H f 10% Fetal K Calf serum, 100 U / ml penicillin streptomycin at 37 ° C with 5% CO second THP xenograft tumor models 1 All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Henan Province, People, People’s Republic of China. M Nnlich athymic BALB / c nude, 4, 5 weeks old, were obtained from the animals Vital River Company. Five Mice Per K Housed in a pathogen-free facility fig for all experiments. All Mice were treated with CO60, 2.7 Gy per mouse 24 hours before inoculation with irradiated THP 1 cells and then subcutaneously 1107 脳 THP 1 cells in the tumor volume reached right flank.When 100,300 mm injected 3, wherein the tumor BALB / c were Nacktm Mice Feeder llig into four groups with five M mice in each group divided. Intraperitoneal injections of SC 203 048 203 048 SC alone PTL alone in combination with PTL, or 100% dimethyl sulfoxide under Similar conditions have been initiated in Mice of each group, and the treatment was administered every 2 days for a total of seven times. The tumor size E was measured with calipers at the Cinacalcet 364782-34-3 indicated times. Tumor volume was calculated using the following formula: length of this volume / 2 All Mice were get 2 days after the last treatment Tet and tumor tissues were separated. After Pr Para tion and distance were fixed some of the tumor tissue in 10% formalin-L Solution for Phosphate-TUNEL assay, w While others quickly frozen in liquid nitrogen at 80C until analysis.
Real-time reverse transcription of RNA-polymerase chain reaction analysis of total tissue was extracted from the tumor xenograft THP using a tissue kit RNAprep pure and in accordance cDNA using Kit First beach cDNA synthesis RevertAidTM manufacturer’s instructions reverse transcribed. Real-time reverse transcription reaction cha No polymerase was performed to analyze the expression of the FLT3 gene, p65, cyclin D1, Bcl-2 and SMRT using a SYBR Green kit. Actin was used as contr the house. The primers, the real-time in RT-PCR are indicated in Table 1. First heating at 95C for 5 min by 35 cycles at 94 ° C for 30 s, 54C, 56C, 58C, 60C or 30 s followed, and: The PCR was performed according to a 7300 real-time PCR the manual performed 72C for 30 s, with a Phloridzin final phase of dissociation for 15 s at 95C, 54C, 56C, 58C, 60C or 30 s and 15 s 95C. The PCR products were analyzed by electrophoresis best taken into account, And the relative amounts of the products were prepared using the comparative CT method. Western blot analysis of tumor tissue is homogenized and the total cellular Re proteins And nuclear Re proteins Were extractedusing and cytoplasmic extraction reagent kits. The protein concentration was determined using the Bradford method, the samples on 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane by semi-dry transferred. The membranes were blocked at 4 ° C overnight in 5% skim milk in TBST, washed containing 0.05% Tween 20. Then they were labeled with mouse anti-FLT3, mouse anti-p65 antibody Body, mouse cyclin D1-Antique Body, anti-mouse Bcl-2 Antique Body, goat anti-actin antibody and SMRT rpern At incubated room temperature for 2 h,, and three times with TBST and washed once with TBS. It was followed by incubation with HRP corresponding linked seco.