Purpose of siRNA and membrane penetrating peptide for combined treatment of liver cancer
A technology for the treatment of liver cancer and membrane-penetrating peptides, applied in the fields of DNA/RNA fragments, gene therapy, recombinant DNA technology, etc., to achieve great application value, enhanced inhibitory effect, and improved transfection efficiency.
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Embodiment 1
[0027] Example 1 Cell Culture
[0028] 293T cells were placed in 5% CO using DMEM-F12 medium (supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin) 2 Cultured in a cell culture incubator at 37°C, 1 day before transfection, trypsinized 293T cells and counted, 1*10 per well 5 Inoculated in 24-well culture plate, cultured in a cell incubator, so that the cell density was about 55% on the day of transfection.
Embodiment 2
[0029] Example 2 Cell Transfection and Measurement of Transfection Efficiency
[0030] The cells obtained in Example 1 were removed from the complete medium, washed twice with PBS, and 125ul of Opti-MEM medium (invitrogen) was used to dilute the siRNA (labeled FAM) of SEQ ID NO: 1 and the permeable peptide of SEQ ID NO: 3 ( (Shanghai Sangong Synthetic), mix the two evenly, let stand for 20min, then add to the cell culture plate, absorb the transfection medium after 4h, wash twice with PBS, add 500ul complete medium to each well and place in 5% CO 2 The transfected cells were obtained after culturing in a cell incubator at 37°C. Using LipofectamineTM RNAiMAX transfection reagent as a control, transfection was carried out under the same conditions, and the ratios of transfection reagent to siRNA were 1:1, 10:1, 20:1, and 25:1, respectively.
[0031] After 24 hours of transfection, detect the color development of FAM with an inverted fluorescence microscope and take pictures. Ca...
Embodiment 3
[0035] Example 3 Cytotoxicity detection
[0036] According to the method of Example 2, human H9 cells were only transfected with membrane-penetrating peptides, and the cells in each well 24 hours after transfection were digested with trypsin and sucked out respectively, and each well was divided into 1×10 4 Cells were inoculated into a 96-well culture plate and placed in a cell culture incubator with 5% CO2 at 37 degrees Celsius for 24 hours. The 96-well cell plate was removed from the old culture, washed twice with fresh DMEM medium, and 10 microliters of CCK-8 solution was added. Continue to incubate for 4 hours, use untransfected cells as control wells, detect the absorbance at a wavelength of 450nm with a microplate reader, and calculate the cell survival rate (OD of cells in the transfection group / OD of cells in the control group*100%), the results are as follows figure 2 As shown, after 24 hours of transfection, the permeable peptide has no obvious cytotoxicity to human...
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