Human angiogenin antisense oligonucleotide and medicine combination
An antisense oligonucleotide and angiopoietin technology, applied in the field of medicine, can solve the problem of high non-specific toxicity of cells
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Embodiment 1
[0061] acquisition of materials
[0062]The sources of materials used in the present invention are as follows. Human angiogenin is prepared by recombinant ribonucleic acid method. Basic fibroblast growth (bFGF) and epidermal growth factor (EGF) were purchased from Promega Company of the United States; vascular endothelial growth factor (VEGF) was purchased from R and D Company of the United States. Vascular endothelial cell-specific medium (HE-SFM) and cell transformation reagent Lipofectin were purchased from Gibco / BRL, USA. Ribonuclease (RNase A), deoxyribonuclease (DNase I) and proteinase K (Protease K) were purchased from Sigma, USA. Human placental vein vascular endothelial cells (HUVE) and smooth muscle cells (SMC) were purchased from Cell System Company of the United States; human colon adenocarcinoma cell HT-29 and human prostate cancer cell PC-3 were purchased from ATCC Company of the United States; cancer cell culture medium DMEM and fetal bovine serum (FBS) were ...
Embodiment 2
[0064] cell culture
[0065] Human placental vein vascular endothelial cells (HUVE) were cultured in a special medium for vascular endothelial cells (HE-SFM+10%FCS+10ng / ml bFGF) containing 10% fetal bovine serum and 10 ng / ml basic fibroblast growth factor )middle. The culture conditions are constant temperature and humidity at 37 degrees Celsius and 5% carbon dioxide gas phase. The medium was changed every two days, and the culture medium was subcultured at a ratio of 1:3. HUVE cells from passage 3 to passage 15 were used for experiments.
[0066] Human placental vein vascular smooth muscle cells, human prostate tumor cell PC-3, and human colon adenocarcinoma cell HT-29 were cultured in DMEM containing 10% fetal bovine serum. Subculture at a ratio of 1:4.
Embodiment 3
[0068] Antisense oligonucleotide treatment
[0069] HUVE cells were treated with trypsin-EDTA, the number of cells was measured with a hemocytometer or a Coulter counter, and then cultured in a 35-mm cell culture dish at a density of 100,000 cells per square centimeter. After incubating at 37 degrees Celsius for 24 hours, wash with Opti-MEM three times, such cells can be used for antisense oligonucleotide treatment experiments. Antisense oligonucleotides of different concentrations are mixed with an appropriate amount of lipid transfection reagent Lipofectin, pre-incubated at room temperature for 45 minutes, added to the above cells and incubated at 37 degrees Celsius for 5 hours to 72 hours. In some experiments, cells were re-treated with the same antisense oligonucleotides every 24 hours. In other experiments, cells treated with the above antisense oligonucleotides were treated with different angiogenic factors for 48 hours to measure cell division rate.
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