CN114397439B - Application of sonic hedgehog factor in the preparation of auxiliary diagnostic reagents for aortic dissection - Google Patents

Abstract

The invention discloses application of sonic hedgehog in preparing an aortic dissection auxiliary diagnostic reagent. The application of sonic hedgehog as biomarker in preparing auxiliary diagnosis reagent for aortic dissection. The application of the reagent for quantitatively detecting the sonic hedgehog in preparing an aortic dissection auxiliary diagnostic reagent. The reagent for quantitatively detecting the sonic hedgehog is ELISA reagent for detecting the sonic hedgehog. The invention discovers for the first time that the sonic hedgehog (SHH) can be used as a biomarker for the auxiliary diagnosis of aortic dissection. SHH can be used as a biomarker to distinguish healthy persons from aortic dissection patients well. SHH as biomarker has 0.9445 area under ROC curve when differentiating normal control group and aortic dissection group, 74.84 specificity, and has good diagnostic value for aortic dissection.

Description

Application of sonic hedgehog factor in the preparation of auxiliary diagnostic reagents for aortic dissection

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of Sonic hedgehog (SHH) in the preparation of auxiliary diagnostic reagents for aortic dissection.

Background technique

Aortic dissection (Aortic dissection, AD) refers to a fatal vascular disease in which blood flows into the middle layer of the aortic wall through the tear port to form a dissection hematoma caused by various factors, and the false lumen extends along the aortic wall to both sides with the systole of the heart. AD has the characteristics of critical condition and high mortality, which seriously threatens the safety of human life. Therefore, early and accurate diagnosis is the key. At present, the diagnosis of AD mainly relies on imaging methods, but it lacks timeliness, convenience and rapidity. Serological examination has become an important clinical examination method because of its sensitivity, simplicity and economy, and is widely used in the diagnosis of many diseases.

The biomarkers reported in recent years for the diagnosis of AD mainly include: molecular markers related to hypertension such as pro-inflammatory factors; molecular markers related to atherosclerotic inflammatory response such as interleukin-6 (IL-6), Interleukin-8 (IL-8), plasma amyloid A protein (SAA); molecular markers associated with aortic wall matrix degradation such as matrix metalloproteinases (MMPs), extracellular matrix glycoprotein Tenascin-C (TNC); immune-related molecular markers such as soluble ST-2 (soluble ST-2, sST-2), Toll-like receptors (Toll-like receptors, TLRs). However, due to the unsatisfactory sensitivity and specificity, these molecular markers have not been promoted clinically. Therefore, it is imminent to find more accurate and sensitive AD biomarkers.

Sonic hedgehog (SHH) is a secreted protein and an important member of the Hedgehog (Hh) family, which plays an important role in angiogenesis and occurrence, and is involved in pancreatic cancer, liver cancer, atherosclerosis, Abnormal expression can be seen in pathological conditions such as heart disease. However, its diagnostic and predictive role in the development of AD has not been elucidated, and further exploration is needed.

Contents of the invention

The purpose of the present invention is to provide a biomarker for detecting aortic dissection and its application, so as to provide a scientific basis for early clinical diagnosis and prognosis evaluation of AD.

The purpose of the present invention can be achieved through the following technical solutions:

Application of sonic hedgehog factor as a biomarker in the preparation of auxiliary diagnostic reagents for aortic dissection.

The application of the reagent for quantitative detection of sonic hedgehog factor in the preparation of auxiliary diagnostic reagents for aortic dissection.

As a preference of the present invention, the reagent for quantitatively detecting Sonic Hedgehog is an ELISA reagent for detecting Sonic Hedgehog.

Application of a reagent for quantitative detection of sonic hedgehog factor in the preparation of early warning reagents for patients with aortic dissection.

As a preference of the present invention, the reagent for quantitatively detecting Sonic Hedgehog is an ELISA reagent for detecting Sonic Hedgehog.

The method for distinguishing patients with aortic dissection from normal people includes the following steps:

(1) Collect serum samples from patients to be tested, and use normal human serum samples as controls;

(2) Detect the content of biomarkers in the serum samples of the patient to be tested and the normal person, and compare the obtained results;

(3) Indicating whether the subject to be tested is a patient with aortic dissection according to the comparison result; if the content of the biomarker used in the serum sample of the subject to be tested is higher than that of the control, it indicates that the subject to be tested has aortic dissection.

Preferably, ELISA method is used to detect the content of biomarkers in serum samples.

Beneficial effect:

The present invention finds for the first time that Sonic Hedgehog (SHH) can be used as a biomarker for auxiliary diagnosis of aortic dissection. As a biomarker, SHH can well distinguish healthy people from patients with aortic dissection. The area under the ROC curve of SHH as a biomarker in distinguishing the normal control group from the aortic dissection group was 0.9445, the sensitivity was 96.23%, and the specificity was 74.84%, which has a good diagnostic value for aortic dissection.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings required in the embodiments will be briefly introduced below.

Figure 1 is the expression level concentration value of SHH as a biomarker in the normal control group and the aortic dissection group;

Figure 2 is the ROC curve of the comparative analysis of SHH as a biomarker between the normal control group and the aortic dissection group.

Sensitivity is the ordinate: Sensitivity, True positive rate (TPR), the proportion of samples that are predicted to be positive and actually positive to all positive samples. The specificity is the abscissa: Specificity, false positive rate (False positive rate, FPR), the proportion of samples that are predicted to be positive but actually negative to all negative samples.

Detailed ways

Example 1 Serum marker sample test in patients with aortic dissection

Aiming at the possibility that the up-regulation of shh and the activation of the Hedgehog signaling pathway found in our experiments may be involved in the occurrence of AD, we conducted a serum marker sample test and randomly divided them into groups according to gender and age, selecting 160 healthy people as controls and 160 patients with aortic dissection , ELISA experiments were performed to verify whether SHH could determine whether patients had aortic dissection. The experimental steps are as follows:

(1) Preparatory work before testing

①Take out the kit and samples from the refrigerator 1 hour in advance, and equilibrate to room temperature.

②Dilution of 20× washing buffer: dilute with distilled water at 1:20, that is, 1 part of 20× washing buffer plus 19 parts of distilled water.

③ Reagents and consumables required for the experiment:

a. ELISA kit, as shown in Table 1:

Table 1 ELISA kit composition

Kit composition Specifications (96T) Pre-coated microtiter plates 8×12 strips Standard 1 stick Standard/Sample Diluent 15ml Biotinylated Detection Antibody (100×) 120ul Biotinylated Detection Antibody Diluent 12ml SABC complex 12ml TMB Chromogenic Solution 6ml stop solution 12ml 20× concentrated washing liquid 60ml Sealing tape 4 sheets Product Manual 1 copy

b. Required self-provided experimental equipment: microplate reader (450nm); 37 ℃ incubator.

c. Operation steps

a) Take out the strips from the aluminum foil bag after equilibrating at room temperature for 20 minutes.

b) Set up standard wells and sample wells, add 50 μL of standard substances of different concentrations to each of the standard wells; add 50 μL of the sample to be tested into the sample wells; do not add to the blank wells, and incubate for 40 min in a 37°C incubator.

c) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let it stand for 2 minutes, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times.

d) In addition to the blank wells, add 100 μL of biotinylated antibody working solution to each well of standard wells and sample wells, and incubate in a 37°C incubator for 20 minutes.

e) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let it stand for 2 minutes, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times.

f) Add 100 μL of SABC complex working liquid to each well, and incubate in a 37° C. incubator for 20 minutes.

g) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let it stand for 2 minutes, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times.

h) Add 100 μL of TMB mixture to each well, and incubate at 37° C. in the dark for 20 minutes.

i) Add 100 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 30 min.

d. Through the above-mentioned ELISA method, SHH was used as a biomarker to analyze and detect the aortic dissection group and the normal group, and the obtained data was analyzed by Prism8.0 statistical software and the receiver operating characteristic (ROC) curve was drawn to clarify the novel Whether the marker can become a new AD diagnostic marker for clinical application, and at the same time explore the diagnostic value of combined indicators for AD.

(2) Analysis of results

As shown in Figure 1, this figure shows the expression level and concentration value of SHH as a biomarker in the normal control group and the aortic dissection group. The results show that SHH can well distinguish healthy people from patients with aortic dissection.

The ELISA results were analyzed by the ROC curve, as shown in Figure 2 and Table 2, the ROC curve of SHH as a biomarker in distinguishing the normal control group from the aortic dissection group, the results showed that the areas under the curves were greater than 0.9, and the aortic Dissection has good diagnostic value.

Table 2 ROC statistical results when SHH is used to distinguish healthy people from patients with aortic dissection

Claims (5)

1. The application of sonic hedgehog as biomarker in preparing auxiliary diagnosis reagent for aortic dissection.

2. The application of the reagent for quantitatively detecting the sonic hedgehog in preparing an aortic dissection auxiliary diagnostic reagent.

3. The use according to claim 2, wherein the reagent for quantitatively detecting sonic hedgehog is an ELISA reagent for detecting sonic hedgehog.

4. The application of the reagent for quantitatively detecting the sonic hedgehog in preparing early warning reagent for aortic dissection patients.

5. The use according to claim 4, wherein the reagent for quantitatively detecting sonic hedgehog is an ELISA reagent for detecting sonic hedgehog.