CN119215155A - A recombinant amphiregulin vaccine and its application in the treatment of pulmonary fibrosis - Google Patents
A recombinant amphiregulin vaccine and its application in the treatment of pulmonary fibrosis Download PDFInfo
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- CN119215155A CN119215155A CN202411575387.3A CN202411575387A CN119215155A CN 119215155 A CN119215155 A CN 119215155A CN 202411575387 A CN202411575387 A CN 202411575387A CN 119215155 A CN119215155 A CN 119215155A
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- pulmonary fibrosis
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- C—CHEMISTRY; METALLURGY
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Abstract
The invention relates to the technical field of immunotherapy, in particular to a amphiregulin recombinant vaccine and application thereof in pulmonary fibrosis treatment. The vaccine contains polypeptides of amphiregulin antigen epitope, and the amino acid sequence of the polypeptides is shown as SEQ ID No. 1. Research results show that the recombinant vaccine can effectively induce mice to generate specific antibodies aiming at AREG protein in vivo, thereby inhibiting the secretion of the AREG protein and the activation of downstream receptors thereof in the pulmonary fibrosis process. By reducing collagen deposition in lung tissues and alleviating tissue structure damage, the recombinant vaccine significantly reduces gene expression of fibrosis related proteins, restores lung function of a lung fibrosis model mouse, and inhibits further development of diseases. In addition, the recombinant vaccine can be administrated in a mode of aerosol inhalation, and has broad spectrum, high efficiency, long-term stable expression and good safety. The method provides a brand-new targeted treatment means for clinical prevention and treatment of pulmonary fibrosis, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of immunotherapy, in particular to a amphiregulin recombinant vaccine and application thereof in pulmonary fibrosis treatment.
Background
Pathological fibrosis is a common outcome of many chronic inflammatory diseases. It is estimated that in developed countries, almost half of the cases of death are associated with pathological fibrosis. Among them, pulmonary fibrosis is a progressive and irreversible disease, and its prognosis is fatal, although anti-fibrosis treatments exist. Pulmonary fibrosis is composed of a heterogeneous group of pulmonary diseases, and is mainly characterized by progressive and irreversible destruction of the pulmonary structure by scarring, ultimately leading to organ dysfunction, gas exchange dysfunction, and respiratory failure. At present, the incidence and mortality of pulmonary fibrosis rise year by year, significantly increasing the disease burden. However, existing treatments rely primarily on two drugs approved by the U.S. FDA, nidazole and pirfenidone, which can only alleviate but not reverse pulmonary fibrosis. Early diagnosis and exploration of new therapeutic approaches is therefore critical for clinical management of pulmonary fibrosis.
Amphiregulin (Amphiregulin, AREG) is found in the epidermal growth factor family gene cluster of 4q13.3 chromosome band, and contains two gene copies (AREG and AREGB) separated by about 160kb and spanning about 10kb of genomic DNA. AREG gene transcription produces an mRNA of 1.4kb in length containing 6 exons, ultimately encoding a transmembrane glycoprotein precursor (Pro-AREG) of 252 amino acids. Under the action of metalloproteinase TACE or ADAM-17, the protein precursor hydrolytically cleaves, releasing mature soluble AREG containing the EGF motif, followed by induction of autocrine or paracrine activation of its downstream Epidermal Growth Factor Receptor (EGFR). AREG-EGFR signaling can activate multiple pathways such as Ras-Raf/MAPK, PI3K/Akt or STAT, and thus regulate multiple cellular processes such as cell proliferation, apoptosis and migration. Research has shown that a variety of immune cells can produce AREG under different inflammatory stimuli, including basophils, mast cells, type 2 innate lymphocytes, dendritic cells, neutrophils, cd8+ T cells, regulatory T cells (tregs), activated cd4+ T cells, and the like. This suggests that AREG may play a role in immune-related resistance and tolerance mechanisms. Increased AREG expression has been associated with the development of fibrosis in multiple organs (e.g., lung, liver, and heart) and has become a critical role in immunity, inflammation, and tissue repair. However, the specific mechanism of the AREG-EGFR axis in the development of pulmonary fibrosis is not yet clear. Several studies have shown that the knockout of AREG can reduce Bleomycin (BLM) and TGF- β1 transgenic mice induced pulmonary fibrosis. However, there is also literature that tracheal instillation with AREG recombinant protein can alleviate BLM-induced pulmonary fibrosis. Considering the high expression of AREG in patients with idiopathic pulmonary fibrosis, elucidating the pathogenesis of AREG-EGFR axis in pulmonary fibrosis has important public health significance and clinical application value.
Vaccines are common drugs for preventing infectious diseases, and have been developed in recent years for treating autoimmune-related disorders such as hypertension, dyslipidemia, alzheimer's disease, cancer, and inflammatory diseases. If the efficacy and safety of a vaccine were comparable to existing drug therapies, the vaccine would be a potential alternative to treating these lifestyle diseases. Current vaccine treatments for pulmonary fibrosis are not yet mature, and some critical molecules associated with disease (e.g., TGF- β, IL-13, CTGF, etc.) have been identified as potential targets. However, these molecules also play an important role in normal tissue repair and other physiological functions, and treatment against them may trigger side effects or interfere with normal immune response and repair processes. In contrast, the lower expression level of AREG in normal tissues reduces the risk of off-target effects. Furthermore, the AREG knockout and siRNA silencing experiments did not cause significant side effects or reduced life span in animal models. Therefore, AREG is expected to be an ideal target for vaccine development as a key contributor to pulmonary fibrosis. The construction of the vaccine for AREG is not only expected to replace the existing pulmonary fibrosis treatment drug, but also can effectively reduce the medical cost and the disease burden of pulmonary fibrosis, and has important clinical and public health significance.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a amphiregulin recombinant vaccine and application thereof in pulmonary fibrosis treatment.
In order to achieve the above object, the present invention provides the following solutions:
a amphiregulin recombinant vaccine contains polypeptides of amphiregulin antigen epitope, and its amino acid sequence is shown in SEQ ID No. 1.
The vaccine comprises a polypeptide conjugate comprising the polypeptide of claim 1.
The polypeptide conjugate is a polypeptide and a conjugate part as claimed in claim 1, wherein the conjugate part is connected with the polypeptide part, and the conjugate part is at least one selected from keyhole limpet hemocyanin, ovalbumin and bovine serum albumin.
The mass ratio of the polypeptide to the coupling part is 1-10:1, preferably 1-5:1.
The polypeptide conjugate is obtained by incubating the polypeptide of claim 1 and the coupling moiety for 8-12 hours at room temperature, wherein the mass ratio of the polypeptide to the coupling moiety (KLH) to the protein cross-linking agent (Sulfo-SMCC) is 1-10:1-10:1, preferably 1-5:1-5:1.
The vaccine is the polypeptide conjugate and a physiologically acceptable excipient thereof, wherein the mass ratio of the polypeptide conjugate to the excipient is 1-10:25, and preferably 1-5:25.
The vaccine is added with an adjuvant in the using process, and the volume ratio of the immunological adjuvant to the vaccine is 1:1-5, preferably 1:1-2. Wherein the adjuvant is Freund's adjuvant, montana oil adjuvant, RIBI adjuvant, MF59 emulsion adjuvant, or QS-21 adjuvant.
Use of said recombinant vaccine in the treatment of pulmonary fibrosis.
The recombinant vaccine is used for treating pulmonary fibrosis by inducing in vivo generation of neutralizing antibodies against AREG protein through the polypeptide conjugate, so that secretion of the AREG protein and activation of downstream signal channels are reduced, and deposition of collagen fibers and destruction of tissue structures of pulmonary tissues in a fibrosis process are reduced.
The administration mode of the recombinant vaccine is intravenous injection, arterial injection, inhalation administration, intramuscular injection, subcutaneous injection, organ injection and intrathoracic and intraabdominal injection.
The invention discloses the following technical effects:
The recombinant vaccine targeting the AREG protein can effectively induce the generation of specific antibodies aiming at the AREG protein in vivo, thereby reducing the secretion of the AREG protein and inhibiting the activation of a downstream signal channel thereof. By reducing collagen fiber deposition and tissue structure damage in pulmonary fibrosis mouse pulmonary tissue, the vaccine obviously reduces gene expression of fibrosis related proteins, restores pulmonary function, and further realizes the effect of treating pulmonary fibrosis. In a specific implementation, a lung fibrosis mouse model is respectively constructed by adopting a humidifier to perform aerosol inhalation on polyhexamethylene guanidine (PHMG) and tracheal instillation Bleomycin (BLM), and a vaccine is administrated in an aerosol inhalation mode. The results show that the recombinant vaccine effectively reduces collagen deposition in lung tissue and significantly restores lung function in both lung fibrosis mouse models. The vaccine can be used as a candidate medicament for treating pulmonary fibrosis, has no obvious adverse effect, and has good popularization and application prospects. The invention develops or screens the medicine for treating the pulmonary fibrosis by taking the AREG protein as a target spot, and has important clinical application value and research significance.
Drawings
FIG. 1 is a serum antibody titer determination in mouse blood after tracheal instillation of recombinant vaccine.
FIG. 2 is an illustration of AREG protein expression in mouse alveolar lavage fluid following aerosol inhalation PHMG induction of pulmonary fibrosis in a mouse model tracheal instillation of recombinant vaccine.
Figure 3 is a graph showing changes in lung function following tracheal instillation of recombinant vaccine in a mouse model of pulmonary fibrosis induced by aerosol inhalation PHMG.
FIG. 4 is a graph showing collagen fiber deposition in lung tissue after aerosol inhalation PHMG induces tracheal instillation of recombinant vaccine in a mouse model of pulmonary fibrosis.
FIG. 5 shows the pulmonary tissue type I collagen (Col 1a 1), fibronectin (Fn 1) and alpha-smooth muscle actin (Acta 2) expression levels after aerosol inhalation PHMG-induced tracheal instillation of recombinant vaccine in a mouse model of pulmonary fibrosis.
FIG. 6 shows AREG protein expression in mouse alveolar lavage fluid following tracheal instillation of recombinant vaccine in a BLM-induced pulmonary fibrosis mouse model.
FIG. 7 shows changes in lung function following tracheal instillation of recombinant vaccine in a BLM-induced pulmonary fibrosis mouse model.
FIG. 8 is a graph showing collagen fiber deposition in lung tissue after tracheal instillation of recombinant vaccine in a BLM-induced pulmonary fibrosis mouse model.
FIG. 9 shows the expression levels of lung tissue type I collagen (Col 1a 1), fibronectin (Fn 1) and alpha-smooth muscle actin (Acta 2) after tracheal instillation of recombinant vaccine in a BLM-induced pulmonary fibrosis mouse model.
Figure 10 is a schematic of the production of antibodies by a recombinant vaccine into the body.
In the figure, FVC represents forced vital capacity, IC represents deep inspiratory capacity, cchord represents quasi-static compliance, control represents Control group, vaccine represents experimental group, and GAPDH is widely distributed in cells in various tissues as reference gene.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Unless specifically defined otherwise, all technical and scientific terms used herein should be interpreted according to the understanding of one of ordinary skill in the art. Although the invention has been described in terms of preferred methods and materials, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention without departing from the scope or spirit of the invention. The description of the invention may lead the skilled person to other obvious embodiments. These descriptions and examples are for illustrative purposes only.
The recombinant vaccine can effectively induce the generation of specific antibodies aiming at AREG protein in vivo, and reduce the deposition of collagen fibers and the damage of tissue structures in the pulmonary fibrosis process by neutralizing the AREG protein and reducing the secretion of the AREG protein and inhibiting the activation of downstream signal channels. Meanwhile, the vaccine can reduce the gene expression of fibrosis related proteins, restore the lung function and finally achieve the effect of treating the pulmonary fibrosis.
Meanwhile, the recombinant vaccine can effectively induce mice to generate specific antibodies aiming at AREG protein in vivo, so that secretion of the AREG protein and activation of a downstream receptor thereof in the pulmonary fibrosis process are inhibited. By reducing collagen deposition in lung tissues and alleviating tissue structure damage, the recombinant vaccine significantly reduces gene expression of fibrosis related proteins, restores lung function of a lung fibrosis model mouse, and inhibits further development of diseases. In addition, the recombinant vaccine can be administrated in a mode of aerosol inhalation, and has broad spectrum, high efficiency, long-term stable expression and good safety. The method provides a brand-new targeted treatment means for clinical prevention and treatment of pulmonary fibrosis, and has wide application prospect.
EXAMPLE 1 Synthesis of Polypeptides
The research constructs a target polypeptide NH 2-CDCRVNLCY-CONH2 (SEQ ID No. 1) by a solid phase synthesis technology, and the specific process covers the chemical synthesis, purification and final identification analysis of the polypeptide.
1. Polypeptide solid phase synthesis flow
The synthesis of the polypeptides employs a Solid Phase Peptide Synthesis (SPPS) method based on the Fmoc (9-fluorenylmethoxycarbonyl) protecting group strategy. The main steps of the synthesis process include:
1) The carboxyl of the C-terminal amino acid is covalently combined with the solid-phase resin carrier, thereby laying a foundation for the subsequent peptide chain extension.
2) Before each reaction step, the Fmoc protecting group of the amino acid was removed using a piperidine solution, exposing the free N-terminal amino group.
3) Activated amino acids (activated by HBTU or HATU) are introduced to bind to the free amino group of the previous residue, gradually extending the peptide chain. After each round of coupling, the operation of cleaning and deprotection is carried out for a plurality of times, so that the accuracy of synthesis is ensured.
4) After completion of the sequence ligation of all amino acid residues, the protecting groups were cleaved from the resin with a TFA solution (containing the appropriate amount of capture agent) and removed to give the crude polypeptide.
2. Purification of polypeptides
The crude polypeptide is separated and purified by reverse phase high performance liquid chromatography (RP-HPLC) under the following specific operating conditions:
Mobile phase a aqueous solution containing 0.1% trifluoroacetic acid (TFA)
Mobile phase B acetonitrile solution containing 0.1% TFA
Gradient procedure starting from the initial conditions of 90% mobile phase a and 10% mobile phase B, the gradient was transitioned to 40% a and 60% B in 30 minutes. The separation flow rate was set at 1mL/min and the operating temperature was room temperature (about 23 ℃).
The detection mode is that an ultraviolet detector is adopted, and the wavelength is 214nm.
Dissolving the synthesized crude polypeptide in mobile phase A, taking a proper amount (about 20-30mg or 2-2.5 mL) of sample, injecting into a liquid chromatography system, eluting and separating according to set gradient conditions, collecting main peak part, and lyophilizing to obtain purified polypeptide sample, wherein the amino acid is shown in SEQ ID No. 1.
3. Analysis of polypeptide purity
The purified polypeptide is subjected to purity identification by a liquid chromatography-mass spectrometry (LC-MS) technology, and the analysis conditions are as follows:
mobile phase A0.05% TFA in water
Mobile phase B0.1% TFA acetonitrile solution
Gradient elution-starting from an initial ratio of 90% A and 10% B, a gradual transition to 40% A and 60% B was made in 10 minutes, the flow rate was maintained at 1mL/min and the temperature was room temperature (23 ℃).
Detection mode using an ultraviolet detector of 214 nm.
Mass spectrometry detection the molecular weight and purity of the polypeptides were analyzed by atmospheric pressure piezospray ionization (API-ESI) mass spectrometry.
The identification method combines high resolution of HPLC and accurate molecular weight measurement of mass spectrum, can effectively verify the purity and molecular structure of synthesized polypeptide, and ensures the accuracy of experimental results. The purity of the identified polypeptide is more than 95%, so that the purity of the vaccine is achieved.
EXAMPLE 2 preparation of polypeptide conjugates
In this experiment, the polypeptide synthesized in example 1 was subjected to a coupling reaction with Keyhole Limpet Hemocyanin (KLH), and the specific procedures include bed preparation, preparation of a polypeptide mixture, a coupling reaction, and preservation of the final product. The method comprises the following specific steps:
1. Bed preparation
First, the column bed was pretreated with pure water and coupling buffer. The coupling buffer solution is AH solution, the components comprise Na 2HPO4、NaH2PO4, naCl and EDTA, the pH value of the solution is adjusted to 7.2, and the suitability of a reaction system is ensured.
2. Preparation of polypeptide mixtures
The polypeptide synthesized in example 1 was dissolved in N, N-Dimethylformamide (DMF) and allowed to stand at room temperature for 30 minutes until no particles were visible in the solution. Subsequently, AH solution was added to give a mixture of polypeptides, the final concentration of polypeptide in the mixture being 10mg/mL. This step ensures adequate dissolution and uniform distribution of the polypeptide prior to the coupling reaction.
3. Reaction of the coupling moiety with Sulfo-SMCC
Next, the coupling moiety (KLH) was mixed with AH solution to give a first mixture, the final concentration of the coupling moiety was 20mg/mL. At the same time, sodium salt of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic sulfosuccinimidyl ester (sulfoSMCC) was dissolved in DMSO to prepare a second mixture at a concentration of 150 mg/mL. The two were mixed in proportion and reacted at 4 ℃ for 12 hours. After the reaction is completed, the reaction product is separated by a chromatographic column to obtain an intermediate.
4. Generation of polypeptide conjugates
After the intermediate is obtained, it is thoroughly mixed with the polypeptide mixture prepared in step 2, and a vertical mixer is used to ensure uniform dispersion of the reactants. Thereafter, the mixture was allowed to continue to react overnight at room temperature to ensure completion of the coupling reaction. During the reaction, the mass ratio of the intermediate to the polypeptide, the coupling moiety (KLH) and the Sulfo-SMCC in the polypeptide mixture was 10:10:1.
The final product is a polypeptide conjugate, which is stored at-20 ℃ after production to ensure the stability and bioactivity of the polypeptide.
Example 3 mouse immunization experiment
Using the polypeptide conjugate prepared in example 2, an immunization experiment was performed after dilution in physiological saline and emulsification with an equal volume of adjuvant. The subjects were eight week old C57BL/6 mice, which were immunized by random grouping.
1. Immunization dose and strategy
The immunization procedure was performed using a pulmonary fluid quantitative nebulizer, by tracheal instillation, and each mouse received an immunization dose on days 0, 14, 28 and 56, respectively. The two doses were 2 μg or 20 μg of the polypeptide conjugate per dose, respectively, and the injection amount was determined by the experimental design. Control mice were injected with an equal amount of KLH, formulated by mixing with an equal volume of Freund's adjuvant to exclude the effect of the adjuvant on the immune response.
2. Serum collection and antibody detection
During the course of the immunization experiments, serum samples of mice were collected via the tail vein, with time nodes consistent with the immunization dose. Subsequently, the immune response of the mice was assessed by measuring the antibody titer against the immune peptide in the serum using an enzyme-linked immunosorbent assay (ELISA).
Example 4 serum ELISA detection experiments after mice immunization
The mouse serum prepared in example 3 was analyzed by ELISA method to determine its specific antibody level against AREG protein. The specific experimental steps are as follows:
ELISA plate coating
First, 200ng of purified AREG protein (R & D) was added to each ELISA plate well, and 50mM Na 2CO3/NaHCO3 buffer (pH 9.6) was used as a coating solution. ELISA plates were subjected to protein coating overnight at 4 ℃. After the coating is completed, the coating liquid is discarded.
2. Sealing and washing
Mu.L of 5% nonfat dry milk was added to each well and blocked at room temperature for 1 hour to prevent non-specific binding. After blocking, the ELISA plates were washed twice with PBS solution containing 0.05% Tween-20, ensuring adequate removal of the residue.
3. Serum incubation
Subsequently, 100. Mu.L of antigen-immunized serum after gradient dilution or PBS-immunized serum as a control was added to each well, and incubated at room temperature for 1 hour. Thereafter, the supernatant was discarded, and the ELISA plate was washed five times again with PBS solution containing 0.05% Tween-20 to remove unbound antibody.
4. Secondary antibody incubation and color development
100 Μl of horseradish peroxidase-labeled goat anti-mouse IgG antibody (HRP secondary antibody) was added at 1:3000 (v/v) dilution per well and incubation was continued for 1 hour at room temperature. After incubation was completed, the secondary antibody solution was discarded and the plates were washed five times with PBS containing 0.05% Tween-20. Subsequently, 50. Mu. LELISA of the color development liquid was added to each well and developed for 15 minutes.
5. Reaction termination and OD value reading
To terminate the reaction, 50 μl of 2M H 2SO4 was added per well, and finally the optical density (OD 450) values at 450nm wavelength were read by a microplate reader.
6. Antibody titer calculation
The calculation of the antibody titer was based on multiplying the negative control value of the non-added serogroup by 2.1 times to obtain a benchmark value, and taking the log value (log value) of the lowest dilution multiple exceeding the benchmark value as the antibody titer. The results show that the antibody titer of the serum of the mice in the vaccine treatment group, which binds to the AREG antigen, is significantly higher than that of the mice in the control group (figure 1), which indicates that the vaccine has better immune effect.
Example 5 therapeutic Effect of vaccine on nebulized inhalation PHMG induced pulmonary fibrosis mice
The experiment selects C57BL/6 mice, and the mice are evenly divided into four experiment groups according to the weight, wherein 5 mice in each group are specifically divided into the following groups:
Control group (Control) mice in this group received normal treatment without PHMG exposure or vaccine intervention. For observing physiological conditions under normal conditions as a baseline control.
A single intervention group (Vaccine) which mice were not exposed to PHMG and only received the intervention of the recombinant Vaccine during the course of the experiment, in order to evaluate the effect of the Vaccine itself on normal mice.
Fibrosis group (PHMG) this group of mice was exposed to PHMG aerosol to induce pulmonary fibrosis, mimicking the disease model. The group received no vaccine intervention and was used to evaluate PHMG-induced fibrosis effects.
Fibrosis intervention group (PHMG-Vaccine) this group of mice was also exposed to PHMG aerosol to induce pulmonary fibrosis, but began to receive recombinant Vaccine intervention at week 3 of PHMG exposure. This group was used to evaluate the effect of the vaccine on the intervention in the course of fibrosis.
All mice were housed in cages, with 5 mice housed in each cage, corresponding to the four groups of the experiment. The experimental treatment steps for each group were identical, the only difference being whether PHMG exposures and vaccine interventions were performed. By this grouping design, differences between the control group, vaccine intervention group, fibrosis intervention group and fibrosis intervention group can be effectively compared, defining PHMG and individual and combined effects of the vaccine.
PHMG A specific method for inducing pulmonary fibrosis is by dissolving PHMG powder in ultrapure water to prepare 0.1mg/mL solution. The solution was then fed to an aerosol generator and exposed to mice from the fibrotic group and the fibrotic intervention group, while mice from the control group and the simple intervention group were exposed using ultrapure water. The spray amount was set to 30-40mL/h, and the exposure was performed every two days for 4 hours each with an 8 week period. Intervention with the recombinant vaccine was started on week 3 of PHMG exposure. The mice of the single intervention group and the fibrosis intervention group were each injected into the lungs of the mice by tracheal instillation with a pulmonary liquid quantitative nebulizer after dissolving 20 μg of the recombinant vaccine using 50 μl of PBS and an equal volume of freund's adjuvant. The control and fibrosis groups were injected with an equal amount of KLH and an equal volume of Freund's adjuvant. The interventions were 1 time per week for a total of 5 times.
Extracting a mouse alveolar lavage fluid, and detecting the content of AREG protein in alveolar relational fluid by using AREG ELSIA detection kit. The results are shown in FIG. 2, and compared with the Control mice, the AREG protein in the alveolar lavage fluid is slightly reduced, but no statistical difference exists, the AREG protein content in the PHMG alveolar lavage fluid is obviously increased, and the AREG protein content in the PHMG-Vaccine lavage fluid is obviously reduced relative to the PHMG group, so that the Vaccine can effectively reduce the secretion of the AREG protein in the lung tissue of the PHMG-induced pulmonary fibrosis mice after the Vaccine is dried.
The changes in mouse lung function were detected using a small animal lung function meter, and the results are shown in fig. 3. Forced vital capacity (FVC, fig. 3A) is used to assess the ventilation function of the lungs. The results showed that the Vaccine group did not significantly alter FVC compared to the Control group mice, whereas the PHMG group mice had significantly reduced FVC values, suggesting that PHMG inhalation exposure caused impairment of lung ventilation. Compared with PHMG groups, the lung ventilation function of PHMG-Vaccine group mice is obviously recovered. The deep inhalation volume (IC, fig. 3B) was used to assess the ability of the lungs at deep inhalation. Compared with Control mice, the IC of mice in the Vaccine group did not change significantly, while the IC of mice in the PHMG group decreased significantly, indicating that PHMG inhalation exposure resulted in a decrease in lung volume and that PHMG-Vaccine mice recovered significantly. Static compliance (Cchord, fig. 3C) was used to assess the elasticity and distensibility of the lungs under static conditions. The Cchord of the Vaccine group mice did not change significantly compared to the Control group mice, but the Cchord values of the PHMG group were significantly reduced, indicating that PHMG inhalation exposure resulted in a decrease in lung tissue static compliance and that the lung static compliance of the PHMG-Vaccine group mice was significantly restored. Overall results indicate that vaccine intervention is effective in restoring PHMG-induced decline in lung function in mice.
After mice were sacrificed, lung tissue sections were taken and Masson stained to evaluate collagen deposition, and the results are shown in fig. 4A. Analysis results show that compared with Control mice, the lung tissues of the mice in the Vaccine group do not have obvious collagen deposition or pathological structural damage of the lung tissues. By quantitative analysis of collagen fibers in Masson stained sections (fig. 4B), PHMG inhalation exposure was found to result in a significant increase in collagen deposition in mouse lung tissue. The use of vaccine dry prognosis can effectively reduce collagen deposition.
The lung tissue of the above-mentioned mice was ground with TRIzol reagent, and after RNA extraction, qPCR was performed to verify the expression of Col1a1, fn1 and Acta2 genes in the lung tissue of the mice, and the results are shown in FIG. 5. Analysis results show that the expression of fibrosis markers in lung tissues of PHMG-induced pulmonary fibrosis mice can be effectively inhibited after the tracheal instillation of the vaccine.
The above results demonstrate that the vaccine against AREG has highly potent and safe properties against lung tissue. In addition, AREG proteins play a key role in PHMG-induced pulmonary fibrosis. Mice induced with PHMG lung fibrosis can be injected with a vaccine targeting the AREG protein to effectively restore the lung function of the mice and alleviate the progression of lung fibrosis.
EXAMPLE 6 therapeutic Effect of vaccine on mice with tracheal instillation of BLM induced pulmonary fibrosis
The experiment selects C57BL/6 mice, and the mice are evenly divided into four experiment groups according to the weight, wherein 5 mice in each group are specifically divided into the following groups:
Fibrosis group (BLM) the group of mice was exposed to BLM to induce pulmonary fibrosis, mimicking a disease model. The group received no vaccine intervention and was used to evaluate the effects of BLM induced fibrosis.
Fibrosis intervention group (BLM-Vaccine) this group of mice was also exposed to BLM to induce pulmonary fibrosis, but received recombinant Vaccine intervention at the end of week 1 of BLM exposure. This group was used to evaluate the effect of the vaccine on the intervention in the course of fibrosis.
All mice were housed in cages, with 5 mice housed in each cage, corresponding to two groups of experiments. The experimental treatment steps for each group were identical, the only difference being whether vaccine intervention was performed.
The BLM induction of pulmonary fibrosis was performed by dissolving BLM powder using PBS, adding the BLM-containing solution to a pulmonary fluid quantitative nebulizer, and exposing mice of the fibrosis group, fibrosis intervention group, and fibrosis capsid protein optimized intervention group at a dose of 2U/kg. Intervention of the recombinant vaccine on the fibrotic intervention group was started on week 1 of BLM exposure. The mice of the fibrotic intervention group were each injected into the lungs of the mice by tracheal instillation with a pulmonary fluid quantitative nebulizer after dissolving 20 μg of the recombinant vaccine using 50 μl of PBS and an equal volume of freund's adjuvant. The control and fibrosis groups were injected with an equal amount of KLH and an equal volume of Freund's adjuvant. The intervention was performed once a week for a total of three times.
After 4 weeks of exposure, mouse alveolar lavage fluid was extracted and the amount of AREG protein in alveolar relational fluid was detected using AREG ELSIA detection kit. The results are shown in FIG. 6, and the AREG protein content in the lavage fluid of the BLM-Vaccine group is significantly reduced relative to that of the BLM group, which suggests that the Vaccine can effectively reduce the secretion of AREG protein in the lung tissue of a mice with the BLM induced pulmonary fibrosis after the Vaccine is dried.
The change in lung function of mice was detected using a small animal lung function meter. Fig. 6 shows the results of the determination of mouse lung function. Forced vital capacity (FVC, fig. 7A) is used to assess the ventilation function of the lungs. The results show that the lung ventilation function of the mice in the BLM-Vaccine group is obviously recovered relative to that of the mice in the BLM group. The deep inhalation volume (IC, fig. 7B) was used to assess the ability of the lungs at deep inhalation. The results show that the lung volumes of the mice in the BLM-Vaccine group were significantly recovered relative to the BLM group. Static compliance (Cchord, fig. 7C) was used to assess the elasticity and distensibility of the lungs under static conditions. The results show that lung compliance was significantly restored in the BLM-Vaccine group mice compared to the BLM group. Overall results indicate that vaccine intervention is effective in restoring the decline in lung function in mice induced by BLM.
After mice were sacrificed, lung tissue sections were stained for MASSON to assess collagen deposition (fig. 8A). Quantitative analysis of collagen fibers in MASSON stained sections (fig. 8B) showed that BLM inhalation exposure resulted in a significant increase in collagen deposition in mouse lung tissue. Through vaccine intervention, collagen deposition can be effectively reduced.
The above-mentioned mouse lung tissue was taken, and TRIzol was added thereto for milling to extract RNA, followed by qPCR verification, and the expression of Col1a1, fn1 and Acta2 genes in the mouse lung tissue was examined (FIG. 9). The results show that the expression of fibrosis markers in lung tissues of BLM-induced pulmonary fibrosis mice can be effectively inhibited after the mice are subjected to tracheal instillation of the vaccine.
The above results demonstrate that the AREG protein plays a key role in BLM-induced pulmonary fibrosis. Mice with BLM-induced pulmonary fibrosis can be effectively restored in pulmonary function and progression of pulmonary fibrosis relieved by injecting a vaccine targeting the AREG protein.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
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