Objective To investigate the changes in biologic behavior of dendritic cells (DCs) after being modified by human telomerase reverse transcriptase (hTERT) gene. Methods In order to obtain the hTERT gene modified DCs, DCs were transfected with a replication-deficient recombinant adenovirus expression vector of hTERT. Then the expression to hTERT and PCNA was assessed by by Western blot, mature markers on DCs surface were detected by flow cytometry, and the proliferative capacity was determined by MTT method. Results Immunohistochemical staining and Western blotting showed that the expression of hTERT was upregulated obviously in DCs after being modified by hTERT gene. Flow cytometry indicated that the expression of CD83 and CD86 remained unchanged. The DC growth curve showed that the number of DC-hTERT was increased slightly in the first 2 weeks, meanwhile the number of DC/rAd-LacZ and DC was decreased obviously. Although the number in 3 groups was all decreased in the third week, the number of DC/rAd-hTERT was still greater than the other control groups. It was also found that the expression of PCNA was increased in DC/rAd-hTERT compared with that of immature DC, mature DC or DC/rAd-LacZ by Western blot. Conclusion After rAd-hTERT modification, life span of DC in vitro is extended and proliferative capacity is enhanced.

Objective To study the significance of expression of c-met protein in gastric cancer tissue and to explore the correlation between c-met and micro-vessel density (MVD). Methods c-met protein and MVD in gastric cancerous tissue were determined by immunohistochemistry in 47 patients. Results The positive rate of c-met in gastric tissue was 85.1%. The expression of c-met was much higher in patients with tumor diameter larger than 5cm, with deeper invasion, regional lymph nodes metastasis, lymph vessel and venous invasion, and TNM stage Ⅲ-Ⅳ (P

Objective To construct a replication-deficient recombinant adenovirus expression vector of human heparanase (hpa). Methods The hpa gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pDC315 in sense direction, and the resultant recombinant plasmid pDC315-hpa was transfected into HEK293 cell together with plasmid pBHGlox (deltaE1,3) containing adenoviral genome, then the replication-deficient recombinant adenovirus expression vector of hpa (Ad-hpa) was obtained, and it was identified by infection test, electronic microscope observation and PCR amplification. Results After purification and concintration,the titer of Ad-hpa reached 5?10 10pfu/ml. Virus particles could be found in virus concintration solution, and replication of virus was observed in HEK293 cells was observed under transmission electron microscope. Both adenovirus and hpa special fragment could be amplified from Ad-hpa by PCR, whereas hpa special fragment could not be amplified from the control. Conclusion The replication-deficient recombinant adenovirus expression vector of hpa was constructed successfully. This study established a foundation for further study on hpa vaccines and gene therapy for carcinoma.

Objective To study the impact of hepatitis B virus (HBV) infection on the activity of cord hematopoietic stem cells. Methods CD34+ cells were isolated from healthy human cord blood by miniMACS. Cells were cultured in IMDM complete culture medium containing stem cell factor (SCF),fms-like tyrosine kinase 3 ligand (FL), thrombopoietin (TPO), interleukin-3 (IL-3) and 10% fetal bovine serum. High copies HBV were added to the culture system. The proliferation of stem ceils and virus replication were observed. Following the proliferation, dendritic cells (DCs) were induced by adding granulocyte-macrophage colony-stimulating factor and IL-4. Morphous of stem cells and DCs were observed by microscope and the cell surface molecules were detected. Results The proliferation of stem cells infected with HBV was significantly lower than that of healthy stem cells (P＜0.01),and enhanced after adding cytokines (P＜0.01). At the same time, HBV replication was increased after adding cytokines in the culture system (P＜0.01), but the proliferation was still lower than that of healthy stem cells with cytokines in the culture medium (P＜0.05). Dane particles were found in the cytoplasma of stem cells infected with HBV by electron microscope. The expression of CD80,CD86 ,CD1a and HLA-DR on DCs derived from HBV infected stem cells were all lower than those on DCs from non-infected stem cells (P＜0.01). Conclusions HBV could infect CD34+ stem cell and the proliferation of the stem cell could enhance the virus replication. HBV could not only inhibit the proliferation of stem cells,but also down-regulate the immuno-phenotype expression of DCs derived from CD34+stem cells.

Objective To systematically review the efficacy and safety of postoperative radiotherapy and postoperative chemotherapy for patients with endometrial cancer,which may give support for clinical proper selection.Methods The randomized controlled trials (RCTs)comparing postoperative radiotherapy with post-operative chemotherapy for patients with endometrial cancer were searched in EMBase,PubMed,Cochrane Library,Chinese Biomedical Literature Data,China National Knowledge Infrastructure,and VIP database from the inception to August 201 5.Two reviewers independently assessed the quality of included studies and extrac-ted data.We analyzed the statistic data using RevMan 5.1 software.Results Three RCTs concluding 1 1 21 patients were included.Meta analysis showed that there were no significant differences between the two groups in five-year survival rate (RR =0.94,95%CI:0.80-1 .1 0,Z =0.77,P =0.440),five-year progression-free survival rate (RR =0.98,95%CI:0.90-1 .07,Z =0.52,P =0.61 0)and recurrence rate (RR =1 .06, 95%CI:0.91 -1 .24,Z =0.75,P =0.450),but there were significant differences between the two groups in grade 3-4 thrombocytopenia (RR =0.1 3,95%CI:0.07-0.27,Z =5.62,P <0.000 01 )and grade 3-4 neu-tropenia (RR =0.01 ,95%CI:0.00-0.03,Z =8.27,P <0.000 01 ).Subgroup analysis showed that there were significant differences between the two groups in five-year survival rate (RR =0.79,95%CI:0.68-0.91 , Z =3.1 5,P =0.002)and five-year progression-free survival rate (RR =0.82,95%CI:0.69-0.97,Z =2.31 ,P =0.020)for patients with Ⅲ-Ⅳ stage endometrial cancer.Conclusion Current evidence indicates that compared with postoperative radiotherapy,postoperative chemotherapy may improve the survival rate for pa-tients with advanced stage endometrial cancer.The long-term curative effects still need to be confirmed by RCTs with high quality and large sample.

OBJECTIVETo study the HBsAg transient expression in HepG2 or COS-7 cells with eukaryotic expression plasmids inserting HBsAg gene (pCI-S and pcDNA3.1-S) and the efficacy of naked DNA immunization in mice.

METHODSFirstly, the recombinant plasmids of pCI-S and pcDNA3.1-S were constructed by the cloning technique and the accuracy of these constructs was confirmed by restriction enzyme digestion and DNA sequencing. Secondly, plasmids of pCI-S and pcDNA3.1-S were transferred into HepG2 and COS-7 cells, respectively by means of cationic liposome. HBsAg transient expression was assayed by ELISA in cell culture supernatants and cell lysates. Thirdly, plasmids were injected into quadriceps muscles of BALB/C mice and serum samples were obtained from individual immunized or control mice 4 weeks after injection and boost injection, respectively. Anti-HBs were assayed in mice sera by ELISA. HBsAg-specific CTL responses of spleen cells from immunized mice were tested by the LDH method.

RESULTSPlasmids of pCI-S and pcDNA3.1-S allowed HBsAg transient expression in cell culture supernatants and cell lysates of HepG2 or COS-7 cells. Intramuscular immunization of BALB/C mice with plasmids of pCI-S or pcDNA3.1-S elicited the antibody and cytotoxic T lymphocyte responses to HBsAg.

CONCLUSIONSThe vectors used in this study are effective to induce prime antibody and HBsAg-specific-cytotoxic T lymphocyte responses to HBsAg in mice after intramuscular immunization.

Animals ; COS Cells ; Cloning, Molecular ; DNA, Viral ; genetics ; Eukaryotic Cells ; metabolism ; Female ; Gene Expression ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis, Viral, Animal ; immunology ; prevention & control ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Cells, Cultured ; Vaccines, DNA ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

Objective:To investigate the effect of heat shock protein 90 (Hsp90) inhibitor PU-H71 combined with X-ray on radioresistant human cervical cancer cells.Methods:The expression levels of Hsp90 gene between cervical cancer tissues and adjacent tissues were analyzed by bioinformatics. Radioresistant cervical cancer cell lines HeLa RR and SiHa RR were obtained by fractional irradiations (2 Gy per fraction, 30 fractions). The cell lines were divided into the control group (treated with dimethyl sulfoxide), irradiation alone group, PU-H71 group (treated with 0.5 μmol/L PU-H71), and PU-H71+irradiation group (irradiation at 24 h after treatment with 0.5 μmol/L PU-H71). Cell survival was detected by clonal formation assay. Immunofluorescence assay was used to detect γH2AX foci at 1, 6, and 24 h after cell treatment. The expression level of Rad51 protein at 1, 2, 6, 12, and 24 h after cell treatment was detected using Western blot. The expression level of phosphorylated DNA-dependent protein kinase catalytic subunit (p-DNA-PKcs) was measured at 2 h after cell treatment. Cell apoptosis at 48 h after cell treatment was assessed by flow cytometry. Results:PU-H71 enhanced the sensitivity of radioresistant cervical cancer cells to X-ray. Compared with the irradiation alone group, the radiation sensitization ratios (SER) of HeLa RR and SiHa RR cells at 10% survival were 1.36 and 1.27, and the apoptosis rates were increased by approximately 72.1% and 63.1% in the PU-H71+irradiation group, respectively. PU-H71 delayed the duration of γH2AX foci induced by X-ray, inhibited the phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus preventing non-homologous end joining (NHEJ) repair and delaying homologous recombination repair.Conclusion:PU-H71 increases the radiosensitivity of radioresistant cervical cancer cells by inhibiting the repair pathway of DNA double-strand break, which is expected to be a radiosensitizer to enhance the efficacy of radiotherapy for cervical cancer.