动物实验 z-bo 2020-08-25 624

　　The approval determines whether the systemic immunity of the approved personnel against Helicobacter pylori can be achieved. The efficacy of a vaccine combining Helicobacter pylori antigen and aluminum hydroxide (AlOH3) was evaluated in a mouse model of Helicobacter pylori infection. Antigen and -AlOH3 immunity can induce the secretion of interleukin 5, antigen-specific T cells and immune antigens. Complete Freund's adjuvant can induce type II interferon secretion and antigen-specific T cells (determined by ELISPOT). The protection of the two immune responses was evaluated by culturing two histological gastric biopsy specimens, because no bacteria were confirmed after encountering Helicobacter pylori. The protection mechanism is independent of antibodies. The use of CD4+ splenic T cells from antibody-deficient MMT mice (immunoglobulin knockout mice) and immunized mice is sufficient to transfer protective immunity to immunodeficiency receptors. The results indicate that there are other or potential strategies for the rapid development of Helicobacter pylori vaccine. Helicobacter pylori, the most common pathogen in the world, is an extracellular bacteria that can infect the gastric mucosa and cause gastritis and peptic ulcers. It is generally believed that infections are mainly found in young children.

　　Therefore, the use of preventive vaccines may prevent infants from being infected with Helicobacter pylori and prevent adults from receiving long-term vaccine protection. The vaccination strategy is mainly to induce mucosal immunity through oral and nasal passages. These vaccines require bacterial exotoxin adjuvants, which are not safe for human use. Recently, it has been reported that systemic immunity is a possible means of inducing and protecting mouse Helicobacter pylori immunity. The authors put forward the idea that induction of immunity requires Th1-type immune response, a contribution from the pathology of gastric cancer associated with Helicobacter pylori-related reactions. This recommendation is based on immunoglobulin (IgG) class analysis and does not directly measure cytokines. Several reports have confirmed that this protective effect may be mediated by type 2 immunity. We will use parenteral immune adjuvants to investigate this more direct method using the ELISPOT method, which produces a highly polarized Th1 or Th2 response. In this study, we investigated whether systemic immunity with aluminum hydroxide adjuvant (AlOH3) in the absence of antibodies can induce protective Th2CD4 T cell immunity. AlOH3 induces immunity, which brings huge advantages to the development of Helicobacter pylori vaccine. This is because AlOH3 is approved for use in humans. Its safety, stability and low cost promote the large-scale development and application of human Helicobacter pylori. Materials and methods: The mice were purchased from Jackson Laboratory and kept under certain pathogen-free conditions. The mice used were C57BL/6 or C57BL/6J-Rag1tm1Mom (rag1/) lacking mature T and B lymphocytes, or C57BL/6-Igh-6tm1Cgn (mMT) lacking mature B cells.

　　The bacterium H.felis was isolated from a cat stomach biopsy specimen in our laboratory. The Helicobacter pylori HPM6 strain was isolated from human gastric biopsy specimens and adjusted in our laboratory through long-term passage of mice. Detection of cagA by PCR confirmed that HpM6 and inoculation of C57BL/6 mice with HpM6 can cause chronic infection, which lasts for 12 months in 100% infected mice. Two types of Helicobacter pylori were identified by colony morphology, cell morphology, Gram staining, urease, catalase, and oxidase. Bacteria grow on solid medium (agar blood) and are suspended in broth broth. Mouse immunity and pathway: Helicobacter pylori lysate, we and others have proven that this is an effective experimental vaccine antigen by oral route. Ovalbumin (OVA) was purchased from Sigma Chemical Co., Ltd., and AlOH3 was purchased from Pierce. Freund's complete adjuvant (CFA) is made by mixing Mycobacterium tuberculosis H37RA (Difco Laboratories) with 1 mg/mL to make Freund's incomplete adjuvant. The antigen and adjuvant were mixed in water at a ratio of 1:1, and 100 mg antigen and 100 ml injection emulsion were injected intraperitoneally into each mouse on day 0. On day 28, a 0.5 mL culture containing 1×10 7 cfu bacteria was administered through a gastric cannula through an 18-gauge needle. Determine the number of bacteria by setting the optical density to 450m using the growth curve previously established. As an isolate, H. Felis is difficult to grow and reproduce, so the Helicobacter pylori growth curve is used to determine the standard intake value, as described elsewhere. Titer determination of vaccine titer: 28 days later, mice were infected with Helicobacter pylori by tissue silver staining. Euthanize the animal with carbon dioxide. The elongated tissue from the duodenum to the card gate was surgically removed. After fixing the tissue with 10% formaldehyde buffer, the histological examination was performed. Several parts of each mouse sample were stained with silver stain to help identify Helicobacter pylori and Helicobacter pylori by the location and morphology of the bacteria. No Helicobacter pylori was found in the tissues, confirming that the mice were immune protected by silver staining. In addition, gastric biopsy specimens were cultured from mice infected with Helicobacter pylori to check for the presence of bacteria. The sample containing Helicobacter pylori was surgically removed from the gastric antrum and mixed with 200 mL of medium. The homogenate was added to 100 mL of medium containing 7% horse blood. After 96 times of aerobic incubation, the immunized mouse samples were immune protected without growing bacteria. If bacteria are present, the morphology of Helicobacter pylori colony, Gram staining and the production of urease, catalase and oxidase determine the bacteria. H.felis’ culture cannot be manipulated because it is unreliable. H.felis grows in slices, not independently. Pathological evaluation: As in other literature, the intensity of inflammation in the longitudinal section of the large curvature of the mouse stomach was evaluated. This part includes the entire length of the gastric antrum and fundus mucosa. Antrum inflammation grade is 0 to 3, and fundus inflammation grade is 1 to 10. The total score for each mouse is linear range (focal, multifocal, spot or diffuse) depth (superficial and/or spread to the base, submucosa or submucosa or muscle layer) and inflammation. Characteristics of sexual infiltration. The type of infiltrating cells is defined by histological changes. Adoptive transfer: Purify CD4 T cells with a mouse T cell CD4 subgroup column kit, and inject 10 million cells into mice through the tail vein method. The number has been determined and will be used for research in the field of autoimmunity. Enzyme-linked immunosorbent assay (ELISPOT) can prepare a single cell suspension from the spleen. Inoculate 1x106 cells in each well of serum-free HL-1 medium with or without Helicobacter pylori antigen. The medium contains 1 mM glutamine per well. The final concentration is 5 mg/ml. These cultures were added to enzyme-linked immunospot plates incubated with PBS, which specifically captured interferon or interleukin 5, R46-A2 (4 mg/mL) or TRFK5 (5 mg/mL), respectively. .. At room temperature, block with PBS containing 1% bovine serum albumin for 1 hour, wash with PBS 4 times, and then start cell culture for 24 hours. Then, the cells were removed by an elution method, 1 mg/mL XMG1.2-HRP as IFN-g and 4 mg/mL TRFK4 as IL-5 were added to monitor antibodies, and the plate was incubated. For IL-5, add anti-IgG2a-HRP and continue incubating for 2 hours. The antibody in the plate can be confirmed by adding 3-amino-9-ethylcarbazole. Use the Series 1 immunospot image analyzer to evaluate the results. Statistical analysis: ELISPOT analysis between experimental groups was determined by analysis of variance. Fisher's exact test was used to evaluate the infection or non-infection of the immunized mice. Results: The mouse model of Helicobacter pylori infection and immunity used human pathogens isolated from cats or H.felis. Unlike Helicobacter pylori, H.felis uses C57BL/6 mice to induce inflammatory lesions of the gastric mucosa in mouse and human gastritis. In addition, according to the histological section of the mouse stomach, H.felis infection is more serious. Unlike Helicobacter pylori, it is mainly located at the junction of the fundus. It mainly grows and regenerates in the antrum and fundus of the stomach. C57BL/6 mice were immunized with H. felis or Helicobacter pylori antigen and aluminum hydroxide AlOH3 or Freund's complete adjuvant CFA emulsifier. Our recent research shows that these adjuvants can cause type 2 and type 1 immune responses.

　　After 14 days, the spleen cells of these mice were tested for Helicobacter pylori antigen-specific immune response by measuring enzyme-linked immunospot method (type 1 and type 2 cytokines (IFN-γ, IL-5, respectively)). (ELISPOT). Mouse cells immunized with emulsified antigen and aluminum hydroxide may produce IL-5 lacking IFN-r, and the number of IL-5 producing cells is higher than that of antigen-specific IFN-r, both of which are immunized by Helicobacter pylori Inoculate Helicobacter pylori. After immunization with antigen and complete Freund's adjuvant group (ie high IFN-r), the opposite number of cytokines was observed. In addition, the serum of mice immunized with Freund's complete adjuvant CFA has a higher titre of IgG2a antibody. According to the relative number of cells produced and the distribution of the immune response of these antibody subtypes, the antigen emulsifier induced the type 2 immune response induced by aluminum hydroxide, that is, the type 1 immune response of Freund's complete adjuvant. Next, we tested whether induction of type 2 or type 1 Helicobacter pylori immunity can protect the intestinal mucosa from Helicobacter pylori infection. C57BL/6 mice were immunized with Helicobacter pylori or H.felis with aluminum hydroxide or Freund's complete adjuvant. The adjuvant injected in the control mice contained the irrelevant control protein ovalbumin or was not immunized. After 28 days and 28 days, the oral administration of Helicobacter pylori 1×107 was mandatory, and the bacterial count was assessed visually by gastric mucosal silver staining. Determine the presence of Helicobacter pylori by bacterial culture methods. This study of fully immunized mice can provide excellent protection regardless of the adjuvant. By observing that there is no Helicobacter pylori in the silver-stained tissue. However, 12 out of 14 control mice were infected with Helicobacter pylori. Of the eight mice, seven were injected with Helicobacter pylori. No infection after injection of aluminum hydroxide. The protective effect after using aluminum hydroxide adjuvant and Freund's complete adjuvant is very important. Mouse H. felis is more severely infected than Helicobacter pylori. 28 out of 29 mice were infected with strong bacteria attached to the stomach. There are an average of 86 infected glands in each stomach. No bacteria were found in mice using H.feli antigen aluminum hydroxide adjuvant. The use of Freund's complete adjuvant and Helicobacter pylori emulsifier also had a protective effect (5 out of 21 cases were infected). Our research and other studies have shown that cholera toxin must be used with adjuvants to effectively implement mucosal vaccination strategies. The use of aluminum hydroxide and Freund's complete adjuvant together with antigen is also based on an effective mucosal vaccination strategy. After emulsification of aluminum hydroxide and Freund's adjuvant, no bacterial infection was found in mice immunized completely with Freund's adjuvant. In contrast, infection was rarely seen after emulsification with Freund's adjuvant. .. Inducing type 1 or type 2 immunity is an alternative method of mucosal immunity to Helicobacter pylori. HE staining of mouse sections showed that after immunization with aluminum hydroxide or complete Freund's adjuvant, there was the same mucosal inflammation in the gastric antrum and bottom mucosa. The degree of inflammation is not significantly different from the cholera toxin adjuvant immunity in previous studies. The average fundus inflammation of mice immunized with aluminum hydroxide, Freund's complete adjuvant and Helicobacter pylori antigen was 7.2 + 0.9 and 7.4 + 0.7, respectively. The average gastritis of mice immunized with aluminum hydroxide, Freund’s complete adjuvant and Helicobacter pylori antigen was 2.0 + 0 and 2.1 + 0.6, respectively. In all cases, mice immunized with Helicobacter pylori antigen were better than ovalbumin The immunized mice showed more severe inflammation. This type of gastritis after vaccination has also been observed in other experiments. It may be caused by secondary immunity to Helicobacter pylori immune memory.

　　Next, we began to study the immune system against Helicobacter pylori after immunization. Complete Freund’s adjuvants, such as cholera toxin, are highly toxic and should not be used in humans. We focus on the use of aluminum hydroxide adjuvant. In subsequent experiments, the mouse H.felis model was used. Compared with Helicobacter pylori, this organism showed more obvious inflammation in mice and caused heavier infections, making it easier to observe in tissue sections. Immune homologous uMT mice (immunoglobulin knockout mice) with aluminum hydroxide AlOH3 or ovalbumin and H.felis antigen. Whether it can provide protective antibodies after vaccination. The ovalbumin control group was used to test all eight immunoglobulin knockout mice uMT. After feces are injected into the stomach, it is infected by bacteria and shows a high ability to multiply bacteria.

　　However, only one immunoglobulin knockout mouse uMT with aluminum hydroxide adjuvant H.felis showed strong immune protection. In fully immunized B6 mice, these inflammatory responses did not differ significantly. After immunization with aluminum hydroxide and H. felis, the inflammation of the antrum was 7.8 + 0.6, the inflammation of the fundus was 2.38 + 0.5, and the inflammation of the antrum and the fundus were 3.7 + 1.5 and 1.5 + after immunization with ovalbumin and aluminum hydroxide. 0.5. This is consistent with the previous results of using oral immunoglobulin to knock out uMT in mice. These experiments show that there is no need for antibody immunization after vaccination. Next, an experiment was performed to check whether the purified T cells from the vaccinated mice could be protected from adoptive transfer. 28 days after immunization, CD4 T cells were purified with a column in the spleen cells of C57BL/6 mice immunized with H. felis and aluminum hydroxide, ovalbumin and aluminum hydroxide, H. felis and complete Freund's adjuvant. Discussion: In short, it has been shown that the use of aluminum hydroxide AlOH3 as an adjuvant for systemic immunity can induce protective mice against Helicobacter pylori and Helicobacter pylori that mediate CD42-type cell-mediated immunity. These findings may directly affect the development of human Helicobacter pylori vaccine. Approved adjuvants for use in humans may be a viable alternative experiment, which can induce mucosal immunity of Helicobacter pylori through oral administration. To further confirm this, another series of experiments aimed at investigating the impact of parental Helicobacter pylori vaccine on newborn babies will use normal and replicable subcutaneous immunity to obtain protective immunity.

　　Guyetal recently reported this view. He introduced a new method of Helicobacter pylori vaccination, which violates the principle of long-term mucosal immunity. Second, contrary to Guieta's discovery, Helicobacter pylori requires a type 1 immune response. Our data indicate that type 2 immune response is as important as type 1 and may be more effective against Helicobacter pylori infection. Aluminum hydroxide is the only adjuvant available in humans and is an adjuvant that triggers a type 2 immune response. Some recent reports recommend: Helicobacter pylori hepatitis 2 immunization (oral cholera toxin and other experimental immune adjuvants that cause type 2 or mixed type 2 and type 1 reactions) (included).