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rsad2 affects tumor cell temozolomide resistance through Wnt pathway
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A technology of temozolomide and drug resistance, applied in the field of medicine
Inactive Publication Date: 2020-08-11
上海交复生物医药科技有限公司
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However, for patients with high-grade glioma, further chemotherapy is often required
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Embodiment 1
[0120] Example 1: Cell Culture
[0121] Human glioma cell lines U87 and U251 were purchased from Beina Biological Cell Bank. U87 and U251 cells were cultured in DMEM-H containing 10% fetal bovine serum (FBS), glutamine and sodium pyruvate, and placed at 37°C in 5% CO. 2 Incubator cultivation.
[0122] The U87 cells and U251 cells were exposed to increasing concentrations of temozolomide (TMZ), and the drug-resistant cells U87R and U251 cells were cultured. Briefly, cells were cultured in medium containing 2 μM TMZ and passaged. The induction dose of TMZ was increased by 2 μM per generation. The culture was stopped when the concentration reached 20 μM. U87 cells and U251 were treated with DMSO in the same way as resistance control.
Embodiment 2
[0123] Example 2: Cell Line Drug Resistance Analysis
[0124] The cells were prepared into a single cell suspension with a culture medium containing 10% fetal bovine serum, and seeded into a 96-well plate at 1000 cells per well, with a volume of 200 μL per well. 37℃, 5%CO 2 After culturing in a constant temperature incubator for 3-5 days, add 10 μL of MTT solution (5mg / ml in PBS, pH=7.4) to each well and continue to incubate for 4h, stop the culture, and carefully aspirate the culture supernatant in the wells (suspended cells need to be centrifuged before Aspirate and discard the culture supernatant in the well). Add 100 μL of DMSO to each well and shake for 10 min to fully dissolve the crystals. Select 490 nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, record the results, and draw the cell growth curve with the concentration as the abscissa and the absorbance value as the ordinate. And calculate IC50 value from the curve. Th...
Embodiment 3
[0128] Example 3: Real-time quantitative PCR analysis of RSAD2 expression in various cell lines
[0129] use Reagents to extract total RNA from samples. After quantification by NanoDrop 2000 (Thermo Fisher Scientific Inc, USA), 200 ng of total RNA was used for reverse transcription using ReverTra Ace qPCR RT Kit (Toyobo, Japan) according to the instructions. Real-time quantitative PCR amplification was performed with the following primers, primers for the coding region of the RSAD2 gene:
[0130] RSAD2
[0131] Forward: 5'-ATTTGGCCATATGAGGCTGT-3' (SEQ ID NO. 1)
[0132] Reverse: 5'-ATCCTGGATGAGACACGCAC-3' (SEQ ID NO. 2)
[0133] GAPDH
[0134] Forward: 5'-AACAGCCTCAAGATCATCAGC-3' (SEQ ID NO. 3)
[0135] Reverse: 5'-GGATGATGTTCTGGAGAGCC-3' (SEQ ID NO. 4).
[0136] PCR reaction parameters: 95°C for 5 min; 95°C for 30sec, 59°C for 30sec, and 72°C for 30min for a total of 40 cycles. A dissolution curve was drawn at 70°C to 95°C. The above experiment was repeated three time...
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Abstract
The invention belongs to the technical field of medicines and discloses a novel tumor drug resistance marker gene RSAD2 RSAD2 (Radical S-Adenosyl Methionine Domain Containing 2) through massive and deep research, and the gene has close relationship with drug resistance of a tumor chemotherapy temozolomide (TMZ) medicine. Compared with primary cells U87 and U251, the RSAD2 has remarkably increasedexpression levels in temozolomide resistant cells U87R and U251R, and in addition, wnt pathways can be activated. By reducing expression of the RSAD2 gene, the sensitivity of cell strains upon the temozolomide medicine can be improved, and the multiplication capabilities of tumor cells can be inhibited. By adopting the gene, not only is novel information provided to malignant glioma drug resistance mechanisms, but also novel targets are provided for design of anti-tumor drug-resistant medicines.
Description
technical field [0001] The invention belongs to the technical field of medicine, and particularly relates to the effect of RSAD2 on temozolomide resistance of tumor cells through the wnt pathway. Background technique [0002] Gliomas are tumors that originate from glial cells and are the most common primary intracranial tumors. Brain gliomas are classified into astrocytoma, oligodendrocytoma, ependymoma and mixed glioma according to the similarity of their tumor cell morphology to normal glial cells. According to the grading system developed by the World Health Organization (WHO), gliomas can be divided into grades 1 (the least malignant degree and the best prognosis) to grade 4 (the most malignant degree and the worst prognosis). Among them, so-called anaplastic glioma by traditional cytopathology corresponds to WHO grade 3, and glioblastoma corresponds to WHO grade 4. According to this grading system, gliomas can be further divided into low-grade gliomas (WHO grades 1-2)...
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