CN113215247A - Kit for predicting and prognosticating autoimmune thyroid diseases and application thereof - Google Patents

Kit for predicting and prognosticating autoimmune thyroid diseases and application thereof Download PDF

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CN113215247A
CN113215247A CN202110687159.5A CN202110687159A CN113215247A CN 113215247 A CN113215247 A CN 113215247A CN 202110687159 A CN202110687159 A CN 202110687159A CN 113215247 A CN113215247 A CN 113215247A
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aitd
trbv15
trbv29
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kit
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宋荣华
秦秋
张进安
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Zhoupu Hospital Pudong New Area Shanghai (zhoupu Hospital Affiliated To Shanghai Medical College)
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Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a kit for predicting and prognostically judging autoimmune thyroid diseases (AITD) by taking TRBV15 and TRBV29-1 as biomarkers and application thereof. The detection kit of the invention is adopted to carry out combined detection on TRBV15 and TRBV29-1 in human peripheral blood, thereby diagnosing AITD and predicting AITD prognosis, and the detection result has good sensitivity and high specificity. Can be effectively used for early screening and/or prognosis evaluation of AITD, and has good clinical application prospect.

Description

Kit for predicting and prognosticating autoimmune thyroid diseases and application thereof
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a kit for predicting and prognosing autoimmune thyroid disease (AITD) by taking TRBV15 and TRBV29-1 as biomarkers and application thereof.
Background
Autoimmune thyroid disease (AITD) is a chronic autoimmune disease of the thyroid gland, mainly including Graves 'disease (GD) and Hashimoto's Thyroiditis (HT), which may risk thyrotoxicosis and hypothyroidism, respectively, in patients. The incidence of AITD in the human population can be up to 5%, however the factors triggering thyroid autoimmunity are not well understood. The epidemiological data result shows that the common interaction of the genetic susceptibility, the environmental factors and the immune factors leads to the impaired immune tolerance of the organism, thereby promoting the occurrence of diseases. Among them, the immune attack of autoantibodies against thyroid is the basis for the pathogenesis of AITD. In addition, infiltration and accumulation of T cells in thyroid tissue causes destruction of thyroid follicular epithelial cells, resulting in the development of disease. In fact, dysfunction or abnormal number of T cells is extremely complex in AITD, and abnormalities in T cell receptor profiles are thought to play a very important role in the promotion of thyroid cell destruction in AITD diseases. While expression abnormality of T cell receptor related genes is less researched in AITD, the previous research finds that abnormal expression of T cell receptor related genes TRBV15 and TRBV29-1 obviously exists in patients with AITD, and theoretical basis is provided for researching and monitoring the pathogenesis progress of AITD.
The AITD affects the life quality of patients, and serious patients may cause thyropathy, hyperthyroidism heart disease, low-potassium periodic paralysis and the like of the patients to harm the life health of the patients, so the diagnosis, identification and prognosis of the AITD are particularly important. The development of AITD diagnostic and treatment regimens depends on clinical and laboratory findings. The molecular diagnostic technology can provide basis for the diagnosis, differential diagnosis and prognosis of AITD. Because the pathogenesis of AITD is not completely understood, the prognosis effect of the existing AITD related marker for clinical judgment of AITD is poor, and a biomarker or laboratory diagnosis basis for convenient and accurate diagnosis and prognosis judgment is urgently needed.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the application of TRBV15 and TRBV29-1 as an AITD biomarker in preparing or screening AITD detection reagents.
Another objective of the invention is to provide an AITD detection kit using TRBV15 and TRBV29-1 as a combined biomarker.
Another object of the present invention is to provide a method for detecting AITD.
The invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided the use of a combination of TRBV15 and TRBV29-1 as a biomarker in the preparation or screening of AITD detection reagents.
Preferably, the TRBV15 and TRBV29-1 are combined as an AITD peripheral blood biomarker.
Preferably, the AITD detection reagent is used for judgment of AITD, selection of a treatment scheme, and/or prognosis evaluation.
The combination of TRBV15 and TRBV29-1 as an AITD biomarker for preparing an AITD detection reagent means that TRBV15 and TRBV29-1 are combined as a standard or positive control of the AITD detection reagent based on the content of TRBV15 and TRBV29-1 to be used for detecting TRBV15 and TRBV29-1 in a sample.
The combination of TRBV15 and TRBV29-1 as an AITD biomarker for screening the AITD detection reagent refers to the application of TRBV15 and TRBV29-1 as targets in the screening of the AITD detection reagent. In a preferred embodiment, the reagent for specifically recognizing TRBV15 and the reagent for specifically recognizing TRBV29-1 are screened based on the TRBV15 and TRBV29-1, so that the level of TRBV15 and TRBV29-1 in the sample can be detected as the reagent for detecting AITD.
In a preferred embodiment, a primer specifically recognizing TRBV15 and a primer specifically recognizing TRBV29-1 are selected based on the TRBV15 and the TRBV29-1, and thus serve as primers for detecting AITD.
In a preferred embodiment, an antibody specifically recognizing TRBV15 and an antibody specifically recognizing TRBV29-1 are selected based on said TRBV15 and TRBV29-1, thereby serving as a reagent for detecting AITD.
In a second aspect of the invention there is provided the use of an agent which specifically recognizes TRBV15 and an agent which specifically recognizes TRBV29-1 in the manufacture of an AITD detection kit.
In a preferred embodiment, the agent that specifically recognizes TRBV15 is selected from antibodies that specifically recognize TRBV 15. The agent specifically recognizing TRBV29-1 is selected from antibodies specifically recognizing TRBV 29-1.
The antibodies include monoclonal antibodies and polyclonal antibodies.
In a third aspect of the invention, there is provided an AITD detection kit comprising reagents specifically recognizing TRBV15 and reagents specific for TRBV 29-1.
Preferably, the AITD detection kit uses TRBV15 and TRBV29-1 as AITD biomarkers.
In a preferred embodiment, the agent that specifically recognizes TRBV15 is selected from primers that specifically recognize TRBV 15. The reagent specifically recognizing TRBV29-1 is selected from primers specifically recognizing TRBV 29-1.
In a preferred embodiment, the agent that specifically recognizes TRBV15 is selected from antibodies that specifically recognize TRBV 15. The agent specifically recognizing TRBV29-1 is selected from antibodies specifically recognizing TRBV 29-1.
The antibodies include monoclonal antibodies and polyclonal antibodies.
In a preferred embodiment, the antibodies that specifically recognize TRBV15 include primary and secondary antibodies for detecting TRBV 15. Antibodies that specifically recognize TRBV29-1 include primary and secondary antibodies for the detection of TRBV 29-1.
Further, the secondary antibody is a fluorescent molecule labeled secondary antibody.
In a preferred embodiment, the kit further comprises reagents commonly used in real-time fluorescence quantitative PCR technology.
In a preferred embodiment, the kit further comprises a positive control and a negative control. The positive controls included TRBV15 and TRBV 29-1. The negative control comprises ddH2O。
Preferably, the AITD detection kit is used for the judgment of AITD, selection of a treatment regimen, and/or prognostic assessment.
In a fourth aspect of the invention, there is provided a method of detecting AITD comprising detecting the level of TRBV15 and TRBV29-1 in a sample.
Compared with the prior art, the invention has the beneficial effects that:
the invention is based on the research result of the inventor, and the severity and prognosis of AITD can be judged more accurately by detecting two newly discovered AITD markers which are mutually related. TRBV15 and TRBV29-1 are simultaneously used as new AITD markers, and can provide basis for molecular pathological diagnosis of AITD. The detection kit provided by the invention is used for carrying out combined detection on TRBV15 and TRBV29-1 in a sample, so that AITD can be diagnosed and detected, and the detection result has good sensitivity and high specificity. Can be effectively used for early screening and/or prognosis evaluation of AITD, and has good clinical application prospect.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in Sambrook et al, MOLEC μ LAR CLONING: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLEC. mu.LAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLEC μ LAR BIOLOGY, Vol.119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.
Terms, abbreviations
qRT-PCR: quantitative real-time fluorescence PCR
Sensitivity: i.e., true positive rate, the ratio of true positive to true positive + false negative.
Specificity: i.e., the rate of true negatives, the ratio of true negatives to true negatives plus false positives.
AITD: the abbreviation of autoimmune thyroid disease mainly includes Graves Disease (GD) and Hashimoto Thyroiditis (HT).
Example 1 detection of the expression levels of TRBV15 and TRBV29-1 in AITD peripheral blood by the qRT-PCR method
In one embodiment, the expression levels of TRBV15 and TRBV29-1 in AITD peripheral blood were determined by qRT-PCR method, which comprises the following steps:
(1) taking peripheral blood of a patient and a normal control group, and separating Peripheral Blood Mononuclear Cells (PBMC);
(2) sorting CD4+ T cells by flow cytometry;
(3) adding Trizol, and extracting total RNA;
(4) the expression levels of TRBV15 and TRBV29-1 of the patients and the normal control groups are obtained by qRT-PCR after primer amplification, thereby assisting in diagnosing AITD and judging the prognosis of the patients. The primers for amplifying the TRBV15 comprise an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.1, and specifically comprises the following steps: GTCCTCAGTCTGCACCTTTCATTCA are provided. The sequence of the downstream primer is shown as SEQ ID NO.2, and specifically comprises the following steps: GGGTAACCTGGTATCTTGG are provided. The primers for amplifying the TRBV29-1 comprise an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.3, and specifically comprises the following steps: CTCCTTCTCCTGGGACTAGGTATGA are provided. The sequence of the downstream primer is shown as SEQ ID NO.4, and specifically comprises the following steps: TAGGTTTGGGCGGCTGATG are provided.
Second, the example is found for the first time: the expression levels of TRBV15 and TRBV29-1 in the peripheral blood of AITD are closely related to the development of AITD, see Table 1. And the expression levels of TRBV15 and TRBV29-1 were positively correlated with the persistence and prognosis of AITD, as shown in Table 2.
TABLE 1 TRBV15 and TRBV29-1 expression differences in AITD and normal controls
Figure BDA0003125020870000051
TABLE 2 expression differences between TRBV15 and TRBV29-1 in refractory and remitting AITD
Figure BDA0003125020870000052
In conclusion, the detection kit provided by the invention is used for carrying out combined detection on TRBV15 and TRBV29-1 in peripheral blood, so that AITD can be diagnosed and detected, and the detection result has good sensitivity and high specificity. Can be effectively used for prediction and/or prognosis evaluation of AITD, and has good clinical application prospect.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims (10)

  1. Use of a combination of TRBV15 and TRBV29-1 as an AITD biomarker in the preparation or screening of AITD detection reagents.
  2. 2. The use according to claim 1, wherein TRBV15 and TRBV29-1 are in combination as an AITD peripheral blood biomarker.
  3. 3. The use according to claim 1, wherein the AITD detection reagent is used for the judgment of AITD, selection of a treatment regimen, and/or prognostic assessment.
  4. 4. The use according to claim 1, wherein the combination of TRBV15 and TRBV29-1 as an AITD biomarker for the preparation of an AITD detection reagent is the combination of TRBV15 and TRBV29-1 as a standard or positive control for an AITD detection reagent based on the detection of TRBV15 and TRBV29-1 content for the detection of TRBV15 and TRBV29-1 in a sample.
  5. 5. The use according to claim 1, wherein the combination of TRBV15 and TRBV29-1 as an AITD biomarker for screening an AITD detection agent means that TRBV15 and TRBV29-1 as targets are applied to the screening of an AITD detection agent, and based on the TRBV15 and TRBV29-1, an agent specifically recognizing TRBV15 and an agent specifically recognizing TRBV29-1 are screened as an AITD detection agent.
  6. 6. Use of a reagent which specifically recognizes TRBV15 and a reagent which specifically recognizes TRBV29-1 in the preparation of an AITD detection kit.
  7. 7. An AITD detection kit, comprising a reagent specifically recognizing TRBV15 and a reagent specifically aiming at TRBV 29-1.
  8. 8. The kit of claim 7, wherein the AITD detection kit uses TRBV15 and TRBV29-1 as AITD biomarkers.
  9. 9. The kit of claim 7, wherein the reagent that specifically recognizes TRBV15 is selected from primers that specifically recognize TRBV15, and the reagent that specifically recognizes TRBV29-1 is selected from primers that specifically recognize TRBV 29-1.
  10. 10. The kit of claim 7, wherein the kit further comprises reagents commonly used in real-time fluorescence quantitative PCR technology.
CN202110687159.5A 2021-06-21 2021-06-21 Kit for predicting and prognosticating autoimmune thyroid diseases and application thereof Pending CN113215247A (en)

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