The Study Of Dentritic Cells Transfected With SLC And Interleukin-2 Gene Aginst Bladder Tumor Cells

Objectives. To investigate whether dentritic cells (DC) transfected with secondary lymphoid tissue chemokine(SLC) and interleukin-2 (IL-2) genes is capable of improving the potency and efficiency of propagation and cytotoxicity against bladder tumor cells in vitro.Methods. SLC and IL-2 gene primer was designed basing on the corresponding gene sequence in GenBank. KpnⅠsite was introduced into the upstream of the primer and XhoⅠsite into downstream. The SLC gene was amplified with the template ofpET32a(+)-SLC (The plasmid is given by Biochemistry and MolecularBiology Laboratory of Third Military Medical University) .then SLC was cloned into pBudCE4.1-IL-2 vector(SLC was removed from pBudCE4.1- SLC-IL-2) to construct recombinant plasmid pBudCE4.1- SLC- IL-2 (The plasmid ispreserved in our Laboratory) . DCs were derived from 10 patients withNormal birth cord blood,Dendritic cell (DC) were transfected with pBudCE4.1/SLC- IL-2 by gene electric transfection.Protein expression was determined by Western blot and enzyme-linked immunosorbent assay. Cytotoxicity of T lymphocyte(TC) and DC_against the human bladder tumor cell were examined by chromium release assay(EC:TC=20:1). Flow cytometric analyses were performed to determine the apoptosis of tumor cells and the percentage of Treg.Results. Results. A high level of expression of the human SLC and IL-2 stable transfected DC was observed. The mean IL-2 production was 19.82±2.48, 511.10 ±52.36, and 549.05±62.02 ng/10~6 cells/24 hours in the DC/parental, DC/IL-2, and DC/SLC+IL-2 genes, respectively. The mean SLC production was 29.82±4.43, 506.10±42.36, and 567.34±52.05 ng/10~6 cells/24 hours in the DC/parental, DC/SLC, and DC/SLC+IL-2 genes, respectively The mean cytotoxicity (effector/target ratio 40:1) of CTL/parental, CTL/IL-2, CTL/SLC, and CTL/SLC+IL-2 against human bladder rumor cell lines in the percentage of cytolysis was 21.2%±4.8%, 32.1%±5.5%, 63.5%±6.6%, and 78.1%±9.63%, respectively.Conclusions. DC/SLC+IL-2 and DC/SLC were expanded by autocrine IL-2 and SLC. DC/SLC+IL-2 and DC/SLC showed significant cytotoxicity that was induced by SLC.IL-2 and CTLs,and demonstrated a potent cytotoxicity against bladder tumor cell lines...