, Ltd., and were bred in the specific pathogen free (SPF)Animal Center, School of Life Science, University of Science and Technology of China. Establishment of a multi-drug resistance cell model based on nude mice liver implantation and subcutaneous implantation A total of 20 male nude mice aged 4-6 weeks were used. Ten mice were anesthesized by an intraperitoneal injection with chloral hydrate (430 mg/kg). A transverse incision was performed under the xiphoid process. A 0.2-ml

Bel-7402 cell suspension (density equal to 1 × 108/ml) was injected into the parenchyma of the right hepatic lobe and the abdomen was closed. The ten mice were randomly divided into the liver implantation experimental group or the control group with equal members (n = 5 for each group). Another 10 animals were subcutaneously injected with 0.2-ml Bel-7402 cell suspension (density AG-014699 in vivo equal to 1 × 108/ml) into the find more right anterior axilla. they were also randomly divided into experimental and control groups (n = 5 for each group). All animals were bred in SPF condition. On the third day, nude mice

in the experimental groups underwent an intraperitoneal injection with ADM at a dose of 1.5 mg/kg each week for 8 weeks. Mice in the control groups underwent an intraperitoneal injection with an equal volume of normal saline solution. Skin reaction, appetite and psychological status were recorded according to the observation in each day. The tumor volume was calculated by the following formula: V = πab2/a (“”a”" represents the long diameter from of the tumor, “”b”" represents the short diameter of the tumor). When the experiment was completed, the nude

mice were sacrificed, the tumor was obtained and levigated in asepsis. A 0.25% trypsin solution was used to digest the cells for 2-3 min and to produce a mono-cell suspension. Cells were inoculated in a 25-ml sterile culture flask for primary culture. After multiple passages and purification, the hepatocellular implantation drug-resistant cell sub-lines Bel-7402/ADML (liver-implanted induction) and the subcutaneous implantation drug-resistant cell sub-lines Bel-7402/ADMS (subcutaneous-implanted induction) were obtained. Tumor tissue was fixed with 1% osmium tetroxide, learn more embedded in resin, and cut into ultra thin sections. After uranyl acetate and citric acid double staining, the sections were observed by an transmission electron microscope (Zeiss 902). Establishment of a multi-drug resistance model by in vitro induction The ADM concentration gradient progressive increase induction method was applied. Bel-7402 cells at a concentration of 5 × 105/ml in the logarithmic phase were inoculated in a 25-ml culture flask and cultured for 24 h. The culture solution was replaced with an ADM culture solution at a low concentration (0.01 μg/ml). After the 24-h culture, the solution containing drugs was discarded. Cells were digested with 0.25% trypsin and centrifuged at 1000 rpm for 3 min.