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Exosome loading ABCA1 mRNA and construction method and application thereof

A construction method and technology for exosomes, which are applied in the field of exosomes loaded with ABCA1 mRNA and their construction, can solve the problems of increasing the expression of ABCA1 protein in the liver, the inability to load ABCA1, and the large molecular weight, and achieve the effect of improving efficiency.

Inactive Publication Date: 2019-01-18
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem in the prior art that ABCA1 with large molecular weight cannot be loaded into exosomes, the present invention provides a method for constructing exosomes loaded with ABCA1 mRNA, successfully packaging ABCA1 mRNA into exosomes, and the obtained loaded ABCA1 mRNA The exosomes can effectively increase the expression of liver ABCA1 protein, promote HDL synthesis from the source, and can be used in the preparation of drugs for the prevention or treatment of atherosclerosis

Method used

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  • Exosome loading ABCA1 mRNA and construction method and application thereof
  • Exosome loading ABCA1 mRNA and construction method and application thereof
  • Exosome loading ABCA1 mRNA and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] (1) After the 3' end of the CDS sequence (nucleotide sequence shown in SEQ ID No.1) of ABCA1, a specific binding sequence AREs of the RNA binding protein HuR (shown in SEQ ID No.2) is fused Nucleotide sequence), namely ABCA1-AREs gene (nucleotide sequence shown in SEQ ID No.3), ABCA1-AREs gene was synthesized according to the nucleotide sequence shown in SEQ ID No.3.

[0085] (2) Using pcDNA3.1(-) as the backbone, cut the pcDNA3.1(-) vector with the restriction endonuclease EcoRV, mix and connect the linear vector obtained after digestion with the synthetic ABCA1-AREs gene, and screen , get pcDNA3.1-ABCA1-AREs expression vector.

[0086] (3) Trizol extracted the total RNA of mouse muscle cells; the total RNA was reverse-transcribed using Promega M-MLV reverse transcriptase to obtain mouse fibroblast cDNA:

[0087] The reverse transcription system is: 2 μg of RNA template, 4 μl of 5×RT buffer, 2 μl of dNTP mixture (10 mM), 2 μl of 0.1MDTT and 1 μl of M-MLV reverse trans...

Embodiment 2

[0106] This experiment verified the efficiency of ABCA1 mRNA loading exosomes:

[0107] Detection method: Exosomes were lysed by Trizol, RNA was extracted according to the instructions of the RNA extraction kit, glycogen was added to precipitate RNA according to the instructions during the extraction process, followed by reverse transcription, and then qPCR was used to detect the loading efficiency of target RNA molecules.

[0108] The formula for calculating the relative expression of ABCA1 mRNA was as follows: β-actin was selected as an internal reference gene, and 2-ddCt was used to calculate the relative expression of ABCA1 mRNA in each exosome.

[0109] Sample: Exosomes expressed by hepatocytes that were not transfected with any vector were used as a control (Empty vector); the pcDNA3.1-CD9-HuR expression vector was prepared according to steps (3) to (6) described in Example 1; The pcDNA3.1-ABCA1-AREs expression vector was prepared by steps (1)-(2) described in Example 1....

Embodiment 3

[0113] 100 μL of exosomes prepared in Example 1 was injected into C57 mice through the tail vein, and after 48 hours, the mice were sacrificed to further collect liver tissue. At the same time, PBS was used as the control group, which was the same as the above operation.

[0114] Using WB to detect the expression of ABCA1 and the control group in the liver, the primers used are shown in Table 1, and the results are as follows figure 2 shown.

[0115] Table 1 Reaction primers used in WB detection

[0116]

[0117]

[0118] Wherein β-actin is an internal reference, PBS represents the expression level of ABCA1 in the liver of mice treated in the control group, and Exo-ABCA1-AREs represents the expression level of ABCA1 in the liver of mice injected with exosomes prepared in Example 1. Depend on figure 2 It can be seen that the exosomes loaded with ABCA1 mRNA constructed in the present invention significantly increased the expression of ABCA1 in mouse liver cells.

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Abstract

The invention provides a construction method for an exosome loading ABCA1 mRNA, belonging to the technical field of gene medicine. The method comprises constructing an ABCA1 fusion expression vector and a CD9 fusion expression vector, wherein the ABCA1 fusion expression vector is a CDS sequence of ABCA1 and a tandem RNA binding protein recognition sequence. The CD9 fusion expression vector is a CD9 CDS sequence and a corresponding RNA binding protein encoding gene, and the ABCA1 fusion expression vector and the CD9 fusion expression vector are co-transfected into the liver cells, and the exosomes are collected after the culture, so that the ABCA1 mRNA is excreted. body. The construction method can improve the efficiency of loading ABCA1 mRNA into exosomes, overcome the problem that the large molecular weight ABCA1 mRNA molecule cannot be efficiently loaded into the exosomes, and obtain a large amount of exosomes containing ABCA1 mRNA.

Description

technical field [0001] The present invention relates to the technical field of gene medicine, in particular to an exosome loaded with ABCA1 mRNA and its construction method and application. Background technique [0002] Atherosclerosis (AS) is the main cause of coronary heart disease, cerebral infarction and peripheral vascular disease. Lipid metabolism disorder is the basis of atherosclerosis, which is characterized by the involvement of arterial lesions starting from the intima, usually with accumulation of lipids and complex carbohydrates, hemorrhage and thrombosis, followed by fibrous tissue hyperplasia and calcium deposition, followed by There is gradual degeneration and calcification of the arterial media, resulting in thickening and hardening of the arterial wall and narrowing of the vascular lumen. Lesions often involve large and medium muscular arteries, and once they develop enough to block the lumen of the artery, the tissues or organs supplied by the artery will...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61K35/407A61P9/10
CPCA61K35/407A61P9/10C07K14/47C12N5/067C12N2510/00
Inventor 袁丽君李者龙柏丹娜杨薛康杨国栋周雪莹孙汶齐赵联璧
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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