Complex capable of inhibiting genetic function in exosome, and cancer proliferation and/or metastasis suppressor
An inhibitor, exosome technology, applied in the field of genetically functional conjugates
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Embodiment 1
[0066] Example 1: Capturing fluorescently labeled antibodies into cells ( figure 2 )
[0067] It was verified whether antibodies recognizing exosome surface antigens were incorporated into cells together with exosomes.
[0068] Hela cells (cervical cancer cells) were inoculated onto porous glass-bottomed dishes (Matsunami GlassInd., Ltd) at an amount of 9000 / well in 5% CO 2 Incubator at 37°C for 24 hours. Add fluorescently labeled antibodies (anti-CD63 antibody, anti-CD9 antibody, anti-CD81 antibody, and anti-TSG101 antibody) to each well, and then use 5% CO 2 The incubator was incubated at 37°C for 24 hours. Remove the supernatant and wash the cells with 1X PBS. Add 100 μL of 4% paraformaldehyde followed by incubation for 5 min at room temperature to fix the cells. Wash cells twice with 1X PBS. Viable cells were stained by adding 200 μL of Hoechst 33342 in 1×PBS (final concentration=5 μM), followed by incubation at room temperature for 10 minutes. Cells were washed tw...
Embodiment 2 and comparative example 1
[0071] Example 2 and Comparative Example 1: Capturing antibody / nucleic acid conjugates into cells ( Figure 4 )
[0072] It was verified whether antibodies recognizing exosome surface antigens were incorporated into cells together with exosomes.
[0073] Hela cells (cervical cancer cells) were inoculated onto porous glass-bottomed dishes (Matsunami GlassInd., Ltd) at an amount of 9000 / well in 5% CO 2 Incubator at 37°C for 24 hours. An anti-CD63 antibody-9r / nucleic acid conjugate (anti-CD63 IgG-9r+anti-miR(Cy5)) was added to each well. The anti-miR(Cy5) used here was 5'-Cy5-aguca auagggugug ugaga gacuu acug-3' (FASMAC, SEQ ID NO: 1).
[0074] As Comparative Example 1, anti-CD63 IgG and anti-miR (Cy5) were added instead of the anti-CD63 IgG-9r / nucleic acid conjugate.
[0075] Phalloidin was used to stain the cytoskeleton. Phalloidin is an oligopeptide that specifically binds to the polymeric actin (F-actin) that makes up the cytoskeleton.
[0076] Use 5% CO 2 Incubator at 3...
Embodiment 3
[0077] Example 3: MicroRNA function inhibitory effect of anti-CD63 antibody / anti-miR nucleic acid conjugate ( Figure 6 )
[0078] The inhibitory effect of microRNA function upon incorporation of anti-CD63 antibody / anti-miR nucleic acid conjugates into cells was assessed.
[0079] Hela cells (cervical cancer cells) were seeded on 96-well plates at an amount of 4500 / well, and 5% CO was used to 2 Incubator at 37°C for 24 hours. MicroRNA (miR-Luc) targeting luciferase mRNA was introduced into each well (LipofectamineRNAiMAX). Use 5% CO 2 The incubator was incubated at 37°C for 18 hours, and luciferase-expressing plasmids (pGL4.13 and pGL4.73) were introduced (Lipofectamine 2000). Use 5% CO 2 The incubator was incubated at 37°C for 6 hours, and anti-CD63 antibody / anti-miR nucleic acid conjugate (anti-CD63 IgG-9r+anti-miR-Luc), anti-miR-Luc only (300nM) or anti-CD63 antibody only (600nM) was added, and use 5% CO 2 The incubator was incubated at 37°C for 24 hr; firefly lucife...
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