Application of anisomycin to preparation of medicine for preventing/treating ovarian cancer
A technology for anisomycin and ovarian cancer, applied in the direction of drug combinations, antineoplastic drugs, active ingredients of heterocyclic compounds, etc., can solve the problems of expression up-regulation and achieve great social benefits and good clinical application prospects
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Embodiment 1
[0022] Example 1 Isolation of tumor stem cells from ovarian cancer patients
[0023] Undifferentiated mixed epithelial ovarian cancer in 7 patients with ovarian cancer. The age, stage, degree of differentiation, and histological distribution of all ovarian cancer patient populations are shown in surface 1 .
[0024] Isolation of tumor stem cells from ovarian cancer patients (7 ovarian cancer patients underwent surgical resection of ovarian cancer tissue. In this experiment, the surgically removed tissue was collected in a sterile centrifuge tube, digested with 0.25% trypsin to form a single cell suspension, each 100 μL 4 μL of CD44 and CD117 monoclonal antibodies (mouse anti-human CD117-FITC; rabbit anti-human CD44-PE, eBioscience) were added to the ovarian cancer cell suspension, and the cells were incubated at 4°C for 30 minutes. After washing once with salt-buffered saline (PBS), the CD44+ / CD117+ OCSCs cell subsets were isolated by flow cytometry (BD FACS Aria, BD Biosc...
Embodiment 2
[0028] Example 2 Anisomycin significantly inhibits the proliferation and invasion of OCSCs
[0029] Detection of cell proliferation by MTT method (thiazolan method)
[0030] Cells were seeded on (2 × 10 3 / ml) 96-well plate and incubate at 37°C, 5% CO 2 The cells were cultured in DMEM (modified high glucose cell culture medium) medium containing 10% FBS (fetal bovine serum) under conditions until 85% of the cells were confluent. MTT (3(V / V)-diphenyl bromide; Sigma) reagent (5 mg / ml) was added to the cell culture medium at various time points and incubated at 37°C for 4 hours. The reaction was stopped by the addition of 150 μL of dimethyl sulfoxide (DMSO, Sigma Chemicals) per well. Cells were lysed for 15 min, and the 96-well plate was gently shaken for 5 min. The absorbance value was measured by enzyme-linked immunosorbent assay (ELISA) at 490 nm (model 680, Bio-Rad Bio-Rad).
[0031] Annexin V-FITC / PI (cell membrane phosphatidylserine-fluorescein isothiocyanate / propidium...
Embodiment 3
[0038] Example 3 Anisomycin stimulates the expression of apoptosis-related protein and lncRNA BACE1-AS (long non-coding ribonucleic acid β-degrading enzyme translation nucleic acid sequence) in OCSCs
[0039] RNA extraction and analysis by qRT-PCR (real-time quantitative polymerase chain reaction)
[0040] Total RNA was isolated from each cell using Trizol (cell / tissue total RNA extraction buffer) reagent. RNA samples were treated with Dnase I (Sigma-Aldrich) (type I deoxyribonuclease (Sigma Reagent Co., Ltd.)), and revertraα (α-type ribonucleic acid reverse transcription) first-strand cDNA synthesis enzyme kit (TOYOBO) was used Quantify and reverse transcribed into cDNA. Real-time fluorescence quantitative RT-PCR adopts realplex4 (fluorescence 4-color channel) real-time fluorescence quantitative RT-PCR detection system of Eppendorf (Germany), and SYBR green fluorescence quantitative PCR Master Mix (Toyobo) is used as detection dye. Amplification was performed for 40 cycles ...
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