Application of anisomycin in the preparation of drugs for the prevention/treatment of ovarian cancer
Anisomycin, ovarian cancer technology, applied in the directions of drug combination, antitumor drug, heterocyclic compound active ingredient, etc., can solve problems such as expression up-regulation, achieve the effect of great social benefit and good clinical application prospect
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Embodiment 1
[0022] Example 1 Isolation of tumor stem cells from patients with ovarian cancer
[0023] Seven patients with undifferentiated mixed epithelial ovarian cancer. The age, stage, degree of differentiation, and histological distribution of all ovarian cancer patient populations are shown in Table 1.
[0024] Isolation of tumor stem cells from ovarian cancer patients (7 ovarian cancer patients underwent surgery to remove ovarian cancer tissues. In this experiment, the excised tissues were collected in sterile centrifuge tubes, digested with 0.25% trypsin into a single-cell suspension, and each 100 μL Add 4 μL of CD44 and CD117 monoclonal antibodies (mouse anti-human CD117-FITC; rabbit anti-human CD44-PE, eBioscience) to the ovarian cancer cell suspension, and incubate the cells at 4°C for 30 minutes. Wash once with salt-buffered saline (PBS), and then use flow cytometry (BD FACS Aria, BD Bioscience, CA, USA) to separate CD44+ / CD117+ OCSCs cell subpopulation)
[0025] Table 1 Char...
Embodiment 2
[0028] Example 2 Anisomycin significantly inhibited the proliferation and invasion of OCSCs
[0029] MTT method (thiazolan method) to detect cell proliferation
[0030] Cells were seeded in (2×10 3 / ml) 96-well plate and at 37°C, 5% CO 2 Under the conditions, the cells were cultured with DMEM (modified high glucose cell medium) medium containing 10% FBS (fetal bovine serum) until 85% of the cells were confluent. MTT (3(V / V)-diphenylbromide; Sigma) reagent (5mg / ml) was added to the cell culture medium at different time points and incubated at 37°C for 4 hours. The reaction was terminated by adding 150 μL of dimethyl sulfoxide (DMSO, Sigma Chemicals) per well. The cells were lysed for 15 minutes, and the 96-well plate was shaken gently for 5 minutes. The absorbance value was determined by enzyme-linked immunosorbent assay (ELISA) at 490nm (model680, Bio-Rad Bole Biological Company).
[0031] Annexin V-FITC / PI (cell membrane phosphatidylserine-fluorescein isothiocyanate / prop...
Embodiment 3
[0038] Example 3 Anisomycin stimulates the expression of OCSCs apoptosis-related proteins and lncRNA BACE1-AS (long non-coding ribonucleic acid β decomposing enzyme translation nucleic acid sequence)
[0039] RNA extraction and analysis by qRT-PCR (real-time fluorescent 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 deoxyribonucleolytic enzyme (Sigma Reagent Co., Ltd.)), and were treated with reverseraα (α-type ribonucleic acid reverse transcription) enzyme kit for first-strand cDNA synthesis (TOYOBO) Quantification and reverse transcription into cDNA. Real-time fluorescence quantitative RT-PCR adopts the realplex4 (fluorescence 4-color channel) real-time fluorescence quantitative RT-PCR detection system of Eppendorf Company (Germany), and uses SYBR green fluorescence quantitative PCR Master Mix (Toyobo) as the det...
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