Nucleic acid-derived neovascularization inhibitor and preparation method and application thereof
A new blood vessel and inhibitor technology, applied in the field of biomedicine, can solve the problem of insignificant effect of miRNA-410 recombinant expression plasmid, and achieve the effect of improving the therapeutic effect
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Embodiment 1
[0035] Embodiment 1 is an example of carrier construction, which only needs to be implemented once in the same batch. Examples 2-6 are treatment examples. In actual operation, the product of Example 1 can be preserved and amplified indefinitely without reconstitution before each treatment.
[0036] Example 1 Construction of the miR-410 expression vector pLKO-miR-410 of the present invention
[0037] The main content of this example is: design and synthesize the nucleic acid sequence containing miR-410, and use T4 DNA polymerase to phosphorylate the 5' end, and prepare the processed single-stranded DNA fragments into double-stranded DNA fragments by annealing and bonding method , and connected to the vector pLKO.1 using the cohesive ends of AgeI and EcoRI, and verified by sequencing. All enzymes were purchased from ThermoFisher, among which T4DNA polymerase product number is EP0062, AgeI product number is FD1464, EcoRI product number is ER0271, and pLKO.1 was introduced from A...
Embodiment 2
[0053] Example 2 Use the plasmid injection of the present invention to treat nude mouse tumor-bearing mouse models of prostate cancer 1. Take 20 nude mice and subcutaneously inject 1*10 6 Tumor cells DU145, three weeks later, all nude mice subcutaneous tumors in the injection site. When these tumors grew to about 0.5cm in diameter, they were randomly divided into 2 groups, 10 in each group. One group is the control group, named group A. The other group is the treatment group, named as group B.
[0054] 2. Mass culture the DH5a strain containing the plasmid pLKO-miR-410, and extract the plasmid. The obtained plasmid was purified with polyethylene glycol and dissolved in double distilled water.
[0055] 3. Prepare the pLKO-miR-410 injection according to the table below, and use the pLKO.1 empty plasmid to prepare the control injection according to the same formula, and the solvent is ultrapure water (18.2 MΩ).
[0056]
[0057]
[0058] 4. Use pLKO-miR-410 injection an...
Embodiment 3
[0060] Embodiment 3 uses the plasmid injection of the present invention to treat Pb-CRE + , PTEN LoxP Transgenic mice 1. Take Pb-CRE + , PTEN LoxP Twenty transgenic mice (introduced from the Model Animal Center of Nanjing University) were randomly divided into 2 groups, 10 in each group. One group is the control group, named group A. The other group is the treatment group, named as group B.
[0061] 2. Mass culture the DH5a strain containing the plasmid pLKO-miR-410, and extract the plasmid. The obtained plasmid was purified with polyethylene glycol and dissolved in double distilled water.
[0062] 3. Prepare pLKO-miR-410 injection according to the table below, and the solvent is ultrapure water (18.2MΩ).
[0063] Element Final concentration NaCl 137mM KCl 2.7mM Na 2 HPO 4
10mM K H 2 PO 4
2mM CaCl 2
1mM pLKO-miR-410 plasmid 0.1mg / ml
[0064] 4. Specific administration method:
[0065] The injectio...
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