A kit for predicting aortic aneurysm growth and rupture risk based on protein lactylation levels

Abstract

The present invention discloses a kit for predicting the growth and rupture risk of aortic aneurysms based on protein lactylation levels. The present invention constructs an aortic aneurysm and dissection mouse model, detects the protein lactylation level in its vascular tissue, and divides it into a high lactate level group and a low lactate level group. It is found that the formation of aortic aneurysms and the occurrence of aortic dissection are reduced in mice with low lactate levels. Further survival analysis shows that the survival curves between the two groups are significantly different. Therefore, on the basis of aortic aneurysm detection indicators, with protein lactylation level as the entry point, by identifying protein lactylation level, the protein lactylation level is associated with the growth and rupture risk of aortic aneurysms, thereby achieving the purpose of early prediction of aortic aneurysm deterioration and dissection occurrence, which is helpful for the prediction and early diagnosis and treatment of clinical critical events, and reduces the occurrence of serious adverse events such as dissection.

Description

Technical Field

[0001] The present invention relates to a kit for predicting the growth and rupture risk of aortic aneurysms based on protein lactylation levels, and belongs to the technical field of biomedical detection. Background Art

[0002] Aortic aneurysm / dissection (AA / AD) is a clinical emergency with high morbidity and mortality. AA / AD occurs when the inner layer (intima) of the aortic wall ruptures, allowing blood to enter the aortic media through the tear. The global prevalence of AA / AD is 1.3%-8%. Early screening of high-risk individuals (including those with high-risk factors such as hypertension) has been shown to be effective in preventing AA-related deaths, making early diagnosis and screening of AA / AD particularly necessary.

[0003] Post-translational protein modification (PTM) is the further regulation of proteins after translation. Recent studies have identified lactate-derived protein lactylation as a newly discovered important modification that can affect protein function. Interestingly, lactate significantly impacts protein structure and function. In tumors, it has a profound impact on immune cells, affecting the differentiation and maturation of anti-inflammatory macrophages, thereby impairing tumor immune evasion and killing. Lactate levels are closely associated with tumor development and poor prognosis. However, there are currently no reports on predicting aortic aneurysm growth and rupture risk by measuring protein lactate levels. Summary of the Invention

[0004] The purpose of the present invention is to solve the technical problem of how to predict the growth and rupture risk of aortic aneurysm to achieve early screening of aortic aneurysm / dissection.

[0005] In order to achieve the purpose of solving the above technical problems, the technical solution adopted by the present invention is to provide a reagent for detecting protein lactylation level for use in preparing a kit for predicting the growth and rupture risk of aortic aneurysms.

[0006] Preferably, the test sample is diseased vascular tissue.

[0007] Preferably, the reagent for detecting the protein lactylation level is a reagent used for detecting the total protein lactylation level using a pan-lactylation antibody or an LC-MS / MS method.

[0008] Preferably, the reagents used for detecting the total protein lactylation level using pan-lactylation antibodies include: a protein extraction kit and detection antibodies, and the detection antibodies include a primary anti-lysine residue lactylation antibody and a goat anti-rabbit secondary antibody.

[0009] Preferably, the reagents used in detecting the total protein lactylation level using the LC-MS / MS method include: a protein extraction kit and an internal standard.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention is based on the correlation between protein lactylation levels and the growth and rupture risk of aortic aneurysms, providing a theoretical basis for the application of lactation level detection in the clinical prediction of aortic aneurysm prognosis. In clinical practice, the total protein lactylation level in the diseased vascular tissue of aortic aneurysm patients is detected to screen for high cardiovascular event risks, which can supplement previous clinical indicators and achieve the purpose of early prediction of aortic aneurysm deterioration and dissection. It is helpful for the prediction and early diagnosis and treatment of clinical critical events, and reduces the occurrence of serious adverse events such as dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the survival difference curve between the high lactate level group and the low lactate level group of mice (the survival rate of the high lactate level group was significantly lower than that of the low lactate level group, P < 0.05);

[0013] Figure 2 Receiver operating characteristic (ROC) curve for predicting aortic aneurysm growth and rupture risk by protein lactylation level. DETAILED DESCRIPTION

[0014] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0016] Among them, the method for detecting the level of protein lactylation is as follows:

[0017] (1) Use a protein extraction kit to extract total protein and determine the protein concentration;

[0018] (2) Measure the total protein lactylation level by western blot. The primary antibody can be a lysine residue lactylation antibody (in QuickBlock TM Western blot was performed using a primary antibody dilution of 1:1000 in Western blot buffer and incubated overnight at 4°C. The secondary antibody was a goat anti-rabbit IgG (H+L) peroxidase-conjugated antibody diluted 1:10,000 in TBST buffer with 5% blocking milk for 2 hours at room temperature. Bands were quantified using a VILBER Fusion Solo S to determine pan-lactylation levels.

[0019] Example

[0020] 1) Experimental animals and animal model preparation: SPF-grade adult male C57BL / 6 mice (8 weeks old, 20-25g) were used. The mice were subcutaneously infused with AngⅡ (2500ng / kg·min) and 0.9% saline with an osmotic volume of 0.5μl / hr using a subcutaneously implanted micro-osmotic sustained-release pump. The pump was continuously pumped for 28 days to construct an aortic aneurysm and dissection model. The mice were divided into WT+Control group and WT+AngⅡ drug group, and the survival curve of the mice was recorded daily. After 28 days, the mice were euthanized and the tissues were fixed and embedded for paraffin sectioning and H&E and EVG staining were performed. The aortic elastic fiber tear and false lumen formation were observed under a microscope. The experimental protocol was approved by the Animal Care and Ethics Committee of Fudan University.

[0021] 2) Sample collection to observe aortic dilatation and tumor formation and detect protein lactic acid levels

[0022] Blood vessels were collected by opening the abdominal cavity and removing the blood vessels. The blood vessels were placed in a 10 cm culture dish containing pre-cooled PBS buffer on ice, and the excess tissue was quickly cut off. The excess PBS was slightly absorbed on gauze, and then weighed and recorded. Care was taken to avoid squeezing, bruising, and drying. Evidence was taken using a Leica developing microscope to observe the aortic aneurysm formation and vascular dilation. A tissue of about 0.5 cm in size was collected from the same part of each mouse's individual vascular tissue, and the total protein lactate level was detected after protein extraction. According to the test results, the mice were divided into two groups according to the median lactate modification level: low-level group (standardized value 1±0.23) vs high-level group (standardized value 2.34±0.45). The results showed that mice with low lactate levels had reduced aortic aneurysm formation and reduced aortic dissection. Further survival analysis showed that the survival curves of the high lactate level group and the low lactate level group were significantly different, as shown in the following figure: Figure 1 shown.

[0023] By calculating the relationship between lactic acid level and aortic growth and rupture, an ROC curve was generated. The growth of aortic aneurysms was predicted by lactic acid level, with a C value of 0.76 and an optimal cutoff value of (1.56 mg / ml). Figure 2 This indicates that the detection of lactic acidification level has good sensitivity and specificity in predicting the growth and rupture risk of aortic aneurysm.

[0024] By testing the protein lactation level in the patient's lesion tissue after surgery and following up (for one year) to compare the actual patient conditions, it was found that patients with higher lactation levels (greater than 1.56) had a higher risk of aortic aneurysm regrowth and rupture, with an incidence rate of 15%. Patients with lower lactation levels (less than 1.56) had a lower risk of aortic aneurysm growth and rupture, with an incidence rate of 0%, with a statistically significant difference (P < 0.05). Therefore, the present invention accurately and reliably assesses the prognosis of aneurysm regrowth and rupture risk by testing the lactation level in the patient's vascular lesion site, and can be used as an early screening method for re-rupture risk detection.

[0025] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the total protein lactylation level in diseased vascular tissue in the preparation of a kit for predicting the growth and rupture risk of aortic aneurysms.

2. The use according to claim 1, characterized in that The reagent for detecting the total protein lactylation level in diseased vascular tissue is a reagent used for detecting the total protein lactylation level using a pan-lactylating antibody or an LC-MS / MS method.

3. The use according to claim 2, characterized in that The reagents used for detecting the total protein lactylation level using the pan-lactylation antibody include: a protein extraction kit and a detection antibody, wherein the detection antibody includes a primary anti-lysine residue lactylation antibody and a goat anti-rabbit secondary antibody.

4. The use according to claim 2, characterized in that The reagents used in the LC-MS / MS method for detecting the total protein lactylation level include: a protein extraction kit and an internal standard.

Images