CN114563562A

CN114563562A - Application of PRDX6 in combination with iron ion detection in ARDS process monitoring

Info

Publication number: CN114563562A
Application number: CN202210194782.1A
Authority: CN
Inventors: 杨冬; 王晓岑; 魏婷婷; 郎克; 宋燕莎; 顾兆麟; 宋元林
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2022-05-31

Abstract

The invention relates to an application of PRDX6 in combination with iron ion detection in the monitoring of ARDS processes, belonging to the technical field of biological medicines. The application of PRDX6, iron ions and PRDX6 combined with the iron ions in preparing a diagnostic kit for monitoring the occurrence, development and prognosis of ARDS; monitoring the development process and prognosis of ARDS by detecting the content of PRDX6, iron ions and PRDX6 combined iron ions in the blood or bronchoalveolar lavage fluid of a patient; the medicine can help clinical arrest the progress of ARDS diseases as soon as possible, assist the ARDS process management, and have profound significance for ARDS treatment and prognosis.

Description

Application of PRDX6 in combination with iron ion detection in ARDS process monitoring

Technical Field

The invention relates to application of PRDX6(Peroxiredoxin 6) and iron ion detection in monitoring an ARDS process, belonging to the technical field of biological medicines.

Background

ALI/ARDS (acute lung injury, ARDS) is a common clinical critical condition characterized by protein-rich edema fluid and alveolar space formation as a result of lung and non-lung injury, and is also receiving much attention and attention as a major cause of death in patients with COVID-19 critical condition. In Berlin definition, ARDS is divided into light, medium and heavy degrees, and is defined as when positive end-expiratory pressure (PEEP) or Continuous Positive Airway Pressure (CPAP) is 5cmH or more₂O and 200 < partial oxygen pressure/inhaled oxygen fraction (partial pressure of intrinsic oxygen/fraction of amplified oxygen, PaO)₂/FiO₂) Mild ARDS is not more than 300, when PEEP is not less than 5cmH₂O and 100 < PaO₂/FiO₂Moderate ARDS at 200 or less, when PEEP is 5cmH or more₂O and PaO₂/FiO₂The weight ratio of ARDS is less than or equal to 100. The mortality rate of ARDS is high, especially severe ARDS, and can reach 46 percent. However, at present, there is no early warning index capable of effectively predicting occurrence and development of ARDS clinically, and the progress of the disease cannot be inhibited as soon as possible, so that the patient misses the gold period of treatment, and the death rate of the ARDS patient is increased. Therefore, predicting the onset and progression of ARDS disease is of profound significance for clinical ARDS treatment and prognosis.

Disclosure of Invention

The invention aims to solve the technical problem of how to effectively predict the occurrence and development of ARDS.

In order to solve the problems, the technical scheme adopted by the invention is to provide the application of PRDX6 in preparing a diagnostic kit for monitoring the occurrence and development process of ARDS.

Preferably, the use comprises detecting the level of PRDX6 in the patient's blood or bronchoalveolar lavage fluid.

The invention provides application of PRDX6 in preparing a diagnostic kit for monitoring ARDS prognosis.

The invention provides application of iron ions in preparing a diagnostic kit for monitoring the occurrence and development process of ARDS.

Preferably, the use comprises detecting the level of iron ions in the patient's blood or bronchoalveolar lavage fluid.

The invention provides application of iron ions in preparing a diagnostic kit for monitoring ARDS prognosis.

The invention provides an application of PRDX6 in combination with iron ions in preparing a diagnostic kit for monitoring the occurrence and development process of ARDS.

Preferably, the use comprises detecting the level of PRDX6 and iron ions in the patient's blood or bronchoalveolar lavage fluid.

The invention provides an application of PRDX6 combined with iron ions in preparing a diagnostic kit for monitoring ARDS prognosis.

Compared with the prior art, the invention has the following beneficial effects:

the present invention assists the ARDS process management by detecting the PRDX6 and iron ion content in the patient's blood and BALF (bronchoalveolar lavaging fluid, BALF). The invention can effectively predict the occurrence and development conditions of ARDS, assist the clinical suppression of the disease development as early as possible, improve the survival rate of ARDS patients and have far-reaching significance for ARDS treatment and prognosis. The detection method (1) adopted by the invention has small sample amount required by detection; (2) safety and no toxicity; (3) the accuracy is high.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail as follows:

the technical scheme adopted by the invention is to provide the application of PRDX6 in preparing a diagnostic kit for monitoring the occurrence and development process of ARDS.

The above applications include detecting the level of PRDX6 in the patient's blood or bronchoalveolar lavage fluid.

The invention provides an application of PRDX6 in preparing a diagnostic kit for monitoring ARDS prognosis.

The above applications include the detection of the iron ion content in the patient's blood or bronchoalveolar lavage fluid.

The above applications include the detection of PRDX6 and iron ion content in patient blood or bronchoalveolar lavage fluid.

PRDX6 is a subtype of PRDXs family, and is mainly present in alveolar type II epithelial cells and Clara cells, and catalyzes H in cells₂O₂Reduction of fatty acid hydroperoxides, phospholipid hydroperoxides, and the like, regulate the intracellular oxidation-antioxidant balance. However, more and more studies have found that PRDXs, which originally exert a protective effect inside cells, are likely to aggravate the progression of the disease once they are released outside the cells.

The experimental research result of the invention shows that the level of PRDX6 in BALF of ARDS patients is obviously increased and changed along with the severity of the disease, and the PRDX6 plays an important role in the progress of ARDS.

The results of the clinical study showed that the level of PRDX6 in BALF was significantly increased at different time points, with a peak at day 1 of ARDS, a return to normal at day 3 of ARDS, a return to normal at day 7 of ARDS, and a substantial maintenance of normal control levels at day 14 of ARDS. This curve corresponds to the pathological course of ARDS. PRDX6 is abundant in alveolar type II epithelial cells, and PRDX6 can be secreted into alveolar cavities due to acute-phase (1-6 days) epithelial injury and death, resulting in increased BALF level. In the subacute phase (7-14 days), alveolar edema fluid is absorbed, and PRDX6 secreted into the alveolar space proceeds simultaneously with the absorption. In addition, in subacute conditionsDuring sexual phase, alveolar type ii epithelial cells proliferate, resulting in reduced secretion of PRDX6, therefore, levels of PRDX6 in BALF were similar to the control at day 7 and day 14 of ARDS, suggesting a common result of more absorption and less secretion. Thus, based on data from clinical studies, levels of PRDX6 in BALF were identified to reflect the progression of lung injury. Data on the other hand show that in PaO₂/FiO₂In more severe ARDS patients with lower rates, PRDX6 levels were higher in BALF and PRDX6 concentrations and PaO in BALF₂/FiO₂In negative correlation, PaO₂/FiO₂The lower the ARDS, the more severe the ARDS, the higher the concentration of PRDX6 in BALF. These results suggest that PRDX6 participates in the progression of ARDS, and reflects the severity of ARDS.

In animal experiments, Prdx6 in BALF of ALI mice is significantly higher than that of control mice, and is consistent with the trend of protein concentration in BALF, and the protein concentration can indirectly reflect the severity of lung injury, generally, the higher the protein concentration in BALF is, the more serious lung injury is, and experimental data show that the Prdx6 concentration of the group with higher protein concentration is also high, and the trends of the two are consistent, which indicates that the Prdx6 concentration can reflect the severity of ARDS.

In addition, studies have shown iron ion (Fe) in lung tissue in a mouse model of Acute Lung Injury (ALI)²⁺,Fe³⁺) The content of the iron ion chelate is obviously higher than that of a control group mouse, and the lung injury is obviously reduced after the iron ion chelate, which indicates that the iron ion plays a certain role in promoting ALI development. Ferritin is a form of stored iron and is a key protein for maintaining iron metabolic balance in vivo and regulating iron ion content. Suggesting that there should be some correlation between serum iron content and ARDS severity.

In conclusion, the extracellular PRDX6 and the iron ions are involved in the occurrence and development of ARDS and are positively correlated with the severity of ARDS diseases, and the two are used as combined early warning indexes to predict the ARDS progress more accurately.

PRDX6 concentration detection is based on Enzyme Linked immunosorbent Assay (ELISA) principle. The detection basis of ELISA is immobilization of antigen or antibody and enzyme labeling of antigen antibody. The immobilized antigen-antibody keeps immunocompetence, and the enzyme-labeled antigen-antibody has both immunocompetence and enzyme activity. Specifically combining a sample to be detected with the immobilized antibody or antigen, removing non-binding substances through a washing plate, adding the enzyme-labeled antibody or antigen to be specifically combined with the sample to be detected, removing non-binding substances through washing the plate again, adding a chromogenic substrate combined with the enzyme-labeled antibody, and determining the concentration of the sample to be detected by measuring the absorbance of the chromogenic substance.

The detection of the iron ion concentration is performed based on its own redox characteristics. Iron is a mineral that plays a vital role in many biological life activities, including iron transport and redox reactions. Iron is a transition element that can form a series of redox states, most commonly iron II (Fe)²⁺Or ferrous iron) and iron III (Fe)³⁺Or ferric iron). Iron-containing proteins participate in many reactions, usually chemical reactions that take advantage of the transient changes in the oxidation state of iron.

In the present invention, the sample can be tested directly to measure Fe by releasing the iron by adding an acidic buffer²⁺Or reduced to measure total iron (Fe)²⁺And Fe³⁺). The released iron reacted with the chromium reagent to produce a colorimetric product (593nm) proportional to the iron present. The iron assay kit of the present invention provides a simple and convenient method for measuring iron in various biological samples.

Relevant experiments and results:

1. experimental materials:

2. and (3) experimental detection:

2.1PRDX6 detection:

2.1.1 reagent preparation:

1) the reagents and pre-coated 96-well plates were equilibrated to room temperature before assay.

2) Preparing 1X Wash Buffer: 10X Wash Buffer and ddH₂And fully and uniformly mixing the O according to the proportion of 1:9 to obtain the 1X Wash Buffer. For example, the following steps are carried out: if 50ml of 1X Wash Buffer is required to be prepared, 5ml of 10X Wash Buffer and 45ml of ddH are required₂And (4) fully and uniformly mixing the O.

3) Preparing an Antibody Cocktail: calculating the volume of the actually required Antibody Cocktail according to the sample amount, and uniformly mixing the Capture Antibody, the Detector Antibody and the Antibody Diluent 4BI in the kit according to the ratio of 1:1: 8. For example, 3ml of the Antibody Cocktail may be prepared by gently mixing 300. mu.l of Capture Antibody, 300. mu.l of Detector Antibody and 2.4ml of Antibody dilution 4 BI.

4) Preparing a standard substance:

a. a tube of PRDX6 Recombinant Protein (PRDX6 lysolyzed Recombinant Protein) is taken from the kit, 1ml of Sample Diluent (Sample Diluent NS) is added, and the mixture is gently shaken for 5min at room temperature, namely the PRDX6 Protein standard stock solution with the concentration of 1000 ng/ml.

b. 8 1.5ml EP tubes, numbered 1-8, were removed.

c. 270. mu.l of Sample Diluent (Sample Diluent NS) was added to the EP tube labeled "1", and 150. mu.l of each Sample Diluent was added to the EP tubes labeled 2-8.

d. 30 mu.l of 1000ng/ml PRDX6 protein standard stock solution is added into an EP tube No. 1 and mixed evenly, then 150 mu.l of solution is taken out from the EP tube No. 1 and added into an EP tube No. 2 and mixed evenly, then 150 mu.l of solution is taken out from the EP tube No. 2 and added into an EP tube No. 3 and mixed evenly, and the like.

e. The concentrations of the prepared standard substances are respectively 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.13ng/ml, 1.56ng/ml and 0 ng/ml.

2.1.2. And (3) detection:

1) taking out a proper amount of pre-coated pore plates according to the sample amount;

2) sequentially adding the standard substance and the sample into the pre-coated plate, wherein each well is 50 mu l;

3) adding 50 mul of prepared Antibody Cocktail into each hole;

4) sealing the pore plate by using sealing plate gummed paper, placing on a shaker, rotating at 400rpm, and incubating for 1h at room temperature;

5) washing the plate: mu.l of 1 XWash Buffer was added to each well for 1min each time for 3 washes. The Wash Buffer is discarded completely every time the board is washed, and the board can be patted dry on absorbent paper;

6) adding 100 μ l of TMB into each well, and incubating for 4min in the dark;

7) after the Solution in the well plate turns blue, 100 μ l of Stop Solution is added into each well immediately, and the absorbance is measured at the wavelength of 450nm by a microplate reader.

8) Standard curves were plotted and the concentration of PRDX6 was calculated for each sample based on the standard curve.

Experimental findings indicate that PRDX6 levels in BALF of ARDS patients significantly increased and varied with the severity of the disease.

In PaO₂/FiO₂In more severe ARDS patients with lower rates, PRDX6 levels were higher in BALF and PRDX6 concentrations and PaO in BALF₂/FiO₂In negative correlation, PaO₂/FiO₂The lower the ARDS, the more severe the ARDS, the higher the concentration of PRDX6 in BALF.

The concentration of Prdx6 in BALF of ALI mice is obviously higher than that of control mice, and is consistent with the protein concentration trend in BALF; the experimental data show that the group with higher protein concentration has high Prdx6 concentration, and the trend of the Prdx6 concentration is consistent with that of the group with higher protein concentration.

2.2 iron ion detection:

2.2.1 reagent preparation:

1) the reagents were equilibrated to room temperature before testing.

2) Preparing a standard substance: mu.l of 100mM standard iron ion solution was added to 990. mu.l of water to dilute the solution to 1 mM.

2.2.2 detection:

1) 0, 2, 4, 6, 8 and 10 mu l of 1mM standard iron ion solution are respectively added into a 96-well plate, then the volume of each well solution is made up to 100 mu l by using an iron detection buffer solution, and finally 5 mu l of an iron ion reducing agent is added into each standard well.

2) Adding 1-50 μ l of the sample to be tested to a 96-well plate, and washing each well with iron detection bufferThe volume of the solution was made up to 105. mu.l, and Fe was detected therefrom²⁺The concentration of (c).

3) Adding 1-50 μ l of sample to be detected into a 96-well plate, supplementing the volume of solution of each well to 100 μ l with iron detection buffer solution, and finally adding 5 μ l of iron ion reducing agent into each sample well to detect the total iron concentration.

4) The 96-well plate was placed on a shaker and incubated for 30min at room temperature in the dark.

5) Mu.l of iron ion probe was added to each of the standard well and the sample well, and incubated for 60min at room temperature in the dark.

6) Absorbance was measured at a wavelength of 593nm with a microplate reader.

The experiment shows that: in mouse Acute Lung Injury (ALI) model, iron ion (Fe) is in lung tissue² ⁺,Fe³⁺) The content of the iron chelate is obviously higher than that of a control group of mice, and the lung injury is obviously reduced after the iron ions are chelated.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalents to the disclosed technology without departing from the spirit and scope of the present invention, and all such changes, modifications and equivalents are intended to be included therein as equivalents of the present invention; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims

Use of PRDX6 for the preparation of a diagnostic kit for monitoring the development of ARDS.
2. Use according to claim 1, characterized in that: the use comprises detecting the level of PRDX6 in the patient's blood or bronchoalveolar lavage fluid.
Use of PRDX6 in the manufacture of a diagnostic kit for monitoring the prognosis of ARDS.
4. Use of ferric ions for the manufacture of a diagnostic kit for monitoring the development of ARDS.
5. Use according to claim 4, characterized in that: the use comprises detecting the level of iron ions in the blood or bronchoalveolar lavage fluid of a patient.
6. Use of iron ions in the manufacture of a diagnostic kit for monitoring the prognosis of ARDS.
Use of PRDX6 in combination with iron ions for the manufacture of a diagnostic kit for monitoring the development of ARDS.
8. Use according to claim 7, characterized in that: the use comprises detecting the levels of PRDX6 and iron ions in the patient's blood or bronchoalveolar lavage fluid.
Use of PRDX6 in combination with iron ions for the manufacture of a diagnostic kit for monitoring the prognosis of ARDS.