siRNA aiming at human annexin A2 acceptor gene and application thereof

A human annexin and receptor technology, applied in the field of genetic engineering, can solve the problems of short duration and multiple treatments, and achieve the effect of inhibiting proliferation and inducing block.

Inactive Publication Date: 2015-02-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the short duration of action of vascular endothelial growth factor monoclo

Method used

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  • siRNA aiming at human annexin A2 acceptor gene and application thereof
  • siRNA aiming at human annexin A2 acceptor gene and application thereof
  • siRNA aiming at human annexin A2 acceptor gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Synthesis of siRNA

[0027] Search NCBI GeneBank to obtain the full sequence of AXIIR and mRNA sequence, use the existing network resources and commonly used software for biological analysis of AXIIR, and select the coding region as the target sequence for siRNA design. Refer to the siRNA design principle, and compare it with the human genome sequence through the blast function of the GeneBank database to ensure that there is no homology; exclude potential siRNAs that have 8 consecutive bases at the 5' end of the aitisense chain paired with other genes; exclude any consecutive 14 bases base-pairing potential siRNAs with other genes.

[0028] The siRNA sequence designed and synthesized in this embodiment is

[0029]

[0030] From figure 1 It can be seen that siRNA2 can inhibit the expression of AXIIR by about 20%; siRNA2 can inhibit the expression of AXIIR to more than 50%, and siRNA3 can inhibit the expression of AXIIR by about 20%.

[0031] Therefore, ...

Embodiment 2

[0032] Example 2: Cell Transfection

[0033] (1) One day before transfection, collect the cells in the logarithmic growth phase and inoculate them in a 12-well plate, and the inoculated number is about 5×10 4 cells, add 1 mL of culture medium.

[0034] (2) Add 4 μL liposome transfection reagent to 100 μL Opti-MEM medium, pipette gently, and let stand at room temperature for 5 minutes.

[0035] (3) Add 60Nm to 100μL Opti-MEM medium and mix gently.

[0036](4) Mix the transfection reagent and the siRNA diluent, blow evenly, and let stand at room temperature for 20 minutes.

[0037] (5) Change the medium 8 hours after transfection, and continue to cultivate for 48 hours before performing other operations after transfection.

Embodiment 3

[0038] Example 3: Detection of mRNA and protein expression of AXIIR in umbilical vein endothelial cells by real-time fluorescent quantitative RT-PCR and western blotting

[0039] The specific process is as follows:

[0040] 1 Real-time fluorescent quantitative RT-PCR

[0041] (1) Extraction of total RNA: Collect cells into a 1.5 mL RNase-free centrifuge tube, add 0.5 mL Trizol, mix well on ice and pipette, and let stand for 10 min. Add 0.125mL chloroform, shake vigorously for 20s, and let stand on ice for 5min. Centrifuge at 4°C, 12000r / min×15min, pipette 0.2mL supernatant to another 1.5mL, then add the same amount of isopropanol as the supernatant, mix gently, and let stand on ice for 10min. Centrifuge at 4°C, 12000r / min×15min, discard the supernatant, add 1mL pre-cooled 75% ethanol, gently wash the precipitate, centrifuge at 4°C, 12000r / min×15min. Discard the supernatant, dry it, and dissolve it in 20 μL DEPC water. A multifunctional microplate reader was used to determi...

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Abstract

The invention belongs to the field of gene engineering technology, and provides a small interfering RNA specifically inhibiting annexin A2 acceptor, and application of the siRNA to prepare medicines for treating neovascular diseases. Experiments prove that the siRNA aiming at AXIIR gene expression is capable of inhibiting propagation, migration and blood vessel formation of umbilical vein endothelial cells, and inducing retardance of cell cycle. The siRNA is applicable to prepare stable effective low-toxicity biological targeting preparations or medicines for inhibiting AXIIR expression and treating neovascular diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a small nucleic acid interference molecule that specifically inhibits annexin A2 receptors and its application in the preparation of drugs for treating neovascular diseases. Background technique [0002] Neovascular disease is a process of growing new blood vessels on the basis of existing blood vessels, which has important physiological and pathological significance. Angiogenesis after birth is conducive to the growth of tissues and organs, but in adulthood, most blood vessels remain in a quiescent state. Under pathological stimuli, vascular endothelial cells can restore their proliferative ability to further promote angiogenesis. Pathological angiogenesis involves the secretion and uncontrolled regulation of various cytokines, which are involved in the occurrence and development of various diseases, such as tumors, diabetic retinopathy, rheumatoid arthri...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P29/00A61P35/00A61P27/02A61P19/02A61P9/00
Inventor 赵世红宋洪元张月露
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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