per day effectively cured the injury of gastric mucosa caused by H.pylori infection, and the effect has no significant difference compared with antibiotics (P>0.05).CONCLUSION:We establish a BALB/c mouse mod-el infected with H.pylori successfully.Sucralfate (30%) is an ideal protectant for HP1188-IgY, which might decrease H. pylori infection in the stomach of BALB/c mice by oral inoculation .

BACKGROUNDTo study the effects of dendritic cells (DC) transfected with recombinant adenovirus encoding Epstein-Barr virus(EBV)latent membrane protein 2A(LMP2A)gene, and to provide evidence for further investigation on the therapeutic vaccine against EBV-associated malignancies.

METHODSDCs were transfected with EBV-LMP2A recombinant adenovirus (Ad5-LMP2A), which was generated by homologous recombination. The expression of LMP2A protein on mature DC transfected with Ad5-LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS), and the dead cells were counted by trypan blue staining. The alteration of surface markers on mature DC including CD1a, CD83, CD40, CD80, HLA?DR was detected by means of FACS before and after transfection. Meanwhile, the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H-thymidine uptake and fluorescent semi?quantitative polymerase chain reaction (PCR), respectively.

RESULTSAbout 80% mature DC expressed LMP2A protein and >92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection. Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL-12 P40 mRNA.

CONCLUSIONEBV-LMP2A gene,which was carried by adenovirus vectors, could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5-LMP2A.

Adenoviridae ; genetics ; Cells, Cultured ; Dendritic Cells ; physiology ; Gene Expression ; Genetic Vectors ; Humans ; Recombination, Genetic ; Transfection ; Viral Matrix Proteins ; genetics

OBJECTIVETo explore the effects of micro-elements fertilizers on the quality and yields of Artemisia annua.

METHODField experiments were conducted according to the method of random blocks design. After the harvest the yield was calculated and the content of artemisinin was determined.

RESULTBy applying 0.1%-0.5% Mn and 0.1%-0.5% Zn the dried leaf output and artemisinin content were increased.

CONCLUSIONThe suitable ranges of Mn and Zn can increased the yield and artemisinin content of A. annua.

Artemisia annua ; drug effects ; metabolism ; Artemisinins ; metabolism ; Boron ; pharmacology ; Manganese ; pharmacology ; Zinc ; pharmacology

ObjectiveTo investigate the clinical effect and safety of robotic versus laparoscopic splenectomy in the treatment of nontraumatic splenic diseases. MethodsAccording to the inclusion and exclusion criteria, PubMed, Web of Science, Embase, Cochrane Library, CBM, CNKI, Wanfang Data, and VIP were systematically searched for Chinese and English articles on the comparison of robotic splenectomy and laparoscopic splenectomy in the treatment of nontraumatic splenic diseases published up to March 2019. After quality assessment was performed for the articles included, RevMan 5.0 provided by Cochrane Library was used for analysis. Mean difference (MD) and rate difference (RD) were used as the effect indicators for continuous variables and binary variables, and pooled value and 95% confidence interval (CI) were calculated. ResultsA total of 7 studies with 374 patients were included, with 160 patients in the robotic splenectomy group and 214 in the laparoscopic splenectomy group. The results of the meta-analysis showed that compared with laparoscopic splenectomy, robotic splenectomy had significantly lower intraoperative blood loss (MD=-127.14, 95%CI: -199.87 to 54.42, P＜0.01), rate of conversion to laparotomy (RD=-0.06, 95%CI: -0.11 to 0.01, P=0.02), and rate of postoperative complications (RD=-0.10, 95%CI: -0.20 to 0.01, P=0.04). There were no significant differences in time of operation and length of hospital stay between the two surgical procedures (both P＞0.05). ConclusionBased on current evidence, robotic splenectomy has better clinical effect and safety than laparoscopic splenectomy in some aspects in the treatment of nontraumatic splenic diseases, and more multicenter large-sample randomized controlled trials are needed in the future for verification.