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Nucleic acid-derived agiogenesis inhibitor as well as preparation method and application thereof

A new blood vessel and inhibitor technology, applied in the field of biomedicine, can solve the problem that the effect of miRNA-410 recombinant expression plasmid is not significant, and achieve the effect of improving the therapeutic effect

Active Publication Date: 2019-04-26
上海亚睿生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, the miRNA-410 recombinant expression plasmid (pLKO.1-miRNA-410) constructed in the above doctoral thesis still has the defect of insignificant effect, and it is necessary to construct an expression vector with a more significant and lasting effect and related inhibitors

Method used

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  • Nucleic acid-derived agiogenesis inhibitor as well as preparation method and application thereof
  • Nucleic acid-derived agiogenesis inhibitor as well as preparation method and application thereof
  • Nucleic acid-derived agiogenesis inhibitor as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 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.

[0038] Example 1 Construction of the miR-410 expression vector pLKO-miR-410 of the present invention

[0039] 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

[0055] 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.

[0056] 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.

[0057] 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).

[0058]

[0059]

[0060] 4. Use pLKO-miR-410 injection an...

Embodiment 3

[0062] 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.

[0063] 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.

[0064] 3. Prepare pLKO-miR-410 injection according to the table below, and the solvent is ultrapure water (18.2MΩ).

[0065] 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

[0066] 4. Specific administration method: ...

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Abstract

The invention relates to a nucleic acid-derived agiogenesis inhibitor as well as a preparation method and application thereof. According to the invention, a high expression vector of miR-410 is constructed and injection and eye drops with reasonable formula are prepared; the vector of the nucleic acid-derived agiogenesis inhibitor disclosed by the invention is proved to significantly inhibit the production of pathologic neovascularization in a prostate cancer nude tumor-bearing mouse model, a PTEN gene prostate specific knockout transgenic mouse and a mouse OIR retina hyperoxia-induced model and effectively treat diseases such as tumor, diabetic retinopathy and the like. The nucleic acid-derived agiogenesis inhibitor has the advantages of obvious effects, safety, no toxic or side effects,simple production technique and low cost.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a nucleic acid-sourced neovascularization inhibitor and its preparation method and application. Background technique [0002] Angiogenesis is the process of sprouting and forming spiral-shaped capillaries on the side walls of normal blood vessels. These capillaries are called neovascularization. This process is an important part of the body's metabolism. It plays an important role in human body damage repair, menstrual cycle, pregnancy and other processes. Under normal circumstances, it is not common for human tissue to develop new blood vessels. However, in some pathological environments, such as tumors, diabetes, and rheumatoid, the human body will also generate new blood vessels in some unnecessary tissues, thereby causing serious damage to normal tissues. Such diseases are called neovascular diseases. For example, in the late stage of diabetes, the imbalance of the internal en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113A61K31/7105A61K48/00A61P35/00A61P9/10
CPCA61P9/10A61P35/00A61K31/7105C12N15/113C12N15/85C12N2310/141
Inventor 童强汪佳祺张丹丹
Owner 上海亚睿生物科技有限公司
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