CN114397439B

CN114397439B - Application of sonic hedgehog in preparing aortic dissection auxiliary diagnostic reagent

Info

Publication number: CN114397439B
Application number: CN202210044219.6A
Authority: CN
Inventors: 季勇; 张佳; 林兰欣; 唐欣; 谢利平
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2023-09-05
Anticipated expiration: 2042-01-14
Also published as: CN114397439A

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 in preparing aortic dissection auxiliary diagnostic reagent

Technical Field

The invention belongs to the technical field of medicine, and particularly relates to application of Sonic hedgehog (SHH) in preparing an aortic dissection auxiliary diagnostic reagent.

Background

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 lacerated orifice to form a dissection hematoma, and the dissection extends to both sides along the aortic wall along with the heart contraction false cavity. AD has the characteristics of critical illness state and high mortality rate, and seriously threatens the life safety of human beings. Thus, early, accurate diagnosis is critical. At present, diagnosis of AD mainly depends on imaging means, but the diagnosis lacks timeliness, convenience and rapidity. Serological examination is widely used for diagnosing various diseases because of its sensitivity, simplicity and economy.

Biomarkers for diagnosing AD reported in recent years are mainly: molecular markers associated with hypertension such as pro-inflammatory factors; molecular markers associated with the atherosclerotic inflammatory response such as interleukin-6 (IL-6), interleukin-8 (IL-8), plasma amyloid a (serum amyloid A protein, SAA); molecular markers associated with aortic wall matrix degradation such as Matrix Metalloproteinases (MMPs), extracellular matrix glycoprotein Tenascin-C (TNC); molecular markers associated with immunity such as soluble ST-2 (sST-2), toll-like receptors (TLRs). However, these molecular markers have not been clinically generalized due to the non-ideal sensitivity and specificity. Thus, it is urgent to search for more accurate and sensitive biomarkers of AD.

Sonic Hedgehog (SHH), a secreted protein, is an important member of the Hedgehog (Hh) family, which plays an important role in angiogenesis and genesis, and is seen in abnormal expression under pathological conditions such as pancreatic cancer, liver cancer, atherosclerosis, ischemic heart disease, etc. However, the diagnostic and predictive roles of the compounds in the development of AD have not been elucidated, and need further investigation.

Disclosure of Invention

The invention aims to provide a biomarker for detecting aortic dissection and application thereof, which provide scientific basis for clinical early diagnosis and prognosis evaluation of AD.

The aim of the invention can be achieved by the following technical scheme:

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.

Preferably, the reagent for quantitatively detecting sonic hedgehog is an ELISA reagent for detecting sonic hedgehog.

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

The method for distinguishing aortic dissection patients from normal persons comprises the following steps:

(1) Collecting serum samples of patients to be tested, and taking normal human serum samples as a control;

(2) Detecting the content of the biomarker in serum samples of a patient to be detected and a normal person, and comparing the obtained results;

(3) Indicating whether the patient to be tested is an aortic dissection patient according to the comparison result; and if the content of the biomarker used in the serum sample of the object to be detected is higher than that of the control, indicating that the object to be detected is an aortic dissection.

Preferably, the amount of biomarker in the serum sample is detected by ELISA.

The beneficial effects are that:

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 a ROC curve area under 0.9445, sensitivity of 96.23%, specificity of 74.84% when distinguishing normal control group from aortic dissection group, and has good diagnostic value for aortic dissection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following description will make brief description of the embodiments using the drawings.

FIG. 1 shows the concentration values of SHH as biomarker expressed in normal control and aortic dissection groups;

fig. 2 is a ROC curve of SHH as a biomarker for comparative analysis between normal control and aortic dissection.

Sensitivity is the ordinate: the Sensitivity, true class ratio (True positive rate, TPR), predicts positive and actually positive samples to all positive samples. Specificity is the abscissa: specificity, pseudo-positive class ratio (False positive rate, FPR), the proportion of samples predicted to be positive but actually negative to all negative samples.

Detailed Description

Example 1 aortic dissection patient serum marker sample test

For the up-regulation of SHH and activation of Hedgehog signal pathway found in our experiments to possibly participate in the occurrence of AD, we performed serum marker sample tests, randomly grouped according to gender and age, selected 160 healthy persons as controls, and performed ELISA experiments to verify whether SHH can determine whether patients have aortic dissection. The experimental procedure was as follows:

(1) Preparation before detection

(1) Taking out the kit and the sample from the refrigerator 1h in advance, and balancing to room temperature.

(2) Dilution of 20 x wash buffer: distilled water was diluted 1:20, i.e., 1 part 20 Xwash buffer plus 19 parts distilled water.

(3) Reagent and consumable material required by experiment:

ELISA kit as in Table 1:

TABLE 1 ELISA kit composition

Kit composition	Specification (96T)
		Pre-coated ELISA plate	8X 12 strips
Standard substance	1 branch
		Standard/sample diluent	15ml
Biotinylated detection antibody (100×)	120ul
		Biotinylation detection antibody diluent	12ml
SABC complex	12ml
		TMB color development liquid	6ml
Stop solution	12ml
		20 Xconcentrated washing liquid	60ml
Sealing plate gummed paper	4 pieces of
		Product instruction	1 part of

b. The required self-contained experimental equipment is as follows: enzyme label instrument (450 nm); a 37 ℃ incubator.

c. Operating procedure

a) The strips were removed from the aluminum foil bags after 20min equilibration at room temperature.

b) Setting a standard substance hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are added into the standard substance hole; adding 50 mu L of a sample to be detected into the sample hole; blank wells were not added and incubated for 40min at 37℃in an incubator.

c) The liquid was discarded, the absorbent paper was dried by beating, each well was filled with washing liquid (350. Mu.L), left to stand for 2min, the washing liquid was thrown off, the absorbent paper was dried by beating, and the plate washing was repeated 5 times.

d) Except for blank wells, 100. Mu.L of biotinylated antibody working solution was added to each of the standard wells and sample wells, and incubated at 37℃for 20min.

e) The liquid was discarded, the absorbent paper was dried by beating, each well was filled with washing liquid (350. Mu.L), left to stand for 2min, the washing liquid was thrown off, the absorbent paper was dried by beating, and the plate washing was repeated 5 times.

f) 100. Mu.L of SABC complex working liquid was added to each well and incubated at 37℃for 20min.

g) The liquid was discarded, the absorbent paper was dried by beating, each well was filled with washing liquid (350. Mu.L), left to stand for 2min, the washing liquid was thrown off, the absorbent paper was dried by beating, and the plate washing was repeated 5 times.

h) 100. Mu.L of TMB mixture was added to each well and incubated at 37℃for 20min in the absence of light.

i) Each well was filled with 100. Mu.L of a stop solution for 30min, and the OD of each well was measured at a wavelength of 450 nm.

d. According to the ELISA method, SHH is used as a biomarker to analyze and detect an aortic interlayer group and a normal group, the obtained data are analyzed by prism8.0 statistical software, a subject working characteristic (ROC) curve is drawn, whether a novel marker can be used as a novel AD diagnosis marker applied to clinic is determined, and meanwhile, the value of a combined index on AD diagnosis is explored.

(2) Analysis of results

As shown in fig. 1, which shows the values of the expression level concentration of SHH as a biomarker in the normal control group and the aortic dissection group, the results show that: SHH can well distinguish healthy from aortic dissection patients.

The ELISA results were analyzed using ROC curve, as shown in fig. 2 and table 2, the ROC curve of SHH as biomarker when differentiating normal control group from aortic sandwich group, and the results showed that: the area under the curve is larger than 0.9, and has good diagnostic value for aortic dissection.

TABLE 2 ROC statistics for SHH used to distinguish healthy from aortic dissection patients

Claims

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.