Application of 20(S)-ginsenoside-Rg3 in reversing drug resistance of glioma cells to chemotherapeutic drugs
A technology of glioma cells and ginsenosides, which can be used in drug combinations, anti-tumor drugs, medical preparations containing active ingredients, etc. Effect
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Embodiment 120
[0081] Example 1 Toxic effects of 20(S)-ginsenoside-Rg3 and TMZ on glioma
[0082] GBM-XX is the primary cell line derived from the surgical specimens of World Health Organization grade IV glioma patients. The resection was performed according to the protocol approved by the ethics committee of our hospital, and the patients' prior informed consent was obtained. The medium consisted of DMEM (Life Technologies / Gibco, Carlsbad, California, USA) and 10% fetal bovine serum (FBS; Life Technologies / Gibco, Carlsbad, California, USA), 100u / ml penicillin and 100 μg / ml streptomycin (Gibco, Grand Isle, NY). cells at 1 x 10 5 Cells / ml inoculated. Culture conditions: temperature 37°C, 5% CO 2 and 100% humidity.
[0083]T98G, U118 and GBM-XX cells were seeded in 96-well flat-bottom plates at 5000 cells / well, cultured in DMEM supplemented with 10% FBS, and then treated with increasing concentrations (0, 60, 120, 240, 360 and 480 μM ), 20(S) ginsenoside-Rg3, (0, 75, 150, 300, 450 and 600...
Embodiment 220
[0086] Example 2 Effect of 20(S)-ginsenoside-Rg3 on the expression of MGMT
[0087] T98G, U118 and GBM-XX were treated with 20(S)-ginsenoside-Rg3 (100 μM) for 72 hours.
[0088] mRNA assay: Total RNA was extracted from cell lines using TRIzol (TaKaRa, Dalian, China) according to the manufacturer's protocol. For mRNA analysis, RNA was reverse transcribed into cDNA using a primer-script one-step RT-PCR kit (TaKaRa, Dalian, China). The cDNA template was amplified by RT-PCR using the SYBR Premix Eraser Kit (TaKaRa, Dalian, China). GAPDH was used as an internal control. Real-time PCR was performed in triplicate. The primer sequences used are as follows: for GAPDH primers are as follows; SEQ ID NO1.: 5'-gtcaacggattggtctggtatt-3' (forward) and SEQ ID NO2.: 5'-agtctgggggcagtgat-3' (reverse); MGMT primers are as follows: SEQ ID NO3.: 5'-gttatgaatgtaggagcccttatg-3' (forward), SEQ ID NO4.: 5'-tgacaacgggaatgaagtaatg-3' (reverse). The result is as Figure 1c As shown, 20(S)-ginsenosi...
Embodiment 320
[0090] Example 3 20(S)-ginsenoside-Rg3 inhibits the expression of MGMT in glioblastoma by regulating the Wnt / β-catenin pathway
[0091] T98G, U118 and GBM-XX were treated with 20(S) ginsenoside-Rg3 (100 μM) for 72 hours, and Wnt / β-catenin pathway-related proteins were determined.
[0092] Western blot analysis Cells were washed with PBS and then lysed with RIPA lysis buffer (Solarbio, China) and protease inhibitors (Roche Applied Science, Switzerland). Protein concentration was measured using BCA protein assay kit (Beyotime Biotechnology, China). Equal amounts of proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were subsequently blocked with 5% skim milk for 2 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were anti-MGMT (1:500, American Abcam Company) and anti-Survivin (1:1000, American Abcam Company), anti-β-catenin, anti-CD44, anti-C-Jun, anti-C-Myc, anti-cyclinD1, an...
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