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Application of LKB1 gene in preparation of anti-abdominal-aortic-aneurysm drugs

A technology for abdominal aortic aneurysm and aortic aneurysm, which can be used in anti-tumor drugs, drug combinations, gene therapy and other directions, can solve problems such as unclear effects, and achieve the effect of reducing the incidence of abdominal aortic aneurysm

Active Publication Date: 2019-04-12
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies have found that LKB1 can regulate endothelial function and blood pressure in endothelial cells, and LKB1 in macrophages can inhibit inflammatory responses, but its role in smooth muscle cells is still unclear

Method used

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  • Application of LKB1 gene in preparation of anti-abdominal-aortic-aneurysm drugs
  • Application of LKB1 gene in preparation of anti-abdominal-aortic-aneurysm drugs
  • Application of LKB1 gene in preparation of anti-abdominal-aortic-aneurysm drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The construction of embodiment 1 mouse abdominal aortic aneurysm model

[0031] LKB1 flox / flox Mouse and smooth muscle-specific expression of Cre recombinase mouse SM22-CreER T2 Mating, get LKB1 flox / flox / Cre + mice, five consecutive days to LKB1 flox / flox / Cre + Mice were intraperitoneally injected with 1 mg of tamoxifen drug (Sigma Company) to induce Cre recombinase to enter the nucleus, thus LKB1 flox / flox / Cre + The sequence between the loxP sites in the mouse is recombined, that is, the LKB1 flox / flox / Cre + The LKB1 gene in the smooth muscle of the mouse is knocked out, and the function of the LKB1 gene is inhibited, thereby obtaining a mouse with a smooth muscle-specific knockout of the LKB1 gene. The mouse is treated with LKB1 SMKO express. figure 1 A strategy for conditional knockout of the LKB1 gene.

[0032] On the 16th day after intraperitoneal injection of tamoxifen, LKB1 SMKO The aorta of the mouse was taken out, and a control mouse (LKB1 injec...

Embodiment 2

[0036] Example 2 Effect of smooth muscle LKB1 gene on mouse abdominal aortic aneurysm

[0037] The double knockout mouse Ldlr that embodiment 1 obtains - / - / LKB1 SMKO and control mice Ldlr without the Cre recombinase gene - / - / LKB1 flox / flox Divided into four groups, with at least 6 mice in each group, processed at the age of 10 weeks. The mice in the four groups and their processing conditions were as follows:

[0038] Group 1: Control mouse Ldlr - / - / LKB1 flox / flox , pump in saline;

[0039] The second group: control mouse Ldlr - / - / LKB1 flox / flox , pumped into AngII;

[0040] The third group: double knockout mouse Ldlr - / - / LKB1 SMKO , pump in saline;

[0041] Group 4: double knockout mouse Ldlr - / - / LKB1 SMKO , pumped into AngII;

[0042] The operation procedure of pumping is as follows: Alzet micropump is subcutaneously implanted on the back of the mouse, and AngII is pumped in at a rate of 1000ng / Kg / min, or the same amount of normal saline is pumped in resp...

Embodiment 3

[0044] Example 3 Preparation of lentivirus against LKB1 gene

[0045] 1. Construction of pFUGW-LKB1 vector:

[0046] 1) The pFUGW plasmid (purchased from Addgene, #14883) was double-digested with endonucleases HindIII and KpnI, and the double-digested product was subjected to agarose gel electrophoresis, and the gel was cut and recovered;

[0047] 2) using human smooth muscle cell cDNA as a template, amplifying and purifying the LKB1 gene fragment by PCR;

[0048] The specific primer nucleotide sequence for PCR amplification is as follows:

[0049] Forward primer: 5'-AAGCTTATGGAGGTGGTGGACCCGCAGC-3';

[0050] Reverse primer: 5'-GGTACCTCACTGCTGCTTGCAGGCCGAC-3';

[0051] The nucleotide sequence of the LKB1 gene is shown in SEQ ID No.1;

[0052] 3) The LKB1 gene fragment obtained in step 2) is double-digested with endonucleases HindIII and KpnI to obtain a double-digestion product of the LKB1 gene;

[0053] 4) Perform agarose gel electrophoresis on the double-digested product...

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Abstract

The invention relates to application of a LKB1 gene in preparation of anti-abdominal-aortic-aneurysm drugs. According to the application of the LKB1 gene in preparation of the anti-abdominal-aortic-aneurysm drugs, important role of the LKB1 gene in regulating expression of matrix metalloproteinase-2 in smooth muscles is disclosed by constructing gene knockout mice with LKB1 gene specifically knocked-out in smooth muscles; and significantly increased incidence of abdominal aortic aneurysm in the LKB1-gene-knockout mice is discovered by constructing abdominal aortic aneurysm models in mice by performing subcutaneous infusion of angiotensin II (Ang II). In addition, lentiviruses with the LKB1 gene are further prepared by the invention. Being injected into tail veins of mice, the lentiviruseswith the LKB1 gene are capable of significantly reducing incidence of abdominal aortic aneurysm in the mice. The anti-abdominal-aortic-aneurysm drugs prepared or screened by using the LKB1 gene as a target according to the invention are of great significance to treatment on abdominal aortic aneurysm.

Description

technical field [0001] The invention relates to the application of LKB1 gene in the preparation of anti-abdominal aortic aneurysm drugs, belonging to the technical field of medicine. Background technique [0002] Abdominal Aortic Aneurysm (AAA for short) refers to a pathological state in which the abdominal aorta exhibits tumor-like expansion locally, and the lumen diameter exceeds 50% of the normal value. It is a potentially fatal disease. Once the tumor ruptures , the mortality rate exceeds 80%. In the United States, the annual incidence of abdominal aortic aneurysm is about 20-40 people / 100,000 people, and about 15,000 people die of abdominal aortic aneurysm every year, accounting for the 13th cause of death. In my country, the incidence of abdominal aortic aneurysm The incidence rate of abdominal aortic aneurysm is also increasing year by year, and the incidence rate of abdominal aortic aneurysm detected by ultrasound in men over 65 years old is as high as 5%. Aneurysma...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K45/00A61P35/00
CPCA61K45/00A61K48/005A61P35/00
Inventor 张文程李洪璇范培红
Owner SHANDONG UNIV QILU HOSPITAL
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