ABSTRACT
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.
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<p><b>OBJECTIVE</b>To investigate the regulatory effect of miR-126 on epidermal growth factor-like domain 7 (EGFL7) in ECV-304 cells.</p><p><b>METHODS</b>The miR-126-expressing plasmid targeting EGFL7 (plegfp-N1-miR-126) was constructed and transiently transfected into ECV-304 cells via liposome. The changes in the mRNA and protein expressions of EGFL7 in the transfected cells were analyzed by fluorescence quantitative RT-PCR and Western blotting.</p><p><b>RESULTS</b>Transfection with the recombinant plasmid plegfp-N1/miR-126 resulted in decreased EGFL7 expression with the passage of time, and the expression reached the lowest level at 48 h after the transfection. The expression of EGFL7 protein was reduced by 67% following the transfection in comparison with the control level, while the transfection with the empty vector resulted in a reduction only by 6.5% relative to the control level.</p><p><b>CONCLUSIONS</b>miR-126 can downregulate EGFL7 expression at the protein level in ECV-304 cells.</p>