Reagent screening method for prognosis effect of acute respiratory distress syndrome

Abstract

The invention provides a reagent screening method for the prognosis effect of acute respiratory distress syndrome, which comprises the following steps: reserving serum specimens of normal subjects and ARDS patients, carrying out bronchofiberscope examination, and reserving alveolar lavage fluid specimens of the normal subjects and ARDS patients; based on the human alveolar lavage fluid specimen, performing ELISA detection on TERT protein; based on an ELISA detection result, comparing the TERT protein of the ARDS patient with the TERT protein of a healthy control; the TERT protein of an ARDS patient is higher than that of a healthy control, and the ARDS patient has the risk of death; the TERT protein of the ARDS patient is equal to the TERT protein of a healthy contrast, and the ARDS patient tends to recover.

Description

Technical Field

[0001] The present invention relates to 生物医药 The technical field relates to a method for screening reagents for the prognostic effect of acute respiratory distress syndrome. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is a clinical syndrome caused by intrapulmonary and / or extrapulmonary causes, with refractory hypoxemia as a significant feature, and has attracted much attention due to its high mortality rate. ARDS was previously also known as acute lung injury (ALI). In 2012, the Berlin Definition used ARDS for clinical use (Acute respiratory distress syndrome: the Berlin Definition, JAMA, 2012, 307(23): 2526-2533), and ALI was mainly used to simulate animal models and basic research of ARDS. Uncontrolled inflammatory response and abnormal accumulation of pulmonary fluid are the core pathophysiological changes of ALI / ARDS, and accurate assessment of ARDS / ALI pulmonary fluid imbalance can provide important evaluation value indicators and reference systems for ARDS / ALI diagnosis and treatment, prognosis assessment and research.

[0003] Under the influence of pathogenic factors inside and outside the lungs, the expression and function of tight junction proteins between pulmonary microvascular endothelial cells are inhibited, the endothelial barrier function is weakened, and a large amount of protein-rich fluid in the blood vessels is extravasated into the alveolar cavity and pulmonary interstitium. At the same time, the expression of cyclic adenosine monophosphate in alveolar epithelial cells is downregulated, the expression and function of ion channel proteins (including sodium ion channels, sodium potassium ATPase, etc.) are impaired, and alveolar fluid reabsorption is impaired. The combined effect of the two leads to an imbalance of lung fluid, which accumulates to form pulmonary edema. The large amount of high-protein fluid that accumulates reduces the effective respiratory area of ​​the alveoli, further aggravates the inflammatory response, and promotes the occurrence of ARDS / ALI.

[0004] Through research, it was found that TERT protein can be upregulated in ARDS through multiple mechanisms, including gene amplification, rearrangement, and mutations within the promoter. TERT is also amplified in about 4% of cancers, especially ovarian cancer, lung adenocarcinoma, lung squamous cell carcinoma, esophageal cancer, and adrenocortical carcinoma. The mechanism by which TERT promoter mutations ultimately promote the severity of ARDS is still being elucidated. Although initial studies used reporter gene analysis to demonstrate increased TERT expression, the increase in TERT expression appeared to be relatively mild, and some studies failed to find a significant association between TERT promoter status and TERT expression. Through patient testing and evaluation, we found that the method of detecting TERT protein in bronchoalveolar lavage fluid has a certain predictive effect on the assessment of lung fluid imbalance and prognosis in ARDS / ALI. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for screening a reagent for the prognostic effect of acute respiratory distress syndrome, which solves the problem of preparing a reagent for detecting TERT protein in alveolar lavage fluid to screen acute respiratory distress syndrome / acute lung injury and / or evaluate the prognostic effect of acute respiratory distress syndrome / acute lung injury.

[0006] To achieve the above object, the present invention proposes a method for screening a reagent for the prognostic effect of acute respiratory distress syndrome, the method comprising:

[0007] Serum samples were collected from normal subjects and ARDS patients, and bronchoalveolar lavage fluid samples were collected by bronchofiberoptic examination.

[0008] Based on the bronchoalveolar lavage fluid specimens of the above-mentioned people, ELISA detection was performed for TERT protein;

[0009] Based on the ELISA test results, the TERT protein in ARDS patients was compared with the TERT protein in healthy controls;

[0010] The TERT protein in ARDS patients is higher than that in healthy controls, and ARDS patients are at risk of death;

[0011] The TERT protein in ARDS patients was equal to that in healthy controls, and ARDS patients tended to recover.

[0012] Furthermore, the ELISA detection step includes:

[0013] Add 100 μL of standard or sample to the 96-well plate (coated with TERT antibody), seal the plate, and incubate at 37 degrees for 2 hours with shaking;

[0014] Wash the plate three times with 300 μL of plate washing buffer;

[0015] Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37 degrees with shaking for 1 hour;

[0016] Wash the plate three times with 300 μL of plate washing buffer;

[0017] Add 50 μL of color development solution to each well and shake to mix at room temperature for 20 min;

[0018] Add 50 μL of stop solution to each well and shake at room temperature to mix;

[0019] The plate was read within 30 minutes (Bio-Rad). The TERT protein level in healthy controls was 0.23±0.05 ng / ml, and that in ARDS patients was 1.25±0.32 ng / ml. Compared with healthy controls, the TERT protein level in ARDS patients was significantly increased (p<0.0001).

[0020] Furthermore, the detection method in the sample includes an immunological method, a fluorescent method, a flow cytometry, a latex turbidimetry, a biochemical method, an enzymatic method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method, or a photoelectric conversion method.

[0021] In the embodiments of the present invention, TERT protein can be used to effectively screen ARDS patients and effectively evaluate the prognosis of ARDS patients. The screening and evaluation method using TERT protein in the present invention has good stability, excellent sensitivity, specificity and accuracy, and good clinical application prospects. DETAILED DESCRIPTION

[0024] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0025] Example 1: Detection and Use of TERT Protein in Bronchoalveolar Lavage Fluid of ARDS / ALI Patients 1. Detection Subjects

[0026] There were 38 normal subjects (i.e. healthy controls) and 41 ARDS patients. After 28 days of follow-up, 12 ARDS patients died. All subjects were informed and agreed before the experiment.

[0027] 2. Sample collection and testing

[0028] (1) Serum samples were collected from normal subjects and ARDS patients, and bronchoalveolar lavage fluid samples were collected by bronchofiberoptic examination.

[0029] (2) Based on the bronchoalveolar lavage fluid specimen of the above-mentioned person, ELISA detection is performed on TERT protein, which specifically includes the following steps:

[0030] (1) Add 100 μL of standard or sample to a 96-well plate (coated with TERT antibody), seal the plate, and incubate at 37°C for 2 hours with shaking;

[0031] (2) Wash the plate three times with 300 μL of washing buffer;

[0032] (3) Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37°C with shaking for 1 hour;

[0033] (4) Wash the plate three times with 300 μL of washing buffer;

[0034] (5) Add 50 μL of color development solution to each well and shake at room temperature for 20 min;

[0035] (6) Add 50 μL of stop solution to each well and shake at room temperature to mix;

[0036] (7) Read the plate within 30 min (Bio-Rad).

[0037] 3. Test results

[0038] (1) TERT protein in ARDS patients was significantly higher than that in healthy controls

[0039] The TERT protein level of healthy controls was 0.23±0.05ng / ml, and that of ARDS patients was 1.25±0.32ng / ml. Compared with healthy controls, the TERT protein level of ARDS patients was significantly higher (p<0.0001). The above experimental results show that the use of TERT protein can effectively screen ARDS patients.

[0040] (2) The TERT protein level of patients who died was significantly higher than that of patients who survived. After 28 days of follow-up, the TERT protein level of ARDS patients who died was 3.31±1.22 ng / ml, while that of ARDS patients who survived was 0.64±0.02 ng / ml. Compared with ARDS patients who survived, the TERT protein level of ARDS patients who died after 28 days of follow-up was significantly increased (p=0.003).

[0041] The above experimental results show that the use of TERT protein can effectively evaluate the prognosis of ARDS patients. When the TERT protein of ARDS patients is higher than 0.85ng / ml, it indicates that their prognosis is poor in the next 28 days.

[0042] In summary, the above experimental results show that the use of TERT protein can effectively screen ARDS patients and effectively evaluate the prognosis of ARDS patients. The method of screening and evaluating using TERT protein in the present invention has good stability, excellent sensitivity, specificity and accuracy, and good clinical application prospects.

[0043] Embodiment 2: kit of the present invention

[0044] 1. Composition of the kit of the present invention

[0045]

[0046]

[0047] Among them, the TERT-specific antibody is the TERT antibody (C-12) produced by Santa Cruz, with the product number: sc-377511.

[0048] 2. How to use the kit

[0049] (1) Add 100 μL of standard or sample to a 96-well plate (coated with TERT antibody), seal the plate, and incubate at 37°C for 2 hours with shaking;

[0050] (2) Wash the plate three times with 300 μL of washing buffer;

[0051] (3) Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37°C with shaking for 1 hour;

[0052] (4) Wash the plate three times with 300 μL of washing buffer;

[0053] (5) Add 50 μL of colorimetric solution to each well and shake at 37°C for 20 min;

[0054] (6) Add 50 μL of stop solution to each well and shake at room temperature to mix;

[0055] (7) Read the plate within 30 minutes (Bio-Rad). When the detection value of TERT protein in the bronchoalveolar lavage fluid is below 0.85 ng / mL (i.e., cut-off value), the risk of death of ARDS patients can be considered low, while when it is above 0.85 ng / mL, ARDS patients can be considered to be critically ill patients with a high risk of death.

[0056] In summary, the kit of the present invention can realize ARDS / ALI screening and rapid prognosis judgment by quantitatively detecting the expression level of TERT protein, provide effective early warning for critically ill patients, and has good application prospects.

[0057] Of course, the protein marker is TERT. The sample is alveolar lavage fluid. The detection method in the sample includes an immunoassay, a fluorescence method, a flow fluorescence method, a latex turbidimetry method, a biochemical method, an enzyme method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method, or a photoelectric conversion method. The reagent includes a reagent that can be used to detect the expression level of the protein marker TERT, including a kit, a test paper, or a chip.

Claims

1. A method for screening a reagent for the prognostic effect of acute respiratory distress syndrome, characterized in that: The reagent screening method for the prognostic effect of acute respiratory distress syndrome comprises: Serum samples were collected from normal subjects and ARDS patients, and bronchoalveolar lavage fluid samples were collected by bronchofiberoptic examination. Based on the bronchoalveolar lavage fluid specimens of the above-mentioned people, ELISA detection was performed for TERT protein; Based on the ELISA test results, the TERT protein in ARDS patients was compared with the TERT protein in healthy controls; The TERT protein in ARDS patients is higher than that in healthy controls, and ARDS patients are at risk of death; The TERT protein in ARDS patients was equal to that in healthy controls, and ARDS patients tended to recover.

2. The method for screening the prognostic effect of acute respiratory distress syndrome according to claim 1, characterized in that: The steps of the ELISA test include: Add 100 μL of standard or sample to the 96-well plate (coated with TERT antibody), seal the plate, and incubate at 37 degrees with shaking for 2 hours; Wash the plate three times with 300 μL of plate washing buffer; Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37 degrees with shaking for 1 hour; Wash the plate three times with 300 μL of plate washing buffer; Add 50 μL of color development solution to each well and shake to mix at room temperature for 20 min; Add 50 μL of stop solution to each well and shake at room temperature to mix; The plate was read within 30 minutes (Bio-Rad). The TERT protein level in healthy controls was 0.23±0.05 ng / ml, and that in ARDS patients was 1.25±0.32 ng / ml. Compared with healthy controls, the TERT protein level in ARDS patients was significantly increased (p<0.0001).

3. The method for screening the prognostic effect of acute respiratory distress syndrome according to claim 2, characterized in that: The detection method in the sample includes an immunological method, a fluorescent method, a flow fluorescence method, a latex turbidimetry method, a biochemical method, an enzymatic method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method, or a photoelectric conversion method.

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