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Objective To investigate the effect of IGF1R β subunit mutants sb-IGF1R and ma-IGF1R on the biological behavior of osteosarcoma 143B cells. Methods We designed and constructed sb-IGF1R and ma-IGF1R fragments. They were cloned into adenovirus AdEasy shuttle plasmid, to obtain Ad-sbIGF1R and Ad-maIGF1R. We observed the proliferation, migration and apoptosis of the osteosarcoma cells transfected with Ad-sbIGF1R, Ad-maIGF1R and Ad-IGF1R. The Ad-sbIGF1R, Ad-maIGF1R and Ad-GFP nude mouse models were constructed to evaluate the tumor growth in vitro. Results By plasmid PCR, IGF-1R β subunit mutant was overexpressed in osteosarcoma cells. Ad-sbIGF1R and Ad-maIGF1R significantly inhibited the proliferation and migration of osteosarcoma cells, and promoted cell apoptosis, and inhibited tumor growth in subcutaneous tumor-bearing nude mouse models. Conclusion IGF1R β subunit mutants inhibit the proliferation and migration of osteosarcoma cells and induce cell apoptosis.
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Objective To explore a new long-term efficient embedding technique of tumor spheres.Methods Tumor spheres,mainly composed of cancer stem cells,were cultured from glioblastoma tissues.The fifth generation of tumor spheres was chosen for egg-white-paraffin embedding and section.Then,those tumor sphere slices were observed by HE staining,immunohistochemistry and immunofluorescence staining.And the immunofluorescence results of these tumor sphere slices were compared with those tumor spheres kept with traditional methods.Results Immunofluorescence results showed that tumor spheres kept with traditional methods looked blurry,and the positive cells and the positive protein expression sites in the cells could not be displayed.HE staining demonstrated that the tumor sphere slices had well-distributed intact spheres and coloring cells with high karyoplasm contrast.Immunohistochemistry and immunofluorescence staining of tumor sphere slices showed clear background,from which positive cells and positive locus could be easily displayed; therefore,semi-quantitative analysis of the positive cells could be performed.Conclusion Egg-white-paraffin embedding technique of the tumor sphere slices can reduce experimental errors and cut down the costs,which enjoys its advantage as compared with traditional embedding technique of the tumor sphere slices.
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Objective To establish the imatinib (STI-571)-resistant subline in vitro and investigate its biological characteristics. Methods Human glioblastoma multiform drug-resistant cell line (named U251AR) was established in vitro by successively increasing the concentration of imatinib in a cell culture medium. The 50％ inhibitory dose (IC50) values and the resistance indexes ([IC50U251/STI-571]/[IC50 U251]) for other chemotherapeutic agents were evaluated using cell counting kit-8 assays. Expressions of acquired multidrug resistance P-glycoprotein (MDR 1, ABCB 1; MDR3, ABCB4),breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated protein 1 (MRP1,ABCC1) were detected by QRT-PCR. Flow cytometry was employed to detect the protein expression of ABCG2. Results The U251AR was developed after culture for 12 months and similar morphologies of U251 and U251/STI-571 cells were determined. The resistance coefficient of U251AR cells to imatinib was 20.41 times more than that of the parent cells, and U251AR cells showed cross-resistance to many anti-tumor agents (P＜0.05). The resistance coefficients of U251AR cell line to doxorubicin and cisplatin were 5.06 and 10.28 times, respectively, more than those of U251 cells (P＜0.05). QRT-PCR indicated that the mRNA levels of MDR1, MRP1, BCRPandABCB4 (P-g4) in the U251/STI571 resistant cells were significantly higher than those in the U251 cells (P＜0.05). The protein expression of ABCG2 in U251AR cell line was significantly increased as compared with that in the parent cells (P＜0.05).Conclusion We have successfully established multidrug resistant cell line U251AR, and the drug resistance of U251/STI571 is associated with over-expressions of ABCC1, ABCB1, ABCB4, and ABCG2 mRNA, and ABCG2 protein.