CN109324179B - Method for establishing PDIA3 specific autoimmune reaction mouse model, model and application - Google Patents
Method for establishing PDIA3 specific autoimmune reaction mouse model, model and application Download PDFInfo
- Publication number
- CN109324179B CN109324179B CN201811178777.1A CN201811178777A CN109324179B CN 109324179 B CN109324179 B CN 109324179B CN 201811178777 A CN201811178777 A CN 201811178777A CN 109324179 B CN109324179 B CN 109324179B
- Authority
- CN
- China
- Prior art keywords
- pdia3
- ait
- protein
- specific autoimmune
- model
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Rehabilitation Therapy (AREA)
- Rheumatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a PDIA3 specific autoimmune reaction mouse model, a model constructed by the method, and application of the model. According to the invention, a mouse specific autoimmune reaction model is established by applying PDIA3, a possible mechanism that thyroid and brain functions are damaged due to an autoimmune reaction induced by PDIA3 is directly discussed, and laboratory evidence is provided for the application of PDIA3 as a target for clinical diagnosis and treatment of autoimmune thyroiditis (AIT) and AIT-related encephalopathy.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a protein disulfide isomerase A3(PDIA3) specific autoimmune reaction mouse model, a model and application.
Background
Autoimmune thyroiditis (AIT) is one of the most common endocrine diseases. At present, the reports of AIT-related brain damage are increasing year by year, the pathogenesis is not clear, the thyroid autoimmune reaction itself may generate factors causing the central nervous system to be damaged, and the central nervous system is considered to be the most probable pathogenic mechanism involved by the cross autoimmune reaction induced by common antigens existing in thyroid and brain tissues.
Protein disulfide isomerase A3(Protein disulfide-isomer A3, PDIA3) is mainly located in endoplasmic reticulum lumen, can catalyze disulfide bond formation and isomerization, is an important anti-cytotoxic neuroprotective Protein, and plays an important role in hypothalamus-pituitary-thyroid axis. In recent years, researches show that the effect of PDIA3 in brain is wide, whether hypoxia in vitro or transient cerebral ischemia in rats in the cerebral ischemic disease promotes the up-regulation of PDIA3, the up-regulated PDIA3 can play a key role in resisting ischemic injury, and the increase of the level of PDIA3 in brain can generate a beneficial effect on cerebral stroke. Previous studies have shown that anti-PDIA 3 antibody (PDIA3Ab) is elevated in some immune diseases. The PDIA3Ab has great inhibition effect on the activity of PDIA3, for example, PDIA3Ab recognizes and binds to autoantigen PDIA3 expressed in thyroid tissue, may mediate immunological damage of the thyroid tissue in the development process of AIT, and may also act on other extrathyroid target tissues (such as brain and the like) expressing PDIA3 to induce relevant immune response and cause damage.
The prior art has the problems that although the anti-PDIA 3 autoantibody is detected and expressed in patients with individual autoimmune diseases such as autoimmune hepatitis, no report on establishing a PDIA3 specific autoimmune response animal model exists so far, and no research report on the influence of PDIA3 specific autoimmunity on thyroid tissue and brain functions exists.
Disclosure of Invention
The invention provides a method for establishing a mouse specific autoimmune reaction model by applying PDIA3 protein immunization, a model and application thereof, and solves the key means problem of researching a mechanism related to the influence of PDIA3 specific autoimmune reaction on various tissues and organs of an organism, particularly thyroid and brain tissues.
The invention provides a laboratory evidence that PDIA3 can be used as a target point for clinical diagnosis, AIT treatment and AIT related encephalopathy.
The invention further provides a method for establishing a mouse specific autoimmune reaction model by using PDIA3, which comprises the following steps:
step 1, purchasing or constructing a PDIA3 recombinant protein
Step 2, animal immunization
After 1 week of adaptive feeding of CBA/J female mice, passive immunization was performed by multipoint subcutaneous injection of immunizing agent A, which is PDIA3 recombinant protein according to 1: 1 in a volume ratio, fully emulsifying in complete Freund's adjuvant to obtain a mixture;
and injecting an immunizing agent B after 2 weeks, and performing secondary immunization by multipoint subcutaneous injection, wherein the immunizing agent B is PDIA3 recombinant protein and is prepared by the following steps of 1: 1 in a volume ratio sufficient to emulsify the mixture obtained after incomplete Freund's adjuvant.
The method for constructing a mouse specific autoimmune response model using PDIA3 of claim 1, wherein the amount of PDIA3 recombinant protein injected in step 2 is 375 μ g PDIA/mouse.
Preferably, the method for establishing the mouse specific autoimmune reaction model by using PDIA3 successfully establishes the mouse specific autoimmune reaction model after 4 weeks of secondary immunization.
The invention also provides a mouse specific autoimmune reaction model established by the method.
The invention also provides an application of the mouse specific autoimmune reaction model established by the method in the development of AIT and AIT-related encephalopathy diagnosis indexes or treatment medicines.
The invention provides a method for establishing a PDIA3 specific autoimmune reaction mouse model, a model constructed by the method and application of the model for the first time, and the following important benefits can be obtained:
by applying the established PDIA3 specific autoimmune response mouse model, the invention can directly discuss the mechanism of thyroid and brain function damage caused by the autoimmune response induced by PDIA 3. PDIA3 has been found to be expressed in the central nervous system. Our studies also found that PDIA3 is a non-classical autoantigen expressed in thyroid tissue that may act synergistically with other antigens to cause thyroid tissue destruction in the pathogenesis of AIT. The autoimmune reaction mediated by the compound is shown to be one of important factors causing thyroid function damage and AIT related encephalopathy in the process of generation and development of AIT, and a new thought and target point are provided for diagnosis and treatment of AIT and AIT related brain damage.
Drawings
FIG. 1 shows the Western Blot of normal mouse thyroid tissue protein extracts using commercially available PDIA3 monoclonal antibody and normal adult CBA/J female mouse serum as control serum;
the left two bands in fig. 1 are two parallel experiments using PDIA3 monoclonal antibody for detection, suggesting that PDIA3 protein is expressed in thyroid tissue; the two bands on the right side are two parallel experiments stained with control serum, suggesting that autoantibodies against thyroid tissue such as PDIA3 do not exist in the serum of normal mice;
FIG. 2 detection of expression localization of PDIA3 protein in thyroid gland of normal mice using immunohistochemical staining (X200);
wherein FIG. 2A is the immunohistochemical staining of normal adult CBA/J female mouse thyroid paraffin sections incubated with the commercial anti-PDIA 3 antibody as the primary antibody, and FIG. 2B is the immunohistochemical staining of normal adult CBA/J female mouse thyroid paraffin sections incubated with PBS as the negative control;
FIG. 3 is a graph showing the localization of expression of PDIA3 on vascular endothelial cells in thyroid tissue of normal mice by immunofluorescence staining (X400);
wherein, fig. 3A is a staining result of vascular endothelial cells detecting CD34 expression, fig. 3B is a staining result detecting PDIA3 expression, fig. 3C is a staining result of DAPI-labeled cell nucleus, and fig. 3D is an analysis condition under a confocal microscope detecting PDIA3 and CD34 staining results;
FIG. 4 is the level of total IgG of PDIA3Ab in the serum of mice immunized with the PDIA3 protein (4-18 weeks after the last immunization);
FIG. 5 is a mouse thyroid follicular epithelial cell IgG type immune complex deposition multiplex immunofluorescence staining assay (x 400);
wherein, the A row is the deposition of IgG type immune complexes on thyroid follicular epithelial cells of a control group which is not immunized by PDIA3 protein, and the B row is the deposition of IgG type immune complexes on thyroid follicular epithelial cells of mice immunized by PDIA3 protein;
FIG. 6 is a staining result for a common positive case between PDIA3 protein expression and IgG type immune complex deposition in mouse thyroid tissue (. times.400);
wherein row A is the co-positive condition of detecting the expression of PDIA3 protein and the deposition of IgG type immune complexes in the thyroid of control mice not immunized with PDIA3 protein, and row B is the co-positive condition of the expression of PDIA3 protein and the deposition of IgG type immune complexes in the thyroid of mice immunized with PDIA3 protein;
FIG. 7 shows the Western Blot assay of normal mouse brain tissue protein extracts using commercially available PDIA3 monoclonal antibody and normal adult CBA/J female mouse serum as control serum;
the two left bands in fig. 7 are two parallel experiments using PDIA3 monoclonal antibody, indicating that PDIA3 protein is expressed in brain tissue; the two bands on the right side are two parallel experiments using control serum for staining, which suggests that autoantibodies against brain tissue targeted by PDIA3 and the like do not exist in the serum of normal mice;
FIG. 8 is a staining result (. times.200) for the co-positive case between PDIA3 protein expression and IgG type immune complex deposition in mouse brain tissue;
wherein, line A shows the co-positive condition of the PDIA3 protein expression and the IgG type immune complex deposition in the brain tissues of the control group mice which are not immunized by the PDIA3 protein, and line B shows the co-positive condition of the PDIA3 protein expression and the IgG type immune complex deposition in the brain tissues of the mice which are immunized by the PDIA3 protein;
FIG. 9 shows immunofluorescent staining (× 200) of co-deposition of complement C3 and IgG type immunocomplexes in mouse brain tissue;
wherein, the A row is the result of fluorescence staining for detecting co-deposition of IgG immune complex and C3 in the brain tissues of the control group mice not immunized by the PDIA3 protein, and the B row is the result of fluorescence staining for co-deposition of IgG immune complex and C3 in the brain tissues of the mice immunized by the PDIA3 protein;
in fig. 3, 5, 6, 8 and 9, green indicates the result of staining with FITC, red indicates the result of staining with Aleax Fluor555, yellow indicates a common positive stained area, and blue indicates the result of staining with DAPI to mark the nucleus.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.
The invention provides application of PDIA3 as a target for clinical diagnosis and treatment of AIT and AIT-related encephalopathy. Based on the same inventive concept, the invention also provides application of the mouse specific autoimmune reaction model constructed by using the PDIA3 in developing AIT and AIT-related encephalopathy diagnosis indexes or treatment medicines. Based on the same inventive concept, the invention provides a method for establishing a mouse specific autoimmune reaction model by applying PDIA3 protein immunization, which specifically comprises the following embodiments.
Example 1
A method for establishing a mouse specific autoimmune reaction model by applying PDIA3 protein immunization. To investigate the effect of PDIA3 specific autoimmune response on thyroid and brain tissues, we used PDIA3 recombinant protein and freund's adjuvant to immunize adult CBA/J female mice for related studies. CBA/J is an AIT susceptible strain, and AIT can occur after passive immunization by administering thyroglobulin (Tg) and Freund's adjuvant to mice, so adult CBA/J female mice are selected as experimental objects in the study.
The specific method for establishing the PDIA3 specific autoimmune reaction mouse model comprises the following steps:
CBA/J female mice were passively immunized after 1 week of adaptive feeding by multipoint subcutaneous injection of immunizing agent a, which is a commercially available PDIA3 recombinant protein (Abcam, usa, collectively "protein disulfide isomerase A3(PDIA3) recombinant protein"), as shown in 1: 1 in a volume ratio sufficient to emulsify in complete Freund's adjuvant (also known as Freund's complete adjuvant); the purchased injection amount of PDIA3 recombinant protein was 75 μ g/mouse; immunization reagent B was injected 2 weeks later, and secondary immunization was performed by multiple subcutaneous injections, wherein PDIA3 recombinant protein purchased from immunization reagent B was expressed as 1: 1 in a volume ratio sufficient to emulsify the mixture obtained after incomplete Freund's adjuvant (also called Freund's incomplete adjuvant), and the injection amount of the recombinant protein PDIA3 is 375 mu g/mouse. Continuous blood sampling 4 weeks after the last immunization was performed to determine the levels of PDIA3 autoantibodies (PDIA3Ab) to confirm that a specific autoimmune response was established, and corresponding thyroid and brain tissue testing was performed.
To investigate the use of PDIA3 as a clinical diagnostic marker and as a target for the treatment of AIT and AIT-related encephalopathy, the following animal experiments were performed.
A PDIA3 group (immunization group with PDIA3 recombinant protein, n 60) and a control group (control group without PDIA3 recombinant protein for immunization, n 60) were set up, wherein the mice in the PDIA3 group were treated in the same manner as in example 1 except that the purchased PDIA3 recombinant protein was replaced with an equal volume of 0.01mmol/L Phosphate Buffer Solution (PBS) at ph 7.2.
First, the Effect of the autoimmune response induced by PDIA3 immunization on thyroid production
1. Western Blot detection
Total protein was extracted from thyroid tissue of two normal adult CBA/J female mice without any treatment, and then detected by using a commercially available rabbit anti-mouse PDIA3 antibody (PDIA3 monoclonal antibody) as a primary antibody, and Western Blot detection was performed by using serum of adult CBA/J female mice which normally did not receive PDIA3 recombinant protein as a control (PBS was injected, control group), and two parallel experiments were performed. FIG. 1 shows the results of Western Blot using PDIA3 monoclonal antibody and control serum. The results in FIG. 1 show that there is expression of PDIA3 protein in thyroid tissue and that there are no autoantibodies targeting thyroid tissue such as anti-PDIA 3 in normal mouse serum.
2. Confirmation of cellular localization of PDIA3 protein expression in thyroid tissue
Thyroid of normal adult CBA/J female mice is selected to be made into paraffin sections, and immunohistochemical staining is carried out by utilizing an EliVision two-step method. FIG. 2 is a photograph showing immunohistochemical staining for expression of PDIA3 protein in normal mouse thyroid gland (x 200), wherein FIG. 2A is a result of staining normal mouse thyroid paraffin sections with rabbit anti-mouse PDIA3 antibody as primary antibody incubation, and arrows indicate the presence of PDIA3, and FIG. 2B is a result of staining normal mouse thyroid paraffin sections with PBS as a negative control instead of primary antibody incubation. The results showed that thyroid follicular epithelial cells present expression of PDIA3 protein. The location of PDIA3 on vascular endothelial cells (marked by CD 34) is carried out by multiple immunofluorescence on a thyroid tissue frozen section of a normal adult CBA/J female mouse, and the expression of PDIA3 protein in cell membranes and cytoplasm of the thyroid vascular endothelial cells is observed. FIG. 3 is a graph showing the localization of expression of PDIA3 on vascular endothelial cells in thyroid tissue of normal mice by immunofluorescence staining (X400); among them, fig. 3A is a result of CD34 staining, fig. 3B is a result of staining for PDIA3 protein, fig. 3C is a result of staining for DAPI-labeled nuclei, and fig. 3D is a result of staining for co-positive between PDIA3 and CD 34-labeled vascular endothelial cells. Multiple immunofluorescence staining tests show that: aleax Fluor555 labeled PDIA3 (red) localized to FITC labeled vascular endothelial cells (green), DAPI labeled nuclei (blue), and PDIA3 was yellow in color with the co-positive sites of vascular endothelial cells as indicated by the arrows.
3. Detection of PDIA3 specific total IgG levels
The level of PDIA3 Ab-specific total IgG in the serum of mice at each time point after the last immunization was determined by indirect ELISA. FIG. 4 shows the levels of total IgG PDIA3Ab in serum at 4-18W time points after the last immunization, indicating that: after the protein PDIA3 is immunized, the serum PDIA3 specific total IgG level of the mouse is obviously increased compared with that of a control group, starting from 4W and continuing to at least 18W. The PDIA3 recombinant protein and Freund's adjuvant are suggested to secondarily immunize CBA/J female mice to successfully establish a PDIA3 antibody positive mouse model, and the model can provide a stable and good means for researching the influence of PDIA3 specific autoimmune reaction on organs such as thyroid gland, brain tissue and the like.
We detected the presence of apoptotic thyroid tissue in PDIA3 group mice using multiple marker immunofluorescent staining. FIG. 5 is a mouse thyroid follicular epithelial cell IgG type immune complex deposition multiplex immunofluorescence staining assay (x 400); wherein, row A shows the detection of IgG type immune complexes deposited on thyroid follicular epithelial cells in the control group not immunized with PDIA3 protein, and row B shows the deposition of IgG type immune complexes on thyroid follicular epithelial cells in mice immunized with PDIA3 protein. In FIG. 5, column a shows staining of IgG type immune complexes with FITC labeling (green), column b shows staining of follicular epithelial cells with Aleax Fluor555 labeling (red), column c shows labeling of cell nuclei with DAPI (blue), and column d shows that staining of thyroid follicular epithelial cells and staining of IgG type immune complex deposits are positive yellow, as indicated by arrows. Fig. 6 is a staining result (x 400) for a common positive case between PDIA3 protein expression and IgG-type immune complex deposition in mouse thyroid tissue, where row a is a co-positive case of PDIA3 protein expression and IgG-type immune complex deposition in thyroid gland of control group mice not immunized with PDIA3 protein, and row B is a co-positive case of PDIA3 protein expression and IgG-type immune complex deposition in thyroid gland of PDIA3 group mice immunized with PDIA3 protein. Column a is staining of IgG-type immune complexes with FITC label (green), column b is staining of PDIA3 with Aleax Fluor555 label (red), column c indicates labeling of nuclei with DAPI (blue), and column d indicates that PDIA3 protein expression is positive with IgG-type immune complex deposition staining (yellow), as indicated by the arrow. The results in FIGS. 5 and 6 show that immunocomplexes of the IgG type are deposited on follicular epithelial cells (Pan Cytokeratin as a marker) (FIG. 5) and IgG is deposited at the site where PDIA3 protein is expressed (FIG. 6), suggesting that the IgG deposited on thyroid follicular epithelial cells may be PDIA3Ab, providing evidence that PDIA3Ab causes apoptosis in thyroid tissue.
Second, the Effect of PDIA3 specific autoimmune response on brain function
Western Blot analysis using the untreated normal mouse brain tissue protein extract and a commercially available rabbit-derived anti-mouse PDIA3 antibody as a primary antibody revealed that PDIA3 protein was expressed in brain tissue of normal adult mice (FIG. 7). Next, to investigate how PDIA 3-related autoimmune reactions affect brain function by affecting brain tissue cells, we observed brain tissues of mice 10 weeks after secondary immunization of PDIA3 group after saline perfusion by immunofluorescence staining method, and fig. 8 shows the result of co-positive staining of PDIA3 and IgG-type immune complex in mouse brain tissues (x 200); row a shows the co-exposure of PDIA3 to IgG-type immune complexes in the brain tissue of mice in the control group immunized with PDIA3 protein, and row B shows the co-exposure of PDIA3 to IgG-type immune complexes in the brain tissue of mice in the group PDIA3 immunized with PDIA3 protein. Column a is staining of IgG-type immune complexes with FITC label (green), column b is staining of PDIA3 with Aleax Fluor555 label (red), column c indicates labeling of nuclei with DAPI (blue), and column d indicates that PDIA3 protein expression is positive with IgG-type immune complex deposition staining (yellow), as indicated by the arrow. The result shows that obvious IgG type immune complex deposition occurs at the microvascular endothelium and brain cells in the brain tissue, and the deposited IgG is co-positively expressed with PDIA3, which indicates that the IgG deposited at the microvascular endothelium and brain cells in the brain tissue may be PDIA3 Ab.
FIG. 9 shows immunofluorescent staining (× 200) of co-deposition of complement C3 and IgG type immunocomplexes in mouse brain tissue; row a is the result of fluorescence staining to detect co-deposition of IgG immune complexes with C3 in brain tissue of mice in the control group immunized with PDIA3 protein, and row B is the result of fluorescence staining to detect co-deposition of IgG immune complexes with C3 in brain tissue of mice in the PDIA3 group immunized with PDIA3 protein. Column a shows the result of staining with IgG-type immunocomplexes using FITC (green), column b shows the result of staining with C3 using Aleax Fluor555 (red), column C shows the result of staining with DAPI-labeled nuclei (blue), and column d shows that the C3 stain is yellow in color together with the staining with IgG-type immunocomplexes, as indicated by the arrows. Complement C3 was also found locally co-deposited in PDIA3 brain tissue with IgG-type immune complex deposition (fig. 9), whereas it was not found in control.
In addition, the cortex and hippocampus microvasculature and peripheral ultrastructures thereof were observed by transmission scanning electron microscopy, and we found that significant edema was present around the cortex and hippocampus microvasculature in mice immunized with PDIA3, suggesting that autoimmune response to PDIA3 may damage the microvascular endothelial cell function and blood brain barrier of cerebral cortex and hippocampus through complement-mediated cytotoxicity.
Combining the above experimental results to suggest:
(1) the presence of IgG-type immune complexes, complement C3 and the end product of complement system activation was found in PDIA3 antibody positive mouse thyroid follicular epithelial cells: local deposition of the Membrane Attack Complex (MAC), the results of which are not shown here, suggests that autoantibodies to PDIA3 may destroy follicular epithelial cells through complement-mediated cytotoxicity, impair thyroid function, and may lead to hypothyroidism.
(2) The brain tissue of the mouse 10 weeks after the last immunization of the PDIA3 shows that obvious IgG immune complex, complement C3 and MAC deposition of membrane attack complex are found at the position of microvascular endothelium and nerve cells, and apoptosis can be observed, which suggests that after the autoantibody against PDIA3 and complement C3 enter the brain tissue, the autoantibody can also act on the nerve cells to cause apoptosis of the nerve cells, and the brain function change such as the reduction of learning and memory ability can be caused.
In conclusion, PDIA3 is a common antigen existing in thyroid and brain tissues, but unlike the classical thyroid autoantigen (such as Tg), PDIA3 induces an autoimmune response that can affect both the thyroid and brain tissues, which may be an important causative factor in the development of extra-glandular lesions such as encephalopathy during the development of AIT. The discovery has great significance for clinically preventing and treating AIT and AIT-related brain damage and other extraglandular involvement.
It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (3)
- The application of a PDIA3 specific autoimmune response mouse model in developing AIT and AIT related encephalopathy diagnosis indexes or treatment medicines is characterized in that the PDIA3 protein is used for immune establishment of the specific autoimmune response mouse model, and the establishment method comprises the following steps:step 1, purchasing or constructing a PDIA3 recombinant protein;step 2, animal immunization:after 1 week of adaptive feeding of CBA/J female mice, passive immunization was performed by multipoint subcutaneous injection of immunizing agent A, which is PDIA3 recombinant protein according to 1: 1 in a volume ratio, fully emulsifying in complete Freund's adjuvant to obtain a mixture;and injecting an immunizing agent B after 2 weeks, and performing secondary immunization by multipoint subcutaneous injection, wherein the immunizing agent B is PDIA3 recombinant protein and is prepared by the following steps of 1: 1 in a volume ratio sufficient to emulsify the mixture obtained after incomplete Freund's adjuvant.
- 2. The application of the PDIA3 specific autoimmune response mouse model in developing AIT and AIT related encephalopathy diagnosis indexes or treatment drugs according to claim 1, wherein in the step 2, the injection amount of the PDIA3 recombinant protein is 375 μ g of PDIA per mouse.
- 3. The use of the PDIA3 specific autoimmune response mouse model of claim 1 in the development of AIT and AIT related encephalopathy diagnostic markers or therapeutic drugs, wherein the protein specific autoimmune response mouse model is successfully established 4 weeks after secondary immunization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811178777.1A CN109324179B (en) | 2018-10-10 | 2018-10-10 | Method for establishing PDIA3 specific autoimmune reaction mouse model, model and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811178777.1A CN109324179B (en) | 2018-10-10 | 2018-10-10 | Method for establishing PDIA3 specific autoimmune reaction mouse model, model and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109324179A CN109324179A (en) | 2019-02-12 |
| CN109324179B true CN109324179B (en) | 2021-07-20 |
Family
ID=65261737
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811178777.1A Active CN109324179B (en) | 2018-10-10 | 2018-10-10 | Method for establishing PDIA3 specific autoimmune reaction mouse model, model and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109324179B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2991045A1 (en) * | 2015-06-29 | 2017-01-05 | Caris Science, Inc. | Therapeutic oligonucleotides binding c1q |
| CN108218982A (en) * | 2018-02-02 | 2018-06-29 | 暨南大学 | Human cytomegalovirus anti-UL148 polyclonal antibodies and preparation method and application |
| CN108517317A (en) * | 2018-04-04 | 2018-09-11 | 江南大学 | A kind of anti-Clorprenaline monoclonal antibody hybridoma cell strain and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020098194A1 (en) * | 1996-03-21 | 2002-07-25 | Paul V. Lehmann | Methods for inducing immunity |
-
2018
- 2018-10-10 CN CN201811178777.1A patent/CN109324179B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2991045A1 (en) * | 2015-06-29 | 2017-01-05 | Caris Science, Inc. | Therapeutic oligonucleotides binding c1q |
| CN108218982A (en) * | 2018-02-02 | 2018-06-29 | 暨南大学 | Human cytomegalovirus anti-UL148 polyclonal antibodies and preparation method and application |
| CN108517317A (en) * | 2018-04-04 | 2018-09-11 | 江南大学 | A kind of anti-Clorprenaline monoclonal antibody hybridoma cell strain and its application |
Non-Patent Citations (2)
| Title |
|---|
| 《不完全弗氏佐剂诱导的髓样细胞抑制T细胞活性》;刘雨晴等;《中国免疫学杂志》;20151231;第31卷;第344-346页 * |
| 《小鼠自身免疫性甲状腺炎模型构建》;叶登美等;《皖南医学院学报》;20151231;第34卷(第1期);第10-12页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109324179A (en) | 2019-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Giera et al. | Microglial transglutaminase-2 drives myelination and myelin repair via GPR56/ADGRG1 in oligodendrocyte precursor cells | |
| Peng et al. | Cellular plasticity induced by anti–α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor encephalitis antibodies | |
| Lassmann | Comparative neuropathology of chronic experimental allergic encephalomyelitis and multiple sclerosis | |
| CN104185640B (en) | TAU protein in Alzheimer disease mediates therapy based on protein and the diagnosis of lesion | |
| Poopalasundaram et al. | Glial heterogeneity in expression of the inwardly rectifying K+ channel, Kir4. 1, in adult rat CNS | |
| Van Den Born et al. | Distribution of GBM heparan sulfate proteoglycan core protein and side chains in human glomerular diseases | |
| JP5219827B2 (en) | Anti-human Dlk-1 antibody having antitumor activity in vivo | |
| Yang et al. | Silencing Nogo‐A promotes functional recovery in demyelinating disease | |
| JP5615169B2 (en) | Anti-human Dlk-1 antibody having antitumor activity in vivo | |
| Werner et al. | Glutamate excitotoxicity—a mechanism for axonal damage and oligodendrocyte death in multiple sclerosis? | |
| König et al. | The plexin C1 receptor promotes acute inflammation | |
| Park et al. | The conceptual introduction of the “demyelinating Schwann cell” in peripheral demyelinating neuropathies | |
| Hoopes et al. | Characteristics of multi-organ lymphangiectasia resulting from temporal deletion of calcitonin receptor-like receptor in adult mice | |
| O'Neill et al. | Nogo and Nogo‐66 receptor in human and chick: Implications for development and regeneration | |
| Pose-Utrilla et al. | Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1 | |
| JP7327897B2 (en) | Antibody that recognizes the T14 peptide of ACHE | |
| Yamanaka et al. | Increase of close homolog of cell adhesion molecule L1 in primary afferent by nerve injury and the contribution to neuropathic pain | |
| Pecchi et al. | Glial fibrillary acidic protein (GFAP)‐positive radial‐like cells are present in the vicinity of proliferative progenitors in the nucleus tractus solitarius of adult rat | |
| Ohnishi et al. | Claudin domain containing 1 contributing to endothelial cell adhesion decreases in presence of cerebellar hemorrhage | |
| Fang et al. | Myelin oligodendrocyte glycoprotein-IgG contributes to oligodendrocytopathy in the presence of complement, distinct from astrocytopathy induced by AQP4-IgG | |
| WO2006050667A1 (en) | MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF | |
| EP2369936B1 (en) | Compositions and methods for the treatment and prevention of lens fibrotic diseases | |
| CN109324179B (en) | Method for establishing PDIA3 specific autoimmune reaction mouse model, model and application | |
| Fogarty et al. | Angiotensin receptor‐like immunoreactivity in adult brain white matter astrocytes and oligodendrocytes | |
| Bjelobaba et al. | The cortical stab injury induces beading of fibers expressing ecto-nucleoside triphosphate diphosphohydrolase 3 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |