摘要
Objective: To establish an HPLC method for the determination of kaempferitrin in Celastri Orbiculati Fructus and to compare the quality of samples from different habitats in northeast China. Methods: The extracts were obtained by methanol ultrasonic method and kaempferitrin was determinated by HPLC. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) at 30℃ and the mobile phase was a mixture of methanol- 0.01 mol/L solution of potassium dihydrogenphosphate -glacial acetic acid (40:60:15). The flow rate was 1.0 mL/min and the detection wavelength was 254 nm. Results: The linearity range was 0.092 7-0.926 7 μg (r = 0.999 8), average recovery was 100.8%, and RSD = 1.1% (n = 6). Conclusion: This method is validated in terms of selectivity, linearity, precision, and accuracy. It can be used as the effective method for quality control. The determination results show that the longer the fruit sits followed by picking, the less contents it has.
摘要
Objective To analyze the relation between short-term chemotherapy of temozolomide (TMZ) and inhibition of proliferation of U87 glioblastoma cells,and investigate the role and mechanism of P57 in inhibiting U87 cells proliferation under treatment of TMZ.Methods Protein levels of P57 and proliferating cell nuclear antigen Ki-67 in U87 cells accepted TMZ short-term chemotherapy were detected by Western blotting.Protein levels of P57 in clinical primary or recurrent glioblastomas accepted TMZ short-term chemotherapy were compared by immunohistochemistry and Western blotting.After P57 was knocked down,apoptosis,cell cycle and cell vitality changes of U87 cells treated with TMZ and U87 cells with TMZ withdrawal were studied.Results After short-term chemotherapy of TMZ in U87 cells,the expression level of P57 increased but the expression level of Ki-67 decreased.The protein level of P57 in recurrent glioblastoma was higher than that in primary tumor.After P57 was knocked down in U87 cells treated with TMZ,apoptosis was enhanced and the protein level of cycle dependent kinase 2 (Cdk2) increased,which indicated that the inhibition of proliferation induced by TMZ was weakened,while the apoptosis rate increased (31.00±3.48 vs.12.83±1.40) Furthermore,after TMZ was withdrew,the cell cycle and cell vitality in U87 control cells recovered,but in U87 cells with P57 knocked down,the cell cycle was arrested and the cell vitality was reduced.Conclusion P57 inhibits cell cycle progression or proliferation of U87 cells by up-regulating P57 expression and inhibiting Cdk2 expression to reduce the damage caused by TMZ.
摘要
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.
摘要
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.