In this study, we evaluated the tissue distribution and safety of IBM-BMT applied Ad-EGFP-MDR1 in short term. The BMCs were infected with the adenovirus vector with multiplicity of infection (MOI) 50
in 30 μl, and transplantated in tumor-bearing Balb/c mice. Serum total adenovirus antibody, LY2874455 mouse serum adenovirus neutralizing factor (SNF), hematological, histopathology and distribution of human MDR1 were determined to make sure the extent of response caused by this treatment. Materials and methods Mice Balb/c mice (female and male are half-and-half, 16 g-18 g of weight) were purchased from animal center of ChongQing Medical University and maintained under specific pathogen-free conditions until use in animal facilities. Their housing, care, diet and maintenance conformed to the guidelines of China, the “”regulation for the care and Geneticin mw use of laboratory animals”". Cell lines The murine colon carcinoma cell line CT26 and human embryonic kidney (HEK) 293 cell lines (SIBCB, China) were cultured at
37°C and 5% CO2 in RPMI 1640 (Gibco, America) medium, containing 10% fetal calf serum (PAA, Austria). Preparation of donor BMC and IBM injection of BMCs The BMCs were collected from the femurs and tibias of Balb/c mice and injected via IBM, as described in, and then cultured at a density of 2 × 105 cells/ml in IMDM media supplemented with bovine serum albumin and L-glutamine. Cultures were stimulated with a combination of the cytokines, thrombopoietin (10 ng/ml), flt3-ligand (10 ng/ml), stem cell factor (20 ng/ml), granulocyte colony-stimulating factor (15 ng/ml), interleukin (IL)-3 (10 ng/ml) and IL-6 (25 ng/ml). (R&D, America). Cells were populated two days after primary culture, and co-cultured with PDK4 Ad-EGFP-MDR1 (kindly presented by professor Tong-Chuan He, EGFP:enhanced green fluorescence protein) (MOI 50) for two days. The cultured cells were washed by phosphate buffered sodium (PBS) for
three times and harvested. Total RNA of cells was isolated and qualitatively analyzed with a reverse transcription polymerase chain reaction (RT-PCR) kit (Takara, Japan). For MDR1: forward primer: AAAGCTGTCAAGGAAGCCAA, reverse primer: ACTCCATCATCGAAACCAGC. For beta-actin, forward primer: AAGTGTGACGTTGACATCCG, Reverse primer: GAAAGGGTGTAAAACGCAGC. Reverse transcriptase reaction was performed with PCR conditions were as follows: 94°C for 2 min (1 cycle); followed 94°C for 20 sec, 55°C for 1 min, 72°C for 30 sec (30 cycle); followed 72°C for 10 min(1 cycle). P-gp activity was determined by using the daunomycin efflux assay and detected by fluorescence microscope. P-gp in BMCs was investigated by selleck products western bolt. BMCs were washed once with PBS and lysed in lysis buffer.