Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.

Objective To observe the transfection efficiency and anti-fibrotic effect of miR-29b transfected by anti-TGF-β Ⅱ R ScFv/Ck/tP fusion protein (new vector) in hepatic stellate cell (HSC),and to provide a new vector in gene therapy for liver fibrosis.Methods The liposome vector,new vector,and lentiviral vector were used as transfection reagents to transfect miR-29b into HSC.Transfection efficiency was observed under fluorescence microscope and flow cytometry.Collagen α1 (Ⅰ) mRNA and protein expression in different groups were analyzed by real-time RT-PCR and Western Blot,respectively.Results Compared to the control,transfection efficiencies in lentiviral vector,new vector,and liposome vector groups were about 70％,58％,and 29％,respectively.Collagen α1 (Ⅰ) mRNA expression in lentiviral vector,new vector,and liposome vector groups was decreased by about 70％,50％,and 38％,respectively ((t =6.316,P ＜0.01 ; t =4.082,P ＜0.01 ; t =3.014,P ＜0.05).Collagen α1(Ⅰ) protein expression in lentiviral vector,new vector,and liposome vector groups was decreased by about 59％,41％,and 27％,respectively (t =4.209,P ＜0.01; t =4.033,P ＜0.01; t =2.842,P ＜0.05).Conclusions The new vector constructed by us has a high transfection efficiency.MiR-29b transfected by the new vector has a good anti-liver fibrosis effect.

AIM:To investigate the target killing effect of T lymphocytes with chimeric CD20scFv gene on Daudi cells and the activation of T lymphocytes.METHODS:Two kinds of plasmids were transfected into retrovirus-packed PA317 cell lines.The supernatant was collected from successfully transfected PA317 culture and was used to infect peripheral blood T lymphocytes.After one-week screening with G418,the cells were used to kill Daudi and K562 cells.The positive rates of AnnexinⅤ in Daudi cells were measured at different times points respectively by flow cytometry.Meanwhile,the level of IL-2 and IFN-? were determined by ELISA.RESULTS:The Annexin V positive rate was significant higher in Daudi cells compared to control K562 cell lines at 24 h.No difference of AnnexinV in Daudi cells was observed in CD20 modification T lymphocyte groups.The secretions of IL-2 and IFN-? in CD20scFv-CD80-IgGFc-CD28-? gene modified T cells co-cultured with Daudi cells were dramatically higher than that in CD20scFv-IgGFc group at 72 h.CONCLUSION:① The two kinds of genetic modified specific T cells have no significant difference in inducing early apoptosis of Daudi cells.CD28-? can't affect Daudi cell early apoptosis at the CD20scFv target killing.② The increase in the secretions of IL-2 and IFN-? is more obvious in CD20scFv-IgGFc-CD28-? group,indicating that the self-activation takes place in CD3? and CD28 modified T cells without MHC restriction and then increases the activation and killing function of T cells.

Objective To study the decitabine inhibits the proliferation and induces apoptosis of AML1-ETO+ leukemia cells and to explore its possible mechanism. Methods Kasumi-1 cells were commonly cultured in RPMI-1640 medium containing 10％ fetal bovine serum. The proliferation of Kasumi-1 cells was determined by CCK8 assay. Flow cytometry was used to detect Kasumi-1 apoptosis. Related protein expression was measured by Western Blot. The expressions of AML1-ETOand miR-193a were measured by RT-PCR. Results Decitabine could inhibit the proliferation of Kasumi-1 cells with concentration effect and time effect, and could induce apoptosis. The apoptosis rates of control group, 24 hours and 48 hours group were (5. 29±0. 88)％ and (9. 83±1. 71)％ and (19. 47±1. 84)％, respectively. Decitabine could decrease the expression of AML1-ETOprotein, and the AML1-ETOprotein ratios of 0. 1, 0. 5 and 1 μmol/Ldecitabine group to the control group were (0. 85±0. 21), (0. 28土0. 06) and (0. 10±0. 07). The ratios of AML1-ETOprotein of 24 hours group, 48 hours group to control group were (0. 31±0. 21) and (0. 24±0. 11). But decitabin did not affect the expression of AML1-ETOmRNA, and the ratios of AML1-ETOmRNAof 24 hours group and 48 hours group to control group were (0. 96±0. 19) and (0. 84±0. 11). The difference was not significant(F=1. 22, P＞0. 05). Decitabine could up-regulate the expression of miR-193a by (3. 61士0. 06) and (6. 99±0. 74) fold respectively at 24 and 48 hours, and decreased the expression of MDM2 and Cyclin Dl. The protein expression ratios of MDM2 and Cyclin D1 of dosing group and control group were (0. 51土0. 19) and (0. 50±0. 10), respectively. Conclusion Decitabine inhibits the proliferation and induces apoptosis of AML1-ETO+ leukemia cells by up-regulating miR-193a to repress the translation of AML1-ETOand inhibiting the expression of MDM2 and Cyclin Dl.