Construction of the Bifidobacterium infantis -mediated TK/GCV suicide gene therapy system
Herpes simplex virus - thymidine kinase (HSV - TK) gene was PCR amplified and subcloned into pGEX-5X-1, at BamH I and Sal I sites (Takara, Tokyo, Japan), resulting in pGEX-TK. Potential recombinants were first screened by bacterial colony PCR, followed by DNA sequencing verification. After verification, pGEX-TK plasmid was used to transform electrocompetent Bifidobacterium infantis bacterial cells via electroporation, as previously reported [6–11].
Sprague-Dawley (SD) rats (6-8 weeks age, female, weighing 200-250 g) were housed at the Laboratory Animal Center of Chongqing Medical University, Chongqing, China. The animal experiments followed institutional guidelines for the use and care of animals. Animals were housed in microisolator cages under a specific pathogen-free (SPF) condition with 12-hour light-dark cycles.
Bacterial strains and cultivation
Bifidobacterium infantis (Sangon, Shanghai, China) was provided by Molecular Biology Laboratory of Chongqing Medical University. Bifidobacterium infantis bacterial cells were inoculated in MRS (De Man, Rogosa and Sharpe medium) liquid medium, and grown in an anaerobic tank with a mixed-gas (80% N2, 10% CO2, 10% H2) at 37°C overnight.
Establishment of a rat model of bladder cancer andexperimental groups
A rat model of bladder tumor was induced by using MNU (USA, Jersey, Sigma). Fifty four tumor-bearing SD rats were randomly divided into three groups: the normal saline group (n = 18), the Bifidobacterium infantis-pGEX-5X-1 (n = 18), and the Bifidobacterium infantis-pGEX-TK (i.e., BI-TK) group (n = 18). Each group was given tail vein injection of saline, Bifidobacterium infantis-pGEX-5X-1, or Bifidobacterium infantis-TK (containing 4.4 × 109 Bifidobacterium infantis), once every week for two weeks. Each group also received daily intraperitoneal injection of ganciclovir (GCV) (50 mg/kg, Merck, Darmstadt, Germany) for 14 days. On the 15th day after treatment, all rats were sacrificed by the overdose of ketamine (400 mg/kg) and xylazine (50 mg/kg) and necropsy was performed. Total weight of bladders was determined (see below). Tumor tissues were retrieved and embedded in 4% paraformaldehyde for hematoxylin-eosin (H & E) staining.
Determination of bladder total weight
After the rats were sacrificed, the bladders were retrieved by severing the jugular, urethra near the bladder neck and double ureter close to bladder wall. The bladder anterior wall was opened for examining bladder tumor formation; and the liquid was dried with filter papers, The total weight of the bladder was then determined for all animals in the study.
Apoptosis of bladder tumor cells determined by TUNEL assay
The TUNEL assay was carried out according to the manufacturer's instructions (TUNEL kit; Roche, Darmstadt, Germany). Apoptotic cells (approximately 100 cells/field for three non-overlapping fields) were counted. Apoptosis index was calculated as the percentage of apoptosis cells over total counted cells.
Immunohistochemical staining of Caspase3 protein expression in bladder tumor cells
Immunohistochemical staining was conducted according to manufacturer's instructions (Zhongshan Golden Bridge Inc, Shanghai, China). The tumor sections were probed with a biotinylated anti-Caspase 3 antibody, followed by incubation with strapavidin-horseradish peroxidase. The presence of Caspase 3 protein was visualized by adding horseradish peroxidase substrate diaminobenzidine solution. The cells were counterstained with hematoxylin. Positively staining cells were documented under a light microscope and quantitatively analyzed by the Image-Pro Plus Analysis system (Olympus, Tokyo, Japan) from at least five high power fields. The average value of the intensity of positive staining was defined as positive reaction area/field area.
All the experimental data were processed using the SPSS11.0 software. The number of samples of analysis of variance was determined by using SN-K method. α = 0.05.