CN110954701A

CN110954701A - Diagnostic kit for hepatic fibrosis or cirrhosis

Info

Publication number: CN110954701A
Application number: CN201911315897.6A
Authority: CN
Inventors: 夏杰; 黄爱龙; 刘迪娜; 唐霓; 汪凯; 蔡莹; 沙雨; 潘敏刚
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2020-04-03
Anticipated expiration: 2039-12-18
Also published as: CN110954701B

Abstract

The invention belongs to the field of disease diagnosis kits, and particularly discloses a diagnosis kit for hepatic fibrosis and hepatic cirrhosis, which comprises a reagent for detecting Ceacam1+ CSF1R + peripheral blood mononuclear cells. The experiment of the invention shows that the content of Ceacam1+ CSF1R + peripheral blood mononuclear cells relative to healthy people can reflect whether the subject has liver fibrosis or liver cirrhosis, and the sensitivity of the content is higher than that of the current liver function detection index (ALT/AST). The kit has the advantages of few detection indexes, high detection sensitivity and good application prospect.

Description

Diagnostic kit for hepatic fibrosis or cirrhosis

Technical Field

The invention belongs to the field of disease diagnosis reagents.

Background

Hepatic fibrosis is a pathological process in which fibrous nodules are produced as a result of extracellular matrix accumulation during tissue regeneration after the liver structure is damaged; the final stage of its development is cirrhosis, one of the clinically common chronic progressive liver disease and the major lethal disease. 2 to 3 percent of hepatic fibrosis patients in China can develop into cirrhosis each year, the hepatic cirrhosis accounts for 4.3 to 14.2 percent of the total number of the inpatients in internal medicine, the number of people who die of the cirrhosis and complications thereof is up to 30 ten thousand, and the hepatic fibrosis treatment method brings heavy economic and social burden to the nation and people. Therefore, the method has very important significance for early diagnosis and screening of hepatic fibrosis and restraining further development of hepatic fibrosis to cirrhosis.

Common detection means for liver fibrosis include blood biochemical detection (blood routine, blood coagulation function, liver function, APRI score, FIB-4 index, etc.), imaging detection (ultrasound, CT, and magnetic resonance imaging), and pathology detection (liver biopsy). Specific hepatic fibrosis indexes are lacked in blood biochemical detection, a single index has small effect on hepatic fibrosis evaluation, and a plurality of items need to be detected to improve the diagnostic value of the liver fibrosis evaluation in a limited way. Imaging detection usually can only detect severe liver fibrosis, liver cirrhosis and liver space occupying lesion, and has little significance for early stage fibrosis assessment. The pathological detection is the gold standard for liver fibrosis diagnosis, but false negative easily occurs, because liver fibrosis caused by many diseases is unevenly distributed, if a sample of a fibrosis part is not obtained by liver puncture, diagnosis is missed; in addition, liver puncture has great damage to the patient and the detection period is also long.

Therefore, the active development of new means for detecting liver fibrosis is one of the major problems in human health in the 21 st century.

Disclosure of Invention

The invention aims to provide a diagnostic kit for hepatic fibrosis or hepatic cirrhosis.

The technical scheme of the invention comprises the following steps:

a diagnostic kit for hepatic fibrosis or cirrhosis, characterized in that: it comprises a reagent for detecting Ceacam1+ CSF1R + peripheral blood mononuclear cells; the Ceacam1+ refers to cells with Ceacam1 (i.e., CD66 a; uniprot: P13688[ human ], P31809[ mouse ]) on their surface; CSF1R + refers to cells carrying CSF1R (uniprot: P07333[ human ], P09581[ mouse ]) protein on their surface.

As with the previously described kits, the reagents for detecting Ceacam1+ CSF1R + peripheral blood mononuclear cells include fluorescently labeled antibodies for detecting Ceacam1 and CSF1R proteins.

The kit as described above, which is a flow cytometry detection kit.

The kit as described above, which is an immunohistochemical detection kit.

If the peripheral blood is not used as a detection sample, the Mac-1 protein needs to be identified so as to confirm whether the mononuclear cells of the Mac-1 protein are derived from the peripheral blood; when Mac-1 protein is present on the surface of monocytes, it is confirmed that they are derived from peripheral blood. Thus, the aforementioned kit, which may further comprise reagents for detecting the Mac-1 protein (i.e., CD11 b; uniprot: P11215[ human ], P05555[ mouse ]) on the surface of monocytes.

As in the aforementioned kit, the agent for detecting Mac-1 protein includes a fluorescent-labeled antibody against Mac-1 protein.

As in the aforementioned kit, the reagent for detecting Mac-1 protein is a reagent for flow cytometry detection.

As in the aforementioned kit, the reagent for detecting the Mac-1 protein is a reagent for immunohistochemical or immunofluorescent detection.

The application of the reagent for detecting the peripheral blood mononuclear cell surface Ceacam1 and CSF1R proteins in preparing a diagnostic kit for liver fibrosis or cirrhosis.

The reagent may also include a reagent for detecting a monocyte surface Mac-1 protein, for use as described above.

Further, if the detection sample is peripheral blood, the kit also comprises a reagent for detecting Mac-1+ monocytes; the Mac-1+ refers to that the cell surface is provided with Mac-1 (namely CD11 b; uniprot: P11215[ human ], P05555[ mouse ]) protein.

For convenience of presentation, the invention defines Ceacam1+ CSF1R + peripheral blood mononuclear cells as: peripheral blood-fibrosis-promoting mononuclear cells (PFDM) are abbreviated as PFDM. In addition, Mac-1 is a marker for peripheral blood mononuclear cells known in the art, and "Mac-1 + Ceacam1+ CSF1R + monocytes" in the present invention is equivalent to "Ceacam 1+ CSF1R + peripheral blood mononuclear cells".

The invention is different from the existing any hepatic fibrosis or cirrhosis detection technology, and has the following beneficial effects:

the kit disclosed by the invention is simple to use, and only Ceacam1 and CSF1R proteins on the surface of peripheral blood mononuclear cells need to be detected, so that the kit can be compared with healthy people;

the kit has various forms, can be a peripheral blood detection kit, has small damage, and is simple and quick; also can be an immunohistochemical detection kit which is suitable for different requirements.

The kit of the invention is very sensitive, has significantly higher sensitivity compared with the traditional liver function index (AST/ALT), and can detect compensatory cirrhosis which cannot be detected by the liver function index (AST/ALT).

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: immunofluorescence assay of liver tissue from liver fibrosis and healthy mice.

FIG. 2: and detecting the result by flow cytometry.

FIG. 3: PFDM levels between cirrhosis patients and normal.

FIG. 4: comparison of ALT and PFDM levels in serum from patients with compensated and decompensated cirrhosis.

FIG. 5: comparison of AST and PFDM levels in serum of patients with compensated and decompensated cirrhosis.

Detailed Description

Example 1 kit of the invention (flow cytometer detection kit)

1. Kit composition

The core component is as follows: ceacam1, CSF1R fluorescent-labeled antibody (fluorescent primary antibody).

Other components (self-contained): monocyte isolation and extraction kit (commercially available product).

2. Application method

1) Taking peripheral blood of a to-be-detected object, and separating peripheral blood mononuclear cells (a mononuclear cell separation and extraction kit can be used);

2) suspending the mononuclear cells in PBS, and adding a fluorescent primary antibody for incubation;

3) and detecting the fluorescence of the antibody by using a flow cytometer, wherein if the fluorescence of the markers corresponding to Ceacam1 and CSF1R is detected and the intensity is obviously higher than the fluorescence intensity corresponding to a healthy population, the condition that the object to be detected has hepatic fibrosis is indicated.

The kit does not detect Mac-1 because the sample taken is peripheral blood and Mac-1 is a marker for peripheral blood mononuclear cells, which is well known in the art, and Mac-1 does not need to be detected.

Example 2 kit of the invention (immunofluorescence assay kit for liver tissue paraffin section)

1. Composition of the kit

The core component is as follows: fluorescently labeled antibodies (fluorescent primary antibodies) to Mac-1, Ceacam1, CSF 1R.

Other components (self-contained): antigen retrieval solution (Epitope retrieval solution), blocking solution (goat serum), and washing buffer (commercially available product for immunohistochemistry).

2. Application method

1) Sequentially adding xylene-100% ethanol-95% ethanol-85% ethanol-75% ethanol-70% ethanol-double distilled water into paraffin sections of liver tissue, and dewaxing and hydrating (each solvent for 3 min);

2) antigen retrieval: placing deparaffinized and hydrated liver tissue slices into a container, adding the antigen repairing liquid, and performing microwave oven for 8min for 2 times;

3) and (3) sealing: naturally cooling to room temperature, double-steaming and washing with water for 3 times, and sealing with 5% goat serum for 10 min;

4) hatching antibody: recovering goat serum, washing with running water for 2-3min, washing with double distilled water for 3 times, washing with washing buffer solution once, adding fluorescent primary antibody, and incubating at room temperature for 1 h;

5) closing and taking a picture: sealing the piece with a sealing solution containing DAPI and taking a picture under a microscope;

6) and (4) interpretation of results: if the color signals of the Mac-1, Ceacam1 and CSF1R corresponding to the fluorescence primary antibody appear and the intensity is obviously higher than the fluorescence intensity corresponding to the healthy population, the liver fibrosis of the object to be detected is shown.

To further illustrate the beneficial effects of the present invention, the present invention provides the following experimental examples:

experimental example 1 detection of PFDM in the liver of fibrotic mice

1. Method of producing a composite material

Detecting PFDM distribution in liver tissues of liver fibrosis mice and healthy control mice by using immunofluorescence, and the steps are as follows:

1) taking liver tissues of control/hepatic fibrosis mice to perform formalin fixation for 24 h;

2) after dehydration and transparence, paraffin is embedded and sliced to obtain paraffin sections;

3) mac-1, Ceacam1, CSF1R were tested as in example 2.

2. Results

The results showed that normal mouse liver had no PFDM, while fibrotic mouse liver fibers had a large amount of PFDM distributed at intervals (fig. 1).

And (4) conclusion: PFDM occurs in the liver, associated with liver fibrosis; one can detect liver fibrosis by detecting PFDM in the liver.

Experimental example 2 detection of PFDM Using peripheral blood as sample

1. Method of producing a composite material

Using peripheral blood of a liver fibrosis mouse and a healthy control mouse as samples, monocytes were isolated, and Ceacam1 and CSF1R were detected by the method of example 1.

2. Results

The results showed a ratio of 2.45% for Ceacam1+ CSF1R + monocytes in peripheral blood of healthy control mice (fig. 2A); the ratio of Ceacam1+ CSF1R + monocytes in the peripheral blood of liver fibrosis mice was 35.3% (fig. 2B), which was significantly higher than that of healthy control mice.

And (4) conclusion: the PFDM content in peripheral blood is related to hepatic fibrosis, and people can detect hepatic fibrosis by detecting PFDM in peripheral blood.

Experimental example 3 clinical examination

Liver cirrhosis in the decompensation stage is end-stage hepatic fibrosis, and liver injury is obvious and is accompanied by acute elevation of ALT, AST and the like; the compensatory liver cirrhosis is mainly hepatic fibrosis, the degree of liver injury is low, and the elevation of ALT and AST is not obvious. The purpose of this experimental example was to evaluate the indicative effect of PFDM on human liver fibrosis/cirrhosis.

1. Method of producing a composite material

1.1PFDM content comparison

Peripheral blood of clinical cirrhosis patients was collected and PFDM levels were measured and compared to those of normal persons.

1.2 sensitivity of PFDM compared to liver function index

Liver function markers (ALT and AST) of patients in compensatory stage and decompensated stage of liver cirrhosis are detected, and PFDM level difference of patients with hepatic fibrosis corresponding to normal and abnormal liver function markers is compared.

2. Results

2.1 comparison of PFDM levels between patients with cirrhosis and Normal persons

As shown in fig. 3, PFDM was significantly higher in clinical cirrhosis patients than in normal. Indicating that PFDM can be used as a clinical liver cirrhosis diagnosis marker.

2.2 comparison of sensitivity of PFDM to liver function index

As shown in FIGS. 4 and 5, PFDM was significantly elevated in both compensated cirrhosis patients (ALT/AST normal) and decompensated patients (ALT/AST dysregulated). Detection of the liver function markers ALT and AST levels, however, fails to diagnose compensated cirrhosis. It can be seen that PFDM is more sensitive in diagnosing cirrhosis than the liver function markers ALT and AST.

In conclusion, the invention provides an efficient, simple, convenient and sensitive hepatic fibrosis and hepatic cirrhosis diagnostic kit, which has a different principle from the existing kit for detecting hepatic fibrosis and hepatic cirrhosis, can further perfect a hepatic fibrosis/hepatic cirrhosis detection tool library, and has a good application prospect.

Claims

1. A kit for detecting liver fibrosis or cirrhosis, characterized in that: it comprises reagents for detecting Ceacam1+ CSF1R + peripheral blood mononuclear cells.

2. The kit of claim 1, wherein: the reagent for detecting the Ceacam1+ CSF1R + peripheral blood mononuclear cells comprises a fluorescence labeled antibody for detecting Ceacam1 and CSF1R proteins.

3. The kit of claim 2, wherein: the kit is a flow cytometry detection kit.

4. The kit of claim 2, wherein: the kit is an immunohistochemical detection kit.

5. The kit according to any one of claims 1 to 4, wherein: it also includes reagents for detecting Mac-1 protein located on the surface of monocytes.

6. The kit of claim 5, wherein: the agent for detecting the Mac-1 protein includes a fluorescently labeled antibody against the Mac-1 protein.

7. The kit of claim 5, wherein: the reagent for detecting the Mac-1 protein is a reagent for flow cytometry detection.

8. The kit of claim 5, wherein: the reagent for detecting the Mac-1 protein is a reagent for immunohistochemical or immunofluorescence detection.

9. The application of the reagent for detecting the peripheral blood mononuclear cell surface Ceacam1 and CSF1R proteins in preparing a diagnostic kit for liver fibrosis or cirrhosis.

10. Use according to claim 9, characterized in that: the reagent also comprises a reagent for detecting the monocyte surface Mac-1 protein.