Method for constructing TPOAb positive animal model based on adenovirus vector

Abstract

The invention relates to the technical field of biomedicine, and provides an adenovirus vector-based TPOAb positive animal model construction method which comprises the following steps: (1) constructing a TPO overexpression adenovirus vector; (2) injecting the TPO overexpression adenovirus vector into the body of an experimental animal, and inducing the generation of TPOAb; and (3) determining the optimal injection dose through a gradient dose experiment, and constructing a TPOAb positive animal model. The TPOAb positive animal model has the advantages that the generation of TPOAb in an animal body is induced by utilizing the TPO overexpression adenovirus vector through a nucleic acid immune technology, and the TPOAb positive animal model which is high in stability and modeling rate and does not interfere with the thyroid function is constructed.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for constructing a thyroid peroxidase antibody (TPOAb)-positive animal model based on an adenovirus vector. Background Art

[0002] Thyroid peroxidase autoantibodies (TPOAb) are important markers for thyroid autoimmune diseases, and their positive status is closely related to thyroid function abnormalities, pregnancy complications, and offspring development problems. At present, existing animal models of thyroid diseases (such as Hashimoto's thyroiditis models) usually exhibit complex pathological changes such as hypothyroidism and lymphocyte infiltration, and it is difficult to simply simulate the positive status of TPOAb. Therefore, constructing an animal model that can stably and efficiently induce TPOAb positivity without interfering with thyroid function is of great significance for in-depth research on the pathogenesis of thyroid autoimmune diseases and their effects on the mother and offspring.

[0003] In the prior art, there has been research on immunizing mice with recombinant murine thyroid peroxidase (rmTPO) combined with complete Freund's adjuvant (CFA), which successfully induced an increase in TPOAb, but this method may still lead to thyroid function abnormalities. In addition, adenovirus vector-mediated gene delivery technology has been used to construct thyroid disease models, but its application in TPOAb-positive models still needs to be further optimized. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a TPOAb-positive animal model based on an adenovirus vector, which uses nucleic acid immunization technology to induce the production of TPOAb in animals by using a TPO overexpression adenovirus vector, and constructs a TPOAb-positive animal model with high stability, high modeling rate, and no interference with thyroid function.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] A method for constructing a TPOAb-positive animal model based on an adenovirus vector, comprising the following steps:

[0007] (1) Construct a TPO overexpression adenovirus vector;

[0008] (2) Inject the TPO overexpression adenovirus vector into an experimental animal to induce the production of TPOAb;

[0009] (3) Determine the optimal injection dose through a gradient dose experiment to construct a TPOAb-positive animal model.

[0010] As one of the preferred embodiments of the present invention, in the step (1), the method for constructing the TPO overexpression adenovirus vector is as follows: Clone the full-length cDNA of thyroid peroxidase TPO, integrate it with the adenovirus vector GV315, and construct the TPO overexpression adenovirus vector. It can also be obtained through commercial channels.

[0011] As one of the preferred embodiments of the present invention, in the step (1), after the construction of the TPO overexpression adenovirus vector is completed, it further includes the step of "detecting the expression level of the TPO gene by Real-time PCR to verify the effectiveness of the adenovirus vector".

[0012] As one of the preferred embodiments of the present invention, in the step (2), the experimental animals are 3-week-old SPF-grade C57BL / 6N mice.

[0013] As one of the preferred embodiments of the present invention, in the step (3), the optimal injection dose of the TPO overexpression adenovirus vector is: 3×10^9 PFU / time, injected once every 3 weeks, for a total of 3 injections.

[0014] As one of the preferred embodiments of the present invention, in the step (3), it further includes the steps of "verifying the model establishment rate and verifying the impact of the model on thyroid function".

[0015] As one of the preferred embodiments of the present invention, the concentration of TPOAb in the mouse serum is detected by ELISA to evaluate the model establishment rate.

[0016] As one of the preferred embodiments of the present invention, the impact of the model on thyroid function is verified by detecting the levels of thyroid hormones (TSH, FT4, T3, T4) and thyroglobulin antibody (TGAb).

[0017] The advantages of the present invention compared with the prior art are as follows:

[0018] The present invention pioneered the use of nucleic acid immunization, a new approach to establishing an animal model with positive thyroid autoantibodies. By using a TPO overexpression adenovirus vector, a "positive animal model for thyroid peroxidase antibody (TPOAb)" is constructed, avoiding the use of adjuvants in traditional methods and being closer to the natural process of TPOAb production in humans.

[0019] The model provided by the present invention has a high model establishment rate (100%) and stability, can efficiently induce positive TPOAb, and does not interfere with thyroid function, being suitable for studying the independent effects of positive TPOAb on the mother and offspring.

[0020] The present invention determined the optimal injection dose (3×10^9 PFU / time) through gradient dose experiments, avoiding the problem of immunosuppression caused by high-dose injection.

[0021] The present invention verifies that maternal TPOAb positivity is not related to the thyroid function of the offspring. There are no significant differences in TGAb and other thyroid hormones between the TPO adenovirus immunization group and the control group, further verifying that the effect of maternal TPOAb does not depend on the thyroid function of the offspring, providing a basis for the study of the impact of maternal TPOAb positivity on offspring development during pregnancy. Description of the Drawings

[0022] Figure 1 It is the EGFP immunofluorescence staining result of the thyroid gland in Example 3 (in the figure, Figure A is the staining result, PBS: PBS control group, N: empty adenovirus immunization group, TPO: TPO adenovirus immunization group, the image is from the thyroid gland during pregnancy, scale bar 20 μm; Figure B is the corresponding Image J counting of immunofluorescent cells, the data are expressed as mean ± standard deviation, n = 8 / group);

[0023] Figure 2 It is the EGFP immunofluorescence staining result of the placenta in Example 3 (in the figure, Figure A is the staining result, PBS: PBS control group, N: empty adenovirus immunization group, TPO: TPO adenovirus immunization group, the image is from the late pregnancy placenta, scale bar 20 μm; Figure B is the corresponding Image J counting of immunofluorescent cells, the data are expressed as mean ± standard deviation, n = 8 / group);

[0024] Figure 3 It is the EGFP immunofluorescence staining result of the fetal brain in Example 3 (in the figure, Figure A is the staining result, PBS: PBS control group, N: empty adenovirus immunization group, TPO: TPO adenovirus immunization group, the image is from the late pregnancy fetal brain, scale bar 20 μm; Figure B is the corresponding Image J counting of immunofluorescent cells, the data are expressed as mean ± standard deviation, n = 8 / group);

[0025] Figure 4 It is the HE staining of the thyroid gland and the thyroid inflammation score result in Example 3 (in the figure, Figure A is the staining result, PBS: PBS control group, N: empty adenovirus immunization group, TPO: TPO adenovirus immunization group, scale bar: 50 μm; Figure B is the thyroid inflammation score of PND28 pups; Figure C is the thyroid inflammation score of PND56 pups; the data are expressed as mean ± standard deviation, n = 8 / group, during pregnancy, since the thyroid inflammation scores of each group are all 0, the statistical chart is no longer presented). Detailed Description of the Invention

[0026] The following is a detailed description of the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagents used in the present invention are all conventional reagents in the art without special description; the sequences mentioned are all conventional corresponding sequences in the art without special description; the experimental conditions and experimental methods used, without special description, are all conventional conditions and methods in the art and will not be elaborated further.

[0027] Example 1. Construction of an adenovirus vector overexpressing TPO:

[0028] The adenovirus vector overexpressing TPO in this example was purchased from Shanghai Genechem Co., Ltd. (Product name: overexpressing adenovirus particles, product number: GOSA0310875-1). First, the PCR method was used to amplify the target gene TPO fragment. Then, the linearized vector GV315 (adenovirus vector) was obtained by digestion with restriction enzymes. The element order was: CMV-MCS-SV40-EGFP; among them, EGFP is an existing enhanced green fluorescent protein sequence, which can express green fluorescent protein in tissues or cells; the cloning sites are AgeI / NheI. Finally, the TPO fragment was ligated with the linearized vector GV315 to construct an adenovirus vector overexpressing TPO.

[0029] After the construction of the adenovirus vector overexpressing TPO was completed, the plasmid was transfected into 293T cells, and the expression level of the TPO gene was detected by Real-time PCR to verify the effectiveness of the adenovirus vector.

[0030] The results are shown in Table 1.

[0031] Table 1. Expression of the TPO gene

[0032]

[0033] In Table 1, the "CON group" is a 293T cell sample (control group); the "OE group" is a sample after transfection of the 293T cells with the TPO gene plasmid. The calculation by the 2-ΔΔCt method shows that: ΔCt = Ct value of the target gene - Ct value of the internal reference gene, -ΔΔCt = average ΔCt value of the NC group - ΔCt value of each sample. 2-ΔΔCt reflects the relative expression level of the target gene in each sample relative to the control group sample.

[0034] From the results in Table 1, it can be seen that in 293T cells: the TPO expression abundance in the OE group (i.e., the sample after transfection of the 293T cells with the target gene plasmid) is 3,220,178.231 times that of the CON group (i.e., the 293T cell sample control group) (P < 0.05), indicating that the adenovirus vector was successfully constructed and the target gene plasmid was well expressed after transfection into 293T cells.

[0035] Example 2: Preparation of a TPOAb-positive animal model:

[0036] (1) Three-week-old SPF-grade C57BL / 6N mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were selected. After 1 week of adaptive feeding, they were divided into a TPO adenovirus immunization group, an empty adenovirus immunization group, and a PBS control group.

[0037] (2) Gradient pre-experiments were set up to determine the optimal injection dose. During the gradient experiment, the injection doses of TPO adenovirus, empty adenovirus, and PBS were set to 3×10 9 PFU, 5×10 9 PFU, 7×10 9 PFU, 9×10 9 PFU, 1.2×10 10 PFU, 2×10 10 PFU. That is, within the TPO adenovirus immunization group, the empty adenovirus immunization group, and the PBS control group, 6 dose gradients were set, and 6 mice were injected under each gradient. The injection frequency was "injected once every 3 weeks, for a total of 3 injections". After immunization, the concentration of TPOAb in the mouse serum was detected by ELISA at 2 weeks, 4 weeks, and 6 weeks respectively to evaluate the modeling rate of TPOAb positivity in different dose groups; at the same time, by detecting the changes in the levels of thyroid hormones (TSH, FT4, T3, T4) and thyroglobulin antibody (TGAb) in mice after immunization, the effects of different dose groups of the model on thyroid function were verified.

[0038] The results are shown in Tables 2 and 3. At the same time, during the adenovirus injection, 1 mouse died in each of the 9×10 9 PFU and 2×10 10 PFU dose groups in the TPO adenovirus group.

[0039] Table 2. Modeling rate of TPOAb positivity in different dose groups (n / %)

[0040]

[0041]

[0042] Note: The absorbance value of TPOAb in the PBS control group is used as P 95 , when the OD values of the samples are greater than 0.125, 0.118, and 0.128 at 2 weeks, 4 weeks, and 6 weeks respectively, it is defined as an increase in TPOAb.

[0043] Table 3. Dynamic changes in thyroid indexes of mice after immunization (M±SD) (OD 450 nm)

[0044]

[0045] As can be seen from the above results, when the injection dose is 3×10 9 PFU / time, the highest positive modeling rate of TPOAb in the TPO adenovirus group; there is no significant difference in the levels of thyroid hormones and thyroglobulin antibodies among the three groups. The TPO adenovirus model can solely cause an increase in TPOAb without interfering with thyroid function.

[0046] Therefore, the optimal injection dose of the TPO overexpressing adenovirus vector of the present invention is "3×10^9 PFU / time, injected once every 3 weeks, for a total of 3 injections". Based on this, a TPOAb positive animal model was successfully constructed.

[0047] Example 3. Based on the TPOAb positive animal model of the present invention, study the effects of this model on the thyroid function and thyroiditis of the mother during pregnancy and the offspring, and exclude the influence of the adenovirus vector itself:

[0048] 1. Effects of pregnancy on maternal thyroid autoantibodies and thyroid hormones

[0049] Two-way ANOVA was used to explore "the levels of maternal thyroid autoantibodies and thyroid hormones in different pregnancy periods under different modeling groups (refer to Example 2) of the present invention".

[0050] The results are shown in Table 4.

[0051] Table 4. Dynamic changes of thyroid indexes during pregnancy (M±SD)

[0052]

[0053]

[0054] Note: Except for the number of cases (percentage) of the positive situation of TPOAb, which is expressed as n / %, the remaining data are expressed as mean±standard deviation. Taking the absorbance value P of TPOAb in the PBS control group 95 as the critical value, the sample OD value > P 95 , it is determined to be positive for TPOAb.

[0055] As can be seen from the above results, in the analysis of the TPOAb concentration, the interaction term of different pregnancy periods and modeling groups has no statistical significance, F(4, 81) = 2.279, P = 0.068, partial η 2 = 0.101, indicating that there is no interaction between pregnancy and modeling groups in the influence on the TPOAb level.

[0056] Since the modeling was completed before pregnancy, the positive situation of TPOAb and the changes in thyroid function in the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group in the early, middle, and late pregnancy were also compared.

[0057] The results showed (Table 4) that during the entire pregnancy period, the female mice in the TPO adenovirus group always maintained a high positive rate of TPOAb (80% - 100%). There were no significant differences in TGAb, TSH, FT4, T3, T4, and FT3 among the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group in the early, middle, and late pregnancy periods (P > 0.05).

[0058] The above indicates that after the establishment of the TPO adenovirus model, a positive state of TPOAb is continuously formed during the maternal pregnancy, which does not affect thyroid function and is not affected by pregnancy.

[0059] 2. Effects of the TPO adenovirus vector itself on the maternal thyroid, placenta, and fetal brain

[0060] Since the adenovirus vector selected in this study is "GV315 CMV - MCS - SV40 - EGFP", the virus contains the EGFP sequence, which can express green fluorescent protein in tissues, and the EGFP is expressed in the cytoplasm. To determine the infection status after injection of the TPO adenovirus, immunofluorescence staining was performed on sections of the maternal thyroid, placenta, and fetal brain. Eight samples were randomly selected from each group, and three fields of view were randomly selected from each sample section for semi - quantitative evaluation using Image J.

[0061] The results are as Figure 1 、 2 、shown in Figure 3.

[0062] Figure 1 Figure 2 shows the results of EGFP immunofluorescence staining of the thyroid, showing that there were significant differences in the expression of EGFP in the maternal thyroid during pregnancy among the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group (F = 8.127, P = 0.004). Post - hoc pairwise comparison using the Bonferroni method showed that the expression of EGFP in the thyroid of the TPO adenovirus immunization group was higher than that of the empty adenovirus immunization group (34.33 ± 27.73 vs 2.67 ± 1.86, P = 0.011), and the TPO adenovirus immunization group was also higher than the PBS control group (34.33 ± 27.73 vs 1.33 ± 0.52, P = 0.009), and there was no significant difference between the PBS control group and the empty adenovirus immunization group (P = 0.373).

[0063] Figure 2 Figure 3 shows the results of EGFP immunofluorescence staining of the placenta, showing that very little EGFP was expressed in the placenta of the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group. Fluorescent cell counting showed that there was no statistical difference in the number of green fluorescent cells among the three groups (F = 0.652, P = 0.547).

[0064] Figure 3The results of EGFP immunofluorescence staining of fetal brains showed that EGFP was hardly expressed in the fetal brains of the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group. Fluorescent cell counting showed that there was no significant difference in the number of green fluorescent cells among the three groups (F = 0.259, P = 0.778).

[0065] 3. Effects of Maternal TPOAb Positivity on Thyroid Autoantibodies and Thyroid Hormones in the Peripheral Circulation of Offspring

[0066] To clarify whether maternal TPOAb positivity exerts its effects by influencing TGAb and thyroid function in offspring, ELISA was used to compare the concentration levels of serum TGAb, TSH, T3, T4, FT4, and FT3 in pups at PND28 and PND56, and stratified analysis was performed according to the sex of the pups. The results are shown in Table 5.

[0067] Table 5. Dynamic Changes in Thyroid Indexes of Pups at PND28 and PND56 (M±SD)

[0068]

[0069]

[0070]

[0071] Note: *P < 0.05.

[0072] The results showed that at PND28, there was a significant difference in T3 levels among the three groups. Pairwise comparison showed that the T3 level in the pups of the maternal TPO adenovirus immunization group was lower than that in the PBS control group (P = 0.032). At PND56, there was a significant difference in FT4 levels among the three groups. Pairwise comparison showed that the T3 level in the pups of the maternal TPO adenovirus immunization group was lower than that in the empty adenovirus immunization group (P = 0.042). There were no significant differences in TGAb, TSH, T4, and FT3 among the pups of the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group (P > 0.05 for all).

[0073] 4. Effects of Maternal TPOAb Positivity on the Thyroid Morphology of the Mother and Offspring

[0074] Thyroid HE staining was performed on the PBS control group, the empty adenovirus immunization group, and the TPO adenovirus immunization group during pregnancy, and thyroid inflammation scores were evaluated ( Figure 4 ).

[0075] Figure 4 The results of thyroid HE staining and thyroid inflammation scores are shown.

[0076] The results showed that there was no significant difference in the thyroiditis score among the three groups during pregnancy (F = 0.978, P = 0.398), and the thyroid follicles were evenly distributed without atrophy. At PND28 and PND56, there was also no significant difference in the inflammation score of the thyroid glands of the offspring mice in the PBS control group, the adenovirus vector immunization group, and the TPO adenovirus immunization group (F = 2.620, P = 0.089; F = 0.888, P = 0.420).

[0077] In summary, maternal TPOAb positivity is not related to the thyroid function of the offspring. There is no obvious difference in TGAb and other thyroid hormones between the TPO adenovirus immunization group and the control group, further verifying that the effect of maternal TPOAb does not depend on the thyroid function of the offspring. In addition, this model is also different from the "animal model of autoimmune thyroiditis", and no significant difference in the thyroiditis score is found between the TPO adenovirus immunization group and the control group in pregnant mice and their offspring.

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a TPOAb-positive animal model based on an adenovirus vector, characterized in that, It includes the following steps: (1) Construct an adenovirus vector overexpressing TPO; (2) Inject the adenovirus vector overexpressing TPO into experimental animals to induce the production of TPOAb; (3) Determine the optimal injection dose through a gradient dose experiment and construct a TPOAb-positive animal model.

2. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 1, wherein In step (1), the method for constructing the adenovirus vector overexpressing TPO is as follows: Clone the full-length cDNA of thyroid peroxidase TPO, integrate it with the adenovirus vector GV315, and construct an adenovirus vector overexpressing TPO.

3. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 1, wherein, In step (1), after the construction of the adenovirus vector overexpressing TPO is completed, it also includes the step of "detecting the expression level of the TPO gene by Real-time PCR to verify the effectiveness of the adenovirus vector".

4. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 1, wherein In step (2), 3-week-old SPF-grade C57BL / 6N mice are selected as experimental animals.

5. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 1, wherein In step (3), the optimal injection dose of the adenovirus vector overexpressing TPO is: 3×10^9 PFU / time, injected once every 3 weeks for a total of 3 times.

6. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 1, wherein In step (3), it also includes the steps of "verifying the model establishment rate and the impact of the model on thyroid function".

7. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 6, wherein Detect the concentration of TPOAb in the mouse serum by ELISA to evaluate the model establishment rate.

8. The method for constructing a TPOAb-positive animal model based on an adenovirus vector according to claim 6, wherein Verify the impact of the model on thyroid function by detecting the levels of thyroid hormones TSH, FT4, T3, T4 and thyroglobulin antibody TGAb.

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