CN1270773C - Application of chitosan for preparing helicobacter pylorus vaccine adjuvant - Google Patents

Abstract

The present invention discloses a novel purpose of chitosan in the field of pharmacy, particularly the application of the chitosan in the preparation of a helicobacter pylori vaccine adjuvant. The application verifies that the chitosan can be used as a mucous membrane adjuvant of a helicobacter pylori vaccine by animal experiments. The chitosan used as the mucous membrane adjuvant of the helicobacter pylori vaccine has a mechanical action principle that the chitosan has favorable biological characteristics and immunological activity, the immunogenicity of antigen is enhanced, the absorption of stomach intestine mucous membranes to the antigen is promoted, the local immune reaction of the mucous membranes is promoted, and the balance of TH1 and TH2 immune reaction is regulated; thereby, the effects of the adjuvant can be exerted. The application of chitosan also verifies the local immunological regulation action of the stomach mucous membranes infected by helicobacter pylori through the animal experiments.

Description

The application of chitosan in the preparation helicobacter pylorus vaccine adjuvant

Technical field

The present invention relates to the medicinal usage invention field, especially relate to the application of a kind of chitosan in the preparation helicobacter pylorus vaccine adjuvant

Background technology

Chitosan (Chitosan) has another name called chitosan, and chemical name is polymerization β-(1,4)-2-amino-D-glucose, and promptly deacetylate exposes-NH on No. 2 positions of chitin ₂Gene and the product that forms is a high-molecular weight straight-chain polysaccharide, molecular formula is (C ₆H ₁₁NO ₄) n, in the time of 185 ℃, decompose.Its average molecular mass is 1MDa, and strand length is approximately 5000 units, but in actual applications, its molecular weight size is that extent of polymerization changes along with different needs.Chitosan is white or pale yellow powder, is insoluble to neutral and alkaline aqueous solution and most organic solvent, dissolves in unit price mineral acid and organic acid.Its dissolubility depends on the pH value of deacetylation, molecular weight and the solution of chitosan, and generally speaking, its molecular weight is more little, deacetylation is high more, and dissolubility is good more.Along with the difference of physicochemical properties such as deacetylation (DD), molecular weight, the dissociation constant of chitosan (pKa) also changes.When pH value was lower than pKa, the amino group of chitosan molecule was promptly protonated, thereby made whole molecule positively charged, and this is the important feature that chitosan molecule is brought into play its biological characteristics.Chitosan extensively is present in the biological tissues such as crustacean shell, arthropod epidermis, lower animal cell membrane, higher plant cell wall, is the rare positively charged polymer of nature.

Because chitosan safety non-toxic, non-immunogenicity, biodegradable and excellent biological compatibility is arranged almost, and have enhance immunity, promote wound healing, promote epithelial hyperplasia and angiogenesis, hemostasis, antibiotic, suppress multiple physiologically actives such as tumor, blood sugar lowering, blood fat.Therefore be widely used in medical domain in recent years.

Helicobacter pylori (Helicobacter Pylori, Hp) be acknowledged as the important virulence factor of chronic gastritis, peptic ulcer and gastric cancer, World Health Organization (WHO) has classified it as I class cancerigenic factor, thereby effective eradicate helicobacter pylori is for the crucial meaning that has of prevention helicobacter pylori related disease, particularly gastric cancer.Though at present eradicate helicobacter pylori has multiple combined drug therapy, but still has many problems, as the generation of helicobacter pylori Resistant strain, drug price costliness, the big and patient dependence difference of side effect etc.Therefore, immune protection becomes the new way that solves this difficult problem.Helicobacter pylori vaccine comprises whole-bacterial-vaccine, subunit vaccine, dna vaccination and live vector vaccine etc.The former two is protein vaccine, because the immunogenicity of helicobacter pylori protein is lower, need have the participation of immunological adjuvant can bring into play its immune protection effect, and the mucosa adjuvant is the key point that strengthens its immunne response and seek safely and effectively.The most effective and the most frequently used adjuvant is cholera toxin (CT) and escherichia coli heat-labile toxin (LT) at present, but its toxic and side effects to human body is big, can not be applied to human body.Recently people are primarily focused on reconstruction CT, LT to reduce its toxicity, as B subunit, the LTK63 of CT or LT ¹Deng, but still do not have at present a kind of efficient, nontoxic, be applicable to human adjuvant fully.People are also being devoted to study dna vaccination and the live vector vaccine that does not need adjuvant, though they have lot of advantages, still there is defective in itself, can cause the immunoreation of body as live vector itself, thereby suppress carrier bacterin duplicating in vivo; Can dna vaccination can be induced the generation of anti-helicobacter pylori circulating antibody preferably, but effectively induce the partial immunoreation of gastric mucosa, sets up effective mucosa-immune, is still waiting further research.Therefore, protein vaccine still is considered to most promising vaccine at present, and people are actively seeking the novel adjuvant of high effect nontoxic, in the hope of solving this difficult problem.

Summary of the invention

First purpose of the present invention is to provide the new purposes of cost chitosan low, that human body is had no side effect, the i.e. application of chitosan solution in the preparation helicobacter pylorus vaccine adjuvant.

Second purpose of the present invention is to provide the new purposes of cost chitosan low, that human body is had no side effect, the i.e. application of chitosan particle in the preparation helicobacter pylorus vaccine adjuvant.

The 3rd purpose of the present invention is to provide chitosan solution or chitosan particle to be used to protect mammal to comprise that the people avoids the preventative vaccine of helicobacter pylori infections as adjuvant.

The mammal that the 4th purpose of the present invention is to provide chitosan solution or chitosan particle to be used to infect helicobacter pylori as adjuvant comprises people's therapeutic vaccine.

The mechanism of action of chitosan in the preparation helicobacter pylorus vaccine adjuvant is: chitosan has good biological characteristics and immunocompetence, but the immunogenicity of enhancement antigen, promote gastrointestinal mucosa to antigenic absorption, promote the partial immunoreation of mucosa, regulate TH1 and the immunoreactive balance of TH2, thereby bring into play its adjuvant effect.

In order to understand essence of the present invention better, prove the purposes of chitosan below with experiment and result.

It is immanoprotection action and therapeutical effect two parts of the helicobacter pylori vaccine of adjuvant that the present invention divides with the chitosan.

A, materials and methods

One, material

1, medicine and reagent

88.5% deacetylation chitosan (it wins biological product company limited product Shanghai);

Jejunum campylobacter bacterio-agar basis, brucella broth are cultivated basis (Chinese diarrhoeal diseases control agent delivery research center, Shanghai product);

Aseptic sheep blood (south of the River, Shanghai biotechnology Engineering Co., Ltd product);

Sodium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, sucrose, hydrogen peroxide, glycerol (Shantou Xilong Chemical Factory, Guangdong product, analytical pure);

The sheep anti-mouse igg 1 of Radix Cochleariae officinalis peroxide enzyme labelling, IgG2a, IgA (U.S. Zymed-LABORATORIES INC product);

The sheep anti-mouse igg of Radix Cochleariae officinalis peroxide enzyme labelling (U.S. Sigma-ALDRICH, INC. product); Mus IL-2, IL-4, IL-10 quantitative ELISA detection kit (U.S. Bender MedSystems product); The anti-Mus IgA of rabbit, Zymed-LABORATORIES INC product);

Bovine serum albumin (the general Bioisystech Co., Ltd's product that flies in Shanghai);

Helicobacter pylori international standard bacterial strain SS1 (providing) by Chinese helicobacter pylori strain storehouse;

Strepto-avidin-peroxidase reagents series box (Beijing Zhong Shan Bioisystech Co., Ltd product);

Hyclone (south of the River, Shanghai biotechnology Engineering Co., Ltd product);

Tween-20 (U.S. Amresco company product); Cholera toxin (CT) (U.S. Sigma product);

O-phenylenediamine (U.S. Amresco company product); CO ₂, N ₂(Nanchang Gas Plant product);

96 hole polyethylene ELISA Plate (U.S. Corning company product); Experimental water is distilled water.

2, instrument

Hunan instrument---Tianjin, island-200 type electronic analytical balance (Hunan is for the import assembling of balance equipment factory);

MDF-U281-85 ℃ of cryogenic refrigerator (SANYO GS company product);

YJ-1003A type superclean bench (product are given birth to by cleaning equipment company in Suzhou);

TE-A type trace shaker (Jiangsu Tai Cang medical apparatus and instruments factory product);

MCO-175M type three gas incubators (SANYO GS company product);

Olympus microscope (Japanese Olympus company product);

2135 type pathological section machines (German Lycra company product);

TCX-P pathology micro-image analyzer (Nanchang 99 electronics head factory products);

450 type microplate reader (U.S. Bio-Rad company product);

Beckman low temperature supercentrifuge (U.S. Beckman company product);

VC100 type Ultrasonic Pulverization instrument (u s company's product);

-40 ℃ of cryogenic refrigerators (SANYO GS company product);

TS-12E automatic dehydration machine (Hubei Xiaogan Electronic Instruments Plant product);

YB-6 type biological tissue embedding machine (the inferior light medical electronic technology of Hubei Xiaogan institute product);

PIPETMAN micropipettor (1～20ul) (French GILSON company product);

Electrically heated drying cabinet (Shanghai Medical Apparatus and Instruments Factory's product);

Electric heating constant temperature water temperature case (the medical thermostatic equipment in Shanghai factory product);

Electrothermal constant incubator (Shanghai make a leapleap forward medical apparatus and instruments factory's product);

TGLL-18B desk type high speed refrigerated centrifuger (upright letter mechanical investigations institute Jiangsu, Shanghai granary related factory of medical apparatus and instruments factory product);

751-GW type spectrophotometer (Shanghai analytical tool factory product).

3, laboratory animal and SPF level Animal Lab.

Female cleaning level BALB/c mouse, in 6～8 ages in week, average weight 22.5 grams available from Chinese Academy of Sciences's Shanghai Experimental Animal Center (credit number: SCXK (Shanghai) 2002-0010), and are raised in SPF level laboratory (Jiangxi Prov. Medicine Inst).

Two, method

(1), the preparation of main liquid

1, pH value is 7.4, and concentration is the preparation of the phosphate buffer (PBS) of 0.01M:

NaCl 4g, KH ₂PO ₄0.1g, Na ₂HPO ₄12H ₂O 1.45g, KCl 0.1g adds distilled water to 1000ml, and it is standby to sterilize;

2, pH value is 7.4, and concentration is the preparation of the phosphate buffer (PBS) of 0.15M:

Na ₂HPO ₄12H ₂O 268.605g adds the 500ml distilled water and is made into Na ₂HPO ₄12H ₂The O mother solution, NaH ₂PO ₄2H ₂O 117.0075g adds the 500ml distilled water and is made into NaH ₂PO ₄2H ₂The O mother solution, NaCl 7.65g adding distil water 900ml is made into the NaCl mother solution.Get Na ₂HPO ₄12H ₂O mother solution 81ml, NaH ₂PO ₄2H ₂O mother solution 19ml, NaCl mother solution 900ml promptly is made into the phosphate buffer of 1000ml concentration 0.15M, and it is standby to sterilize;

3, the preparation of the required solution of indirect enzyme-linked immunosorbent assay (ELISA) method:

(1), bag is cushioned liquid (pH9.6, the carbonic acid buffer of 0.1mol/L): Na ₂CO ₃1.68g, NaHCO ₃2.86g adding distil water is to 1000ml;

(2), phosphate buffer (0.01mol/L, PH7.2): NaH ₂PO ₄0.345g, Na ₂HPO ₄2.68g, the NaCl8.74g adding distil water is to 1000ml;

(3), cleaning mixture: in above-mentioned phosphate buffer, add Tween-20, be made into the phosphate buffer that contains 0.5%Tween-20, be cleaning mixture;

(4), diluent: be the phosphate buffer that contains 1% bSA;

(5), citric acid-phosphate buffer (0.1mol/L, PH5.0): citric acid 7.3g, Na ₂HPO ₄23.87g adding distil water is to 1000ml;

(6), substrate solution: the substrate o-phenylenediamine is dissolved in citric acid-phosphate buffer with the ratio of 0.4mg/ml, and lucifuge is standby, faces with preceding volume ratio with 0.6ul/ml and adds 30% hydrogen peroxide;

(7) stop buffer: 2mol/L H ₂SO ₄

(2), the cultivation of helicobacter pylori:

Adopt helicobacter pylori reference culture SS1, it is seeded in the campylobacter jejuni selectivity that contains 7.5% aseptic sheep blood cultivates on the agar basis (O under little aerobic condition ₂5%, CO ₂10%, N ₂85%) cultivated 2-3 days for 37 ℃, collect with brucella broth eluting antibacterial, and survey bacterial density under the 660nm wavelength, 1 absorbance (OD value) is 10 ⁸CFU/ml (CFU: colony forming unit).

(3), the preparation of Heliobacter pylori antigen:

The SS1 reference culture was cultivated after 2-3 days, phosphate buffer eluting with 0.15mol/L, PH7.4, and wash 3 times, after the Ultrasonic Pulverization (output 60HZ, the interval is 30 seconds behind the per minute, totally 20 minutes), under 4 ℃ centrifugal 30 minutes with 8000g, get supernatant, survey absorbance, and press following formula: helicobacter pylori protein content (mg/ml)=[(1.45 * OD at 280nm and 260nm ₂₈₀)-(0.74 * OD ₂₆₀)] * extension rate, carry out protein quantification after, it is standby to put-85 ℃ of refrigerators.

(4), the preparation of chitosan particle:

88.5% deacetylation chitosan and normal saline are made into the solution of 10mg/ml, handle 2 times through ultrasonic (output 80HZ) again, each 5 minutes, 1 minute at interval, get its supernatant with 50g after centrifugal 10 minutes, and, collect its precipitate after centrifugal 10 minutes with 1400g again with the filtration of 400 order mesh screens, be the chitosan granule.

(5), the preparation of chitosan acid solution:

With 88.5% deacetylation chitosan in 3% ratio be dissolved in the 0.8% acetic acid saline the chitosan acid solution.

(6), the research of immanoprotection action:

1, laboratory animal grouping

Mice is divided into 9 groups at random by form at random: 1. blank group: phosphate buffer, 15; 2. simple chitosan acid solution group, 12; 3. simple chitosan particle group, 13; 4. helicobacter pylori (HP) antigen group, 15; 5. Heliobacter pylori antigen+chitosan acid solution group, 15; 6. Heliobacter pylori antigen+chitosan particle group, 15; 7. Heliobacter pylori antigen+cholera toxin (CT) is organized, 12; 8. Heliobacter pylori antigen+chitosan acid solution+CTx group, 13; 9. Heliobacter pylori antigen+chitosan particle+CTx group, 14;

2, the dosage of antigen and adjuvant:

Heliobacter pylori antigen 1.2mg//time, chitosan particle 500 μ g//times, cholera toxin 5 μ g//times, the chitosan acid solution is made into the whole solution of chitosan-containing 0.5% in required each group;

BALB/C mice was irritated each immunity of stomach 1 time by above-mentioned grouping and dosage in the 0th, 7,14,21 day, 0.4ml//time, the immunity back gave about 1 * 10 in 4 weeks ⁹The SS1 helicobacter pylori viable bacteria liquid 0.5ml/ of CFU/ml only attacks mice, the next day once, totally 2 times.Contain the chitosan particle group, irritate the preceding elder generation of stomach through ultrasonic output mixing with 20HZ.

3, collection of specimens

In 4 weeks after immunity, each takes out 5 at random from blank group, Heliobacter pylori antigen group, Heliobacter pylori antigen+chitosan acid solution group and Heliobacter pylori antigen+chitosan particle group before attacking, and puts to death, and it is to be measured to get specimen.All the other are all attacked 4 weeks of back in last and put to death.The intraperitoneal injection 2% pilocarpine 0.4ml of elder generation, after collecting saliva, extract eyeball and get blood, put to death mice, under the aseptic condition, dissect and take out the Mus stomach, cut off along greater gastric curvature, rinse out the gastric residue tailing edge longitudinal axis gently with physiological saline solution stomach is cut to three parts, a part is carried out the helicobacter pylori separation and Culture, a part is used for immunology detection, another part gastric tissue dewaters paraffin embedding through 10% formaldehyde fixed, Yihong-haematoxylin (HE) dyeing, Jim Sa (Giemsa) dyeing, SABC detection are done in section.

(7), immunization therapy effect research

1, laboratory animal grouping

Mice is divided into group at random by form at random: 1. blank group: PBS solution, 12; 2. Heliobacter pylori antigen group, 11; 3. Heliobacter pylori antigen+chitosan acid solution group, 12; 4. Heliobacter pylori antigen+chitosan particle group, 12; 5. Heliobacter pylori antigen+CTx group, 11; 6. Heliobacter pylori antigen+chitosan acid solution+CTx group, 11.

2, the dosage of antigen and adjuvant:

Identical with the dosage of antigen in the research of (six), immanoprotection action and adjuvant.

3, the foundation of helicobacter pylori infections animal model and immunity inoculation:

The BALB/c mouse of the cleaning level in age in 6-8 week contains 1 * 10 ⁹The helicobacter pylori standard bacterium pearl SS1 living bacterial liquid of/ml, 0.5ml/ only irritates stomach, the next day once, totally 5 times.Last is irritated stomach after 12 weeks, gets to get after 4 mices kill that gastric mucosa carries out pathologic finding and helicobacter pylori is cultivated, and the two confirms that all the helicobacter pylori infections model sets up.

Established the mice of helicobacter pylori infections, during 12 weeks, irritated each immunity of stomach 1 time, 0.4ml//time in the 0th, 7,14,21 day by above-mentioned grouping and dosage last filling bacterium respectively, contain the chitosan particle group, irritate the preceding elder generation of stomach through ultrasonic output mixing with 20HZ.

4, collection of specimens

In 4 weeks after the last immunity, putting to death all mices, to get specimen to be measured, and the collection of specimens method is with the collection of specimens in (six), the immanoprotection action research.

(8), the mensuration of helicobacter pylori in the gastric mucosa:

Adopt quantitative helicobacter pylori to cultivate and pathology improvement Giemsa staining mensuration.The two all feminine gender be designated as feminine gender, both one of the positive be designated as the positive.

1, the quantitative culture of helicobacter pylori:

Gastric mucosa is weighed under aseptic condition, place transportation liquid (10% hyclone sucrose mixed liquor, be sucrose 10g, hyclone 50ml, distilled water 50ml),-85 ℃ frozen, take out gastric mucosa when waiting to cultivate and add brucella broth 0.3ml homogenate, (1: 1 by a certain percentage, 1: 4,1: 8) dilution, get 0.5ml and coat on the jejunum campylobacter bacterio-agar base that contains 7.5% aseptic Sanguis caprae seu ovis, trimethoprim (TMP) 5mg/L, vancomycin 10mg/L and polymyxin B2500IU/L, little aerobic condition was cultivated 2-3 days down.According to colonial morphology, Gram, ne ar, urease, oxidase and catalase experiment, behind the evaluation antibacterial, calculate the helicobacter pylori clump count, the result represents with clump count/g (weight in wet base tissue).

2, Giemsa dyeing:

Gastric mucosa paraffin section → routine dewaxes to water → Giemsa dye liquor dyeing 20 minutes → 95% ethanol I, II and transparent each the 10 minutes → neutral gum mounting of the differentiation of 100% ethanol each 1～2 second → dimethylbenzene I, II.

Observe under high power lens, see that typical purple blue spiral sample bacterium is a helicobacter pylori, and keep the score according to following standard: 0 is designated as 0 fen, and 1～2 is designated as 1 fen in the-individual gastric pits, and 3～10 are designated as 2 fens, and 11～20 are designated as 3 fens, are designated as 4 fens greater than 21.

(9), the detection of helicobacter pylori antibody: adopt the ELISA method to measure:

1, the concentration of antigen, specimen and ELIAS secondary antibody:

Antigen concentration 20ug/ml, serum 1: 100 dilution, saliva dilution in 1: 5, gastric mucosa homogenate supernatant (gastric mucosa is weighed the back with the homogenate of 1.3ml phosphate buffer, 2000～3000 rev/mins rotating speed, 4 ℃ centrifugal 20 minutes, get its supernatant) dilution in 1: 2.The rabbit anti-mouse igg 1 of peroxidase labelling, IgG2a, IgA dilution in 1: 1000, the dilution in 1: 2000 of the sheep anti-mouse igg of peroxidase labelling.

2, operating procedure

1. with 100ul/ hole Heliobacter pylori antigen liquid bag by 96 hole polyethylene ELISA Plate, add a cover, 4 ℃ of liquid in the hole that spend the night → 2. abandon, wash 3 times → 3. 1% bSA 200ul/ hole, 1 hour → 4. same specimen 100ul/ to be measured the hole that 2. → 5. adds of 37 ℃ of sealings, 2. → 7. 37 ℃ of incubations 1 hour → 6. with adding ELIAS secondary antibody 100ul/ hole, and 2. → 9. 37 ℃ of incubations 1 hour → 8. with adding o-phenylenediamine substrate solution 100ul/ hole, the room temperature lucifuge place 30 minutes → 10. add 2mol/L H ₂SO ₄The 50ul/ hole, cessation reaction is surveyed the OD value under the 492nm wavelength on the microplate reader.

3, the result calculates

Each specimen is done multiple hole, gets its mean value calculation.Every plate is all established blank well, standard female and standard positive hole, is Quality Control to mark the moon/likening to of mark sun, and the coefficient of variation of this value is in 5% between each plate.The result represents with the ratio of specimen OD/ standard female OD to be measured.Anti-helicobacter pylori IgA represents with specimen OD/ standard female OD/g gastric mucosa weight in wet base to be measured in the gastric mucosa.With two immunity and the specimen of not attacking mice be mixed into standard female, the compound sample of mice is positive after attacking through Heliobacter pylori antigen+cholera toxin immunity and by helicobacter pylori with two.Blank well only adds substrate solution and stop buffer, and all the other each steps substitute with diluent.

(10), the detection by quantitative of cytokine: adopt the double-antibody sandwich elisa method to measure

1, the processing of gastric mucosa homogenate supernatant is measured with anti-helicobacter pylori IgA, with dilution in 1: 2, measure helper T cell 1 cytokine (TH1) IL-2 and helper T cell 2 cytokines (TH2) IL-4, IL-10 concentration, undertaken by the test kit operating instruction.Concrete operations are as follows:

1. add every hole with wrapping in the cleaning mixture washing reagent box by the standard substance/specimen 100ul to be measured of 7 kinds of variable concentrations of the polyethylene ELISA Plate of corresponding antibodies 2 times → 2. will dilute in proportion, with the sample diluent as blank → 3. the add biotin labeled corresponding antibodies 50ul/ hole of dilution in proportion, under the room temperature, vibration 2 hours on the agitator → 4. abandon liquid in the hole, wash 3 times-→ 5. add the strepto-avidin 100ul/ hole of Radix Cochleariae officinalis enzyme labelling, under the room temperature, 1 hour → 6. same tmb substrate solution 100ul/ the hole that 4. → 7. adds of vibration on the agitator, under the room temperature, vibration 10 minutes on the agitator → 8. add on the microplate reader of stop buffer 100ul/ hole → 9. under 450nm (measurement wavelength) and 630nm (reference wavelength) dual wavelength survey OD value.

2, standard curve:

IL-2 is with 1000,500,250,125,62.5,31.3,15.6 (pg/ml); IL-4 is with 250,125,62.5,31.3,15.6,7.8,3.9 (pg/ml); IL-10 is with 2500,1250,625,312.5,156.3,78.1,39.1 (pg/ml) preparation standard curve, and each standard substance is surveyed multiple hole, gets its mean.The regression equation of curve is:

IL-2： Y＝-9.8196+344.231X-123.10X ²+42.7796X ³(R＝1.000)，

Y＝-6.8186+333.480X-137.27X ²+44.7122X ³(R＝1.000)；

IL-4： Y＝-5.0590+77.8719X-42.662X ²+16.0525X ³(R＝0.999)，

Y＝-0.3542+59.8328X-10.133X ²+7.2265X ³ (R＝1.000)；

IL-10：Y＝15.4248+983.702X-293.80X ²+114.952X ³(R＝1.000)，

Y＝-5.1157+972.450X-225.08X ²+67.6971X ³(R＝1.000)。

IL-2, IL-4, the IL-10 content of calculating specimen to be measured according to standard curve, and divided by the gastric mucosa weight in wet base, the result represents with pg/mg weight in wet base gastric mucosa.

(11), gastric mucosa pathology detect the inflammation degree:

Adopt HE dyeing, gastric mucosa specimens paraffin embedding slices → routine dewaxes to water → 1% dilute hydrochloric acid ethanol and breaks up respectively transparent respectively 10 minutes → neutral gum mounting of 2 minutes → dimethylbenzene, of anti-blue about 3 seconds → washing → eosin stain dyeing, 5 minutes → 85% ethanol of about 3 seconds → washing → lithium carbonate, 95% ethanol,, 100% ethanol, dehydration.

High power lens is observed 10 visual field lamina propria chronic inflammatory cells (lymphocyte, plasma cell) down and is soaked into, and chronic inflammatory disease is scored.Chronic inflammatory disease: the accidental lymphocyte of 0=lamina propria, 1=lamina propria have lymphocyte, the plasma cell that is dispersed in, and the 2=lamina propria has more lymphocyte, plasma cell, and the 3=lamina propria has a large amount of lymphocytes, plasma cell.

Non-specific SigA sIgA (sIgA) is measured in (12), the gastric mucosa: adopt the strepto-avidin immunohistochemistry staining method (SP SABC method) of peroxidase labelling to measure

Gastric mucosa routine paraffin wax embedded section, take off cured to water → 3% hydrogen peroxide sealing 10 minutes, eliminate endogenous peroxidase activity → 3 times → phosphate buffer of distillation washing (PH7.4, concentration 0.01M) soaks the normal sheep NIS of 5 minutes → dropping, incubated at room 10 minutes, the minimizing background develops the color → inclines behind the serum deprivation, drip the anti-Mus IgA of rabbit of dilution in 1: 1000, negative control replaces anti-→ 4 ℃ of refrigerator overnight → phosphate buffer flushing 3 times → drips biotin labeled goat anti-rabbit igg → 37 ℃ hatching the strepto-avidin working solution of 15 minutes → phosphate buffer flushing 3 times → drip horseradish peroxidase-labeled → 37 ℃ and hatching 15 minutes → phosphate buffer flushing, 3 times → microscopically o-phenylenediamine (DAB) colour developing → tap water and wash with phosphate buffer, haematoxylin is redyed, dehydration, transparent, mounting

The result judges: with lumen of gland dye yellow or pale brown color positive, 100 bodies of gland are calculated in every section, calculate the shared percentage ratio of positive body of gland.

Three, statistical procedures

Enumeration data adopts X 2 test (accurately probabilistic method) and rank test, and measurement data adopts the F-q check.Use public statistical package SPSS 11.0 and SAS 6.12 processing, P value＜0.05 is for there being significant difference.

B, result

First: immanoprotection action result

One, respectively organizes the helicobacter pylori infections situation

1, immune protective rate

Table 1 is respectively organized immune protective rate after attacking

Grouping	N	Helicobacter pylori number positive (positive rate %)	Protective rate (%)
				1. control group 2. the chitosan solution group 3. the chitosan particle group 4. the Heliobacter pylori antigen group 5. Heliobacter pylori antigen+CT organize 6. 7. 8. 9. Heliobacter pylori antigen+CT+ chitosan particle group of Heliobacter pylori antigen+CT+ chitosan solution group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group	10 12 13 10 12 10 10 13 14	10(100) 12(100) 13(100) 10(100) 5(41.67) 4(40) 4(40) 2(15.38) 2(14.28)	0 0 0 0 58.33 a 60 b 60 b 84.62 c 85.71 e
P＜0.001

Annotate: a is that 1.-4. P＜0.01vs organizes; B is that 4. 1. P＜0.05vs organize, and 3. 2. P＜0.01vs organize; C is that 1.-4. P＜0.001vs organizes.

As shown in table 1, immune protective rate of each group has notable difference (P＜0.001), wherein has the protection of the helicobacter pylori vaccine group of adjuvant to be significantly higher than no adjuvant group and no antigen group (P＜0.001-0.05).The protective rate of chitosan and CT use in conjunction group is separately an adjuvant group height than chitosan and CT, but not statistically significant (P＞0.05)

2, helicobacter pylori field planting histological score in the gastric mucosa

Table 2 is respectively organized the helicobacter pylori field planting histological score in the gastric mucosa

Grouping	N	Helicobacter pylori field planting score
						0	1	2	3
		1. 2. chitosan solution group of matched group g3. chitosan particle group g4. Heliobacter pylori antigen group f5. Heliobacter pylori antigen+CT organizes d6. Heliobacter pylori antigen+chitosan solution group c7. Heliobacter pylori antigen+chitosan particle group c8. Heliobacter pylori antigen+CT+ chitosan solution group b9. Heliobacter pylori antigen+CT+ chitosan particle group a	10 12 13 10 12 10 10 13 14	0 1 0 1 7 6 7 12 12	2 8 10 5 2 4 3 1 2					4 2 1 2 1 0 0 0 0	4 1 2 2 2 0 0 0 0
H＝57.181，P＜0.001

Annotate: a is that 1.-5. P＜0.001vs organizes; B is that 1.-4. P＜0.01vs organizes, and 5. P＜0.05vs organizes; C is that 1.-4. P＜0.01vs organizes; D is that 1. P＜0.001vs organizes; F is that 5. 1. P＜0.05vs organize; G is that 1. P＜0.01vs organizes.

As shown in table 2, the helicobacter pylori infections degree of each group has notable difference (P＜0.001), the helicobacter pylori plantation density of selecting a step relatively to find to contain the immunological adjuvant group in twos significantly is lower than no adjuvant group and no antigen group (P＜0.001-0.05), the plantation density of chitosan and CT use in conjunction group is minimum, significantly being lower than single is that (P＜0.001-0.05), the plantation density of simple Heliobacter pylori antigen group and chitosan group significantly is lower than matched group (P＜0.01-0.05) to the adjuvant group with CT.

3, helicobacter pylori quantitative culture plantation density in the gastric mucosa

As table 3 and shown in Figure 1, each organizes the helicobacter pylori plantation density notable difference (P=0.001), the chitosan-containing group is all than the low (P＜0.005-0.05), be that the adjuvant group is than simple chitosan solution group low (P＜0.05) with the CT+ chitosan particle wherein of matched group, simple chitosan particle group, simple Heliobacter pylori antigen group in the adjuvant.

Table 3 is respectively organized the comparison of helicobacter pylori quantitative culture plantation density in the gastric mucosa

Grouping	N	Helicobacter pylori plantation density median (CFU/g gastric tissue)
			1. control group 2. the chitosan solution group 3. the chitosan particle group 4. the Heliobacter pylori antigen group 5. Heliobacter pylori antigen+CT organize 6. 7. 8. 9. Heliobacter pylori antigen+CT+ chitosan particle group of Heliobacter pylori antigen+CT+ chitosan solution group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group	10 12 13 10 12 10 10 13 14	4.74×10 5 0.73×10 5 1.83×10 5 0.48×10 5 0 0 0 0 0
H＝27.43 P＝0.001

Fig. 1 explains: 9. organize P＜0.005vs and 1. and 3. organize, 2. and 4. P＜0.05vs organizes; 6.-8. organize P＜0.01vs and 1. and 3. organize, 4. P＜0.05vs organizes.

Two, respectively organize the inflammation degree of gastric mucosa

As shown in table 4, after the attack, each inflammation degree of organizing gastric mucosa has notable difference (P=0.014), chitosan solution and CT use in conjunction group inflammation degree are significantly higher than matched group, simple chitosan particle group reaches with the chitosan particle is adjuvant group (P＜0.05), is adjuvant group, simple chitosan group and matched group (P＜0.05) with CT for the adjuvant group is higher than with the chitosan particle, and simple Heliobacter pylori antigen group is higher than simple chitosan particle group, matched group and is adjuvant group (P＜0.05) with the chitosan particle.

Each group of table 4 is attacked back gastric mucosa inflammation degree relatively

Grouping	N	The inflammation degree
						0	1	2	3
		1. 2. 3. 4. Heliobacter pylori antigen group of chitosan particle group of chitosan solution group of matched group c5. Heliobacter pylori antigen+CT b6. 7. 8. Heliobacter pylori antigen+CT+ chitosan solution group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group a9. Heliobacter pylori antigen+CT+ chitosan particle group	10 12 13 10 12 10 10 13 14	2 3 4 0 0 2 2 1 0	8 8 8 6 8 7 8 8 12					0 1 1 4 4 1 0 4 2	0 0 0 0 0 0 0 0 0
H＝19.176，P＝0.014

Annotate: attacking back a is that 7. 3. 1. P＜0.05vs organize; B is that 1.-3. P＜0.05vs reaches 7. group; C is that 7. 6. 3. 1. P＜0.05vs organize.

Three, respectively organize serum helicobacter pylori antibody result

Table 5 is respectively organized serum anti-helicobacter pylori IgG, IgG1, IgG2a result before attacking

Grouping	N	IgG	IgG1	IgG2a
					1. 2. 3. 4. Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group of Heliobacter pylori antigen group of matched group	5 5 5 5	1.28±0.48 3.28±2.06 3.78±1.98 4.57±3.07 a	0.79±0.19 1.82±1.02 2.49±1.57 a 2.05±1.00	1.11±0.17 5.50±3.40 5.31±3.22 8.43±5.66 a

Annotate: a is that 1. P＜0.05vs organizes.

As shown in table 5, compare with matched group before attacking, Heliobacter pylori antigen+chitosan particle group serum anti-helicobacter pylori IgG, IgG2a and Heliobacter pylori antigen+chitosan solution group serum anti-helicobacter pylori IgG1 raises (P＜0.05), but serum anti-helicobacter pylori IgG, IgG1, IgG2a rising degree difference do not have significance (P＞0.05) between all the other each groups.

Table 6 is respectively organized serum anti-helicobacter pylori IgG, IgG1, IgG2a result after attacking

Grouping	N	IgG	IgG1	IgG2a
					1. control group 2. the chitosan solution group 3. the chitosan particle group 4. the Heliobacter pylori antigen group 5. Heliobacter pylori antigen+CT organize 6. 7. 8. 9. Heliobacter pylori antigen+CT+ chitosan particle of Heliobacter pylori antigen+CT+ chitosan solution of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group	10 12 13 10 12 10 10 13 14	2.53±1.23 2.96±1.04 2.86±1.72 3.85±1.09 8.47±4.17 c 7.67±5.36 c 7.17±4.71 c 11.04±1.23 a，b 9.53±2.55 a	2.77±1.18 3.96±2.32 4.57±2.09 6.43±4.88 i 9.04±5.35 h 7.71±5.11 e，g 8.02±5.29 e，f 14.91±0.48 d 13.54±0.76 d	4.44±2.09 6.28±3.62 5.56±2.27 9.21±6.80 1 11.22±5.52 k 11.48±5.55 k 16.13±10.20 j 16.00±12.27 j 16.90±10.20 j
F＝11.933 F＝10.308 F＝4.572 P＜0.001 P＜0.001 P＜0.001

Annotate: a is that 1.-4. P＜0.001vs organizes; B is that 5.-7. P＜0.05vs organizes; C is that 1.-4. P＜0.01vs organizes; D is that 1.-7. P＜0.005vs organizes; E is that 1. P＜0.005vs organizes; F is that 2. and 3. P＜0.05vs organizes; G is that 2. P＜0.05vs organizes; H is that 1.-3. P＜0.005vs organizes; I is that 1. P＜0.05vs organizes; J is that 1.-3. P＜0.001vs organizes, and 4. P＜0.05vs organizes; K is that 1. P＜0.05vs organizes.

As shown in table 6, after the attack, each organizes serum anti-helicobacter pylori IgG, helicobacter pylori IgG1, helicobacter pylori IgG2a all has notable difference (P＜0.001), further relatively find in twos, anti-helicobacter pylori IgG is significantly higher than simple Heliobacter pylori antigen group, simple chitosan group and matched group (P＜0.001-0.05), it is adjuvant group (P＜0.05) with CT or chitosan singly that chitosan solution and CT coupling group are significantly higher than containing the adjuvant group.Anti-helicobacter pylori IgG1 is significantly higher than other each groups (P＜0.005) in chitosan and CT coupling group, and single adjuvant group is significantly higher than simple chitosan group and matched group, and (P＜0.005-0.05), the Heliobacter pylori antigen group is significantly higher than matched group (P＜0.05).Anti-helicobacter pylori IgG2a is being that adjuvant group and CT and chitosan coupling group are significantly higher than simple Heliobacter pylori antigen group, simple chitosan group and matched group (P＜0.001-0.05), be that the adjuvant group is significantly higher than matched group (P＜0.05) with CT or chitosan solution with the chitosan particle.

Fig. 2 explains: (1), anti-helicobacter pylori IgG content: be significantly higher than attack preceding (P＜0.05) after 3. group is attacked, though before all being higher than attack after other each group is attacked, no difference of science of statistics (P＞0.05).

(2), anti-helicobacter pylori IgG1 content: be significantly higher than after 2. group is attacked attack before (P＜0.05), be significantly higher than after 3. 4. group is attacked attack before (P＜0.01), though before matched group is attacked the back and all is higher than attack, no difference of science of statistics (P＞0.05).

(3), anti-helicobacter pylori IgG2a content: be significantly higher than after 3. 4. group is attacked attack before (P＜0.05), though before Heliobacter pylori antigen group and matched group are attacked the back and all are higher than attack, no difference of science of statistics (P＞0.05).

As shown in Figure 2, before respectively organizing serum anti-helicobacter pylori IgG, IgG1, IgG2a after the attack and being higher than attack, with the chitosan be that each group of adjuvant is more obvious (P＜0.01-0.05).

Four, respectively organize saliva and gastric mucosa internal specific anti-helicobacter pylori IgA and non-specific sIgA content relatively

1, saliva and gastric mucosa internal specific anti-helicobacter pylori IgA

Before attacking, table 7 respectively organizes anti-helicobacter pylori IgA result in saliva and the gastric mucosa

Grouping	N	IgA
				Gastric mucosa	Saliva
		1. 2. 3. 4. Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group of Heliobacter pylori antigen group of matched group	5 5 5 5			17.67±7.21 22.90±2.43 34.31±14.11 c，d 33.50±3.11 c	0.66±0.23 0.70±0.24 0.99±0.04 b 1.04±0.14 a
F＝4.985，P＝0.012 F＝5.42，P＝0.009

Annotate: a is that 1. P＜0.005vs organizes, and 2. P＜0.05vs organizes; B is that 1.-2. P＜0.05vs organizes; C is that 1. P＜0.01vs organizes; D is that 2. P＜0.05vs organizes.

As shown in table 7, there were significant differences respectively to organize saliva (P＜0.05) and the interior anti-helicobacter pylori IgA of gastric mucosa (P＜0.01) before the attack, before anti-helicobacter pylori IgA attacks in the gastric mucosa be with the chitosan adjuvant group apparently higher than matched group (P＜0.05), be that the adjuvant group also is higher than simple Heliobacter pylori antigen group (P＜0.05) with the chitosan solution; The helicobacter pylori vaccine group that before anti-helicobacter pylori IgA attacks in the saliva with the chitosan is adjuvant is higher than not with adjuvant group (P＜0.05).

After attacking, table 8 respectively organizes anti-helicobacter pylori IgA result in saliva and the gastric mucosa

Grouping	N	IgA
				Gastric mucosa	Saliva
		1. control group 2. the chitosan solution group 3. the chitosan particle group 4. the Heliobacter pylori antigen group 5. Heliobacter pylori antigen+CT organize 6. 7. 8. 9. Heliobacter pylori antigen+CT+ chitosan particle group of Heliobacter pylori antigen+CT+ chitosan solution group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group	10 12 13 10 12 10 10 13 14			25.08±8.98 38.34±18.17 31.50±26.29 33.25±8.95 84.08±31.14 d 85.51±50.22 d 90.50±37.39 c 171.63±97.63 a 131.64±86.06 b	0.67±0.25 0.95±0.18 0.91±0.13 0.94±0.23 2.02±1.14 i 2.15±1.75 h 2.73±1.75 g 4.63±2.30 f 3.11±1.89 e
F＝9.575，P＜0.001 F＝9.991，P＜0.001

Annotate: attacking back a is that 1.-7. P＜0.001vs organizes; B is that 1.-4. P＜0.001vs organizes, and 5. P＜0.05vs organizes; C is that 1.-4. P＜0.05vs organizes; D be P＜0.05vs 1., 3., 4. the group; E is that 1.-4. P＜0.01vs organizes; F is that 1.-6. P＜0.001vs organizes, and 7. and 9. P＜0.01vs organizes; G is that 1.-4. P＜0.01vs organizes; H is that 1.-2. P＜0.05vs organizes; I is that 1. P＜0.05vs organizes.

As shown in table 8, after the attack, each is organized, and anti-helicobacter pylori IgA all has notable difference (P＜0.001) in saliva and the gastric mucosa, further relatively find in twos, anti-helicobacter pylori IgA level in the gastric mucosa, in adjuvant, contain the chitosan group and be significantly higher than Heliobacter pylori antigen group, simple chitosan group and matched group (P＜0.001-0.05), wherein chitosan solution and CT coupling group also are significantly higher than single adjuvant group (P＜0.001), and chitosan particle and CT coupling group to be significantly higher than with CT be adjuvant group (P＜0.05).Anti-helicobacter pylori IgA level is being that adjuvant group and chitosan and CT coupling group are significantly higher than Heliobacter pylori antigen group, simple chitosan group and matched group (P＜0.01) with the chitosan particle in the saliva, chitosan solution and CT coupling group are significantly higher than other adjuvant groups (P＜0.01), for the adjuvant group is significantly higher than matched group and simple chitosan solution group (P＜0.05), the Heliobacter pylori antigen group also is significantly higher than matched group (P＜0.05) with the chitosan solution.

Fig. 3 note: anti-helicobacter pylori IgA content in gastric mucosa and the saliva: be significantly higher than after 3. 4. group is attacked and attack preceding (P＜0.05), no difference of science of statistics (P＞0.05) before and after Heliobacter pylori antigen group and matched group are attacked.

As shown in Figure 3, be before anti-helicobacter pylori IgA all is significantly higher than attack in each group attack back saliva of adjuvant and the gastric mucosa with the chitosan.

2, non-specific secretory IgA (sIgA) in the gastric mucosa

The positive body of gland percentage rate (%) of gastric mucosa sIgA before table 9 is attacked

Grouping	N	sIgA(％)
			1. 2. 3. 4. Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group of Heliobacter pylori antigen group of matched group	5 5 5 5	1.40±0.29 2.80±1.06 9.20±4.06 a，b 10.20±4.91 a，c
F＝7.194，P＝0.003

Annotate: a is that 1. P＜0.005vs organizes; B is that 2. P＜0.05vs organizes; C is that 2. P＜0.01vs organizes.

As shown in table 9, the positive body of gland percentage rate of gastric mucosa sIgA is higher than no adjuvant group and matched group (P＜0.005-0.05) with the chitosan for the adjuvant group before attacking.

As shown in table 10, after the attack, each organizes gastric mucosa sIgA level notable difference (P＜0.001), further relatively find in twos, the chitosan-containing group is significantly higher than no adjuvant group and no antigen group (P＜0.001-0.05) in the adjuvant, wherein also to be significantly higher than only be adjuvant group (P＜0.05) with CT for chitosan and CT coupling group, and be significantly higher than matched group (P＜0.05) with CT for adjuvant group and simple chitosan group.

Table 10 is attacked the positive body of gland percentage rate (%) of back gastric mucosa sIgA

Grouping	N	sIgA(％)
			1. control group 2. the chitosan solution group 3. the chitosan particle group 4. the Heliobacter pylori antigen group 5. Heliobacter pylori antigen+CT organize 6. 7. 8. 9. Heliobacter pylori antigen+CT+ chitosan particle group of Heliobacter pylori antigen+CT+ chitosan solution group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group	10 12 13 10 12 10 10 13 14	5.00±3.62 17.83±9.21 d 16.54±9.24 d 15.20±4.07 25.58±17.48 c 30.30±15.78 b 30.50±15.71 b 37.08±14.74 a 36.57±21.53 a
F＝6.881，P＜0.001

Annotate: a is that 1.-4. P＜0.001vs organizes, and 5. P＜0.05vs organizes; B is that 1. P＜0.001vs organizes, and 2.-4. P＜0.05v8 organizes; C is that 1. P＜0.001vs organizes; D is that 1. P＜0.05vs organizes.

Fig. 4 explains: be significantly higher than attack preceding (P＜0.05) after 2. group is attacked, be significantly higher than after 3. 4. group is attacked and attack preceding (P＜0.01), no difference of science of statistics (P＞0.05) before and after the matched group attack.

As shown in Figure 4, with the chitosan be adjuvant group and Heliobacter pylori antigen group attack back sIgA level all be higher than attack before (P＜0.001-0.01).

Five, respectively organize the gastric mucosa inner cell factor relatively

Before attacking, table 11 respectively organizes IL-2, IL-10 and IL-4 result's (ng/mg weight in wet base tissue) in the gastric mucosa

Grouping	N	IL-2	IL-10	IL-4
					1. 2. 3. 4. Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group of Heliobacter pylori antigen group of matched group	5 5 5 5	19.85±12.6 20.63±3.50 28.64±9.26 27.35±13.87	67.13±32.48 104.33±19.12 255.25±131.83 a 237.05±98.32 a	4.19±2.95 6.49±2.61 14.70±8.73 b 14.48±6.84 b
F＝0.739 F＝6.228 F＝4.189 P＝0.544 P＝0.005 P＝0.023

Annotate: a is that 1. P＜0.01vs organizes, and 2. P＜0.05vs organizes; B is that 1. and 2. P＜0.05vs organizes.

As shown in table 11, respectively organize the interior IL-2 of gastric mucosa before the attack and do not have significant difference (P＞0.05).There were significant differences for IL-10 (P=0.005) and IL-4 (P=0.023), finds relatively that in twos IL-10 and IL-4 are higher than no adjuvant group (P＜0.01-0.05) with the chitosan for the adjuvant group.

After attacking, table 12 respectively organizes IL-2, IL-10 and IL-4 result's (ng/mg weight in wet base tissue) in the gastric mucosa

Grouping	N	IL-2	IL-10	IL-4
					1. control group 2. the chitosan solution group 3. the chitosan particle group 4. the Heliobacter pylori antigen group 5. Heliobacter pylori antigen+CT organize 6. 7. 8. 9. Heliobacter pylori antigen+CT+ chitosan particle group of Heliobacter pylori antigen+CT+ chitosan solution group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group	10 11 10 9 10 10 10 10 10	34.28±11.82 31.79±27.41 51.08±42.60 97.92±64.17 c 80.93±60.21 d 124.26±75.17 a 88.61±57.04 c 86.95±42.08 c 104.10±74.19 b	86.44±38.20 100.75±52.60 88.44±51.7 89.34±29.05 81.78±49.51 108.46±39.02 93.14±39.16 91.78±34.74 224.42±79.78 e	3.87±1.99 4.47±1.89 7.21±4.02 g 4.89±3.15 3.67±1.76 8.78±4.96 f 6.59±1.38 h 4.63±2.71 4.69±2.36
F＝3.370 F＝8.525 F＝3.214 P＝0.002 P＜0.001 P＝0.003

Annotate: a is that 1. and 2. P＜0.001vs organizes, and 3. P＜0.01vs organizes; B is that 1. and 2. P＜0.01vs organizes, and 3. P＜0.05vs organizes; C is that 1. and 2. P＜0.05vs organizes; D is that 1. P＜0.05vs organizes; E is that 1.-8. P＜0.001vs organizes; F is a 1. and 5. group of P＜0.001vs, P＜0.005vs 2., 8., 9. group, 4. P＜0.05vs organizes; G be P＜0.05vs 1., 2., 5. the group; H is that 5. P＜0.05vs organizes..

As shown in table 12, after the attack, each organizes in the gastric mucosa IL-2 (P=0.002), IL-10 (P＜0.001) and IL-4 (P=0.003), and there were significant differences.Find relatively in twos that further the level of IL-2 is that adjuvant group and chitosan particle and CT use in conjunction group are significantly higher than no antigen group (P＜0.001-0.05) with the chitosan solution; With the chitosan particle is that adjuvant group, CT and chitosan solution use in conjunction group and simple Heliobacter pylori antigen group are significantly higher than matched group and simple chitosan solution group (P＜0.05); Be higher than matched group (P＜0.05) with CT for the adjuvant group.The IL-10 level is significantly higher than other groups (P＜0.001) at chitosan particle and CT use in conjunction group.The level of IL-4 is being adjuvant group (P＜0.05) with the chitosan particle for the adjuvant group is significantly higher than with CT; For being significantly higher than, the adjuvant group contains each group of CT (P＜0.001-0.05) with the chitosan solution in matched group, simple chitosan solution group, Heliobacter pylori antigen group and the adjuvant; Simple chitosan particle group is higher than matched group, chitosan solution group and is adjuvant group (P＜0.05) with CT.

Fig. 5 explains: (1), IL-2 content: be significantly higher than after 2.-4. group is attacked and attack preceding (P＜0.05), no difference of science of statistics (P＞0.05) before and after matched group is attacked.

(2), IL-10 content: significantly be lower than after 3. 4. group is attacked and attack preceding (P＜0.05 and 0.001), no difference of science of statistics (P＞0.05) before and after Heliobacter pylori antigen group and matched group are attacked.

(3), IL-4 content: significantly be lower than after 4. group is attacked attack before (P＜0.05), though before 1.-3. all being lower than attack, no difference of science of statistics (P＞0.05).

As shown in Figure 5, attack front and back gastric mucosa interior IL-2, IL-10 and IL-4 level notable difference is arranged.IL-2 all is significantly higher than attack back (P＜0.05) before containing each group attack of Heliobacter pylori antigen.IL-10 contain significantly be lower than after the adjuvant group is attacked attack before (P＜0.001-0.05).IL-4 attacks preceding (P＜0.05) with the chitosan particle for significantly being lower than after the attack of adjuvant group, though before all the other each groups all are lower than attack, no difference of science of statistics (P＞0.05).

Second portion: immunization therapy exercising result

One, the foundation of helicobacter pylori infections animal model

The helicobacter pylori last is irritated stomach after 12 weeks, slaughters 4 mices, gets gastric mucose pylorospirobacillus cultivation and Giemsa dyeing, helicobacter pylori all positive (Fig. 1); Pathology detect and confirm have the gastric mucosa inflammation to have (Fig. 2), confirm helicobacter pylori field planting.

Two, the helicobacter pylori infections situation is respectively organized in immunization therapy

1, immune clearance rate

The helicobacter pylori clearance rate of each group of table 13 immunization therapy

Grouping	N	Helicobacter pylori number positive (positive rate %)	Clearance rate (%)
				1. matched group a2. Heliobacter pylori antigen group b3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	12(100) 10(90.91) 5(41.67) 5(41.67) 6(54.55) 4(36.36)	0 9.09 58.33 58.33 45.45 63.64
P＜0.001

Annotate: a be P＜0.005vs 3., 4., 6. group, 5. P＜0.05vs organizes; B be P＜0.05vs 3., 4., 6. the group.

As shown in table 13, the helicobacter pylori clearance rate of each group has notable difference (P＜0.001), wherein each group of chitosan-containing is significantly higher than matched group and simple Heliobacter pylori antigen group (P＜0.05-0.005), be significantly higher than matched group (P＜0.05) with CT for the adjuvant group.

2, helicobacter pylori field planting histological score in the gastric mucosa

Helicobacter pylori field planting histological score in the gastric mucosa is respectively organized in table 14 immunization therapy

Grouping	N	Helicobacter pylori field planting score
						0	1	2	3
		1. matched group a2. Heliobacter pylori antigen group b3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	0 2 8 7 5 7	5 4 2 4 3 2					4 3 2 1 1 2	3 2 0 0 2 0
H＝19.019，P＝0.002

Annotate: a be P＜0.001vs 3., 4., 6. group, 5. P＜0.05vs organizes; B be P＜0.05vs 3., 4., 6. the group.

As shown in table 14, helicobacter pylori infections degree of each group has notable difference (P＜0.005), further relatively finds to contain adjuvant helicobacter pylori plantation density in twos and significantly is lower than matched group (P＜0.001-0.05); The plantation density that with the chitosan is adjuvant group helicobacter pylori significantly is lower than simple Heliobacter pylori antigen group (P＜0.05).

3, helicobacter pylori quantitative culture plantation density in the gastric mucosa

As shown in Table 15, each organizes the helicobacter pylori plantation density, and there were significant differences (P＜0.001)

The comparison of helicobacter pylori quantitative culture plantation density in the gastric mucosa is respectively organized in table 15 immunization therapy

Grouping	N	Helicobacter pylori plantation density median (CFU/g gastric tissue)
			1. control group 2. the Heliobacter pylori antigen group 3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	5.9×10 5 2.1×10 4 0 0 0.5×10 2 0
H＝28.1 P＜0.001

Two, the inflammation degree of gastric mucosa is respectively organized in immunization therapy

Table 16 immunization therapy is respectively organized gastric mucosa chronic inflammatory disease degree relatively

Grouping	N	The inflammation degree
						0	1	2	3
		1. matched group a2. Heliobacter pylori antigen group a3. 4. 5. Heliobacter pylori antigen+CT group of Heliobacter pylori antigen+chitosan particle group of Heliobacter pylori antigen+chitosan solution group b6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	0 0 0 0 0 0	3 3 10 11 5 7					4 3 2 1 4 4	5 5 0 0 2 0
H-number=22.368 P＜0.001

Annotate: a be P＜0.001vs 3., 4. group, 6. P＜0.005vs organizes; B be P＜0.05vs 3., 4. the group.

As seen in Table 16, each organizes gastric mucosa chronic inflammatory disease degree, and there were significant differences (P＜0.001), and wherein chronic inflammatory disease degree of each group of chitosan-containing significantly is lower than not each group of chitosan-containing (P＜0.001-0.05).

As seen in Table 17, each organizes gastric mucosa acute inflammation degree, and there were significant differences (P＜0.001), and the acute inflammation degree that contains each group of adjuvant significantly is lower than matched group and simple Heliobacter pylori antigen group (P＜0.001-0.05).

Table 17 immunization therapy is respectively organized gastric mucosa acute inflammation degree relatively

Grouping	N	The acute inflammation degree
						0	1	2	3
		1. matched group a2. Heliobacter pylori antigen group b3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	0 1 8 7 5 7	4 4 3 4 3 2					5 4 1 1 2 2	3 2 0 0 1 0
H-number=24.566 P＜0.001

Annotate: a be P＜0.001vs be P＜0.001vs 3., 4., 6. group, 5. P＜0.005vs organizes; B be P＜0.005vs 3., 4., 6. the group, 5. P＜0.05vs organizes.

Three, serum helicobacter pylori antibody result is respectively organized in immunization therapy

Serum anti-helicobacter pylori IgG, IgG1, IgG2a result are respectively organized in table 18 immunization therapy

Grouping	N	IgG	IgG1	IgG2a
					1. control group 2. the Heliobacter pylori antigen group 3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	6.56±3.50 b 7.86±4.02 c 12.73±3.95 12.06±4.84 11.83±4.31 12.71±4.36	3.68±1.67 a 4.52±2.49 a 12.20±5.61 14.35±6.59 13.86±6.23 13.98±6.26	8.46±5.88 a 9.43±3.84 a 24.55±6.91 23.71±4.32 22.06±9.23 23.59±7.43
F＝4.843 F＝10.560 F＝15.639 P＝0.001 P＜0.001 P＜0.001

Annotate: a is that 3.-6. P＜0.001vs organizes; B be P＜0.001vs 3., 5. the group, P＜0.005vs 4., 6. the group; C be P＜0.01vs 3., 5. the group, P＜0.05vs 4., 6. the group.

Shown in table 18, each organizes serum anti-helicobacter pylori IgG, helicobacter pylori IgG1, helicobacter pylori IgG2a all has notable difference (P＜0.001), further relatively find in twos, contain each group of adjuvant and be significantly higher than matched group and simple Heliobacter pylori antigen group (P＜0.001-0.05).

Four, immunization therapy is respectively organized saliva and gastric mucosa internal specific anti-helicobacter pylori IgA and non-specific sIgA content relatively

1, saliva and gastric mucosa internal specific anti-helicobacter pylori IgA

Anti-helicobacter pylori IgA result in saliva and the gastric mucosa is respectively organized in table 19 immunization therapy

Grouping	N	IgA
				Gastric mucosa	Saliva
		1. control group 2. the Heliobacter pylori antigen group 3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11			15.56±6.24 c 27.25±6.56 c 60.18±19.87 63.01±20.92 61.16±22.25 65.66±20.07	1.19±0.63 a 1.32±0.30 b 3.28±1.38 2.81±1.56 3.03±1.52 3.16+165
F＝18.008 F＝6.076 P＜0.001 P＜0.001

Annotate: a be P＜0.001vs 3., 5., 6. group, 4. P＜0.005vs organizes; B for for P＜0.005vs 3., 5., 6. the group, 4. P＜0.01vs organizes; C is that 3.-6. P＜0.001vs organizes

Shown in table 19, each organizes in gastric mucosa and the saliva anti-helicobacter pylori IgA content, and there were significant differences (P＜0.001), wherein contains each group of adjuvant and be significantly higher than matched group and simple Heliobacter pylori antigen group (P＜0.001-0.01).

2, non-specific secretory IgA (sIgA) in the gastric mucosa

Gastric mucosa sIgA SABC result is respectively organized in table 20 immunization therapy

Grouping	N	IgA
			1. control group 2. the Heliobacter pylori antigen group 3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	9.58±6.515 a 15.45±5.627 b 34.67±14.44 33.08±14.54 30.91±16.75 40.90±16.458
F＝9.860，P＜0.001

Annotate: a is that 3.-6. P＜0.001vs organizes; B is that 3.-6. P＜0.005vs organizes.

Shown in table 20, each organizes in the gastric mucosa sIgA content, and there were significant differences (P＜0.001), wherein contains each group of adjuvant and be significantly higher than matched group and simple Heliobacter pylori antigen group (P＜0.001-0.005).

Five, immunization therapy is respectively organized the gastric mucosa inner cell factor relatively

IL-2, IL-10 and IL-4 result's (ng/mg weight in wet base tissue) in the gastric mucosa are respectively organized in table 21 immunization therapy

Grouping	N	IL-2	IL-10	IL-4
					1. control group 2. the Heliobacter pylori antigen group 3. Heliobacter pylori antigen+chitosan solution group 4. Heliobacter pylori antigen+chitosan particle group 5. Heliobacter pylori antigen+CT organize 6. Heliobacter pylori antigen+CT+ chitosan solution	12 11 12 12 11 11	49.83±14.58 a 71.69±21.07 b 108.75±45.00 109.41±41.58 103.91±42.61 115.44±34.39	129.07±44.67 c 215.98±71.85 d 456.09±82.09 459.07±56.19 334.59±72.80 e 468.46±188.98	4.85±0.923 f 5.81±3.35 f 18.25±6.48 17.06±8.78 10.59±3.35 k 21.12±4.22
F＝4.739 F＝20.793 F＝17.668 P＝0.001 P＜0.001 P＜0.001

Annotate: a be P＜0.001vs 3., 4., 6. group, 5. P＜0.005vs organizes; B is that 3.-6. P＜0.05vs organizes; C is that 3.-6. P＜0.001vs organizes; D be P＜0.001vs 3., 4., 6. the group, 5. P＜0.01vs organizes; E be P＜0.01vs 3., 4., 6. the group; F be P＜0.001vs 3., 4., 6. the group, 5. P＜0.05vs organizes; G be P＜0.05vs 3., 4., 6. the group.

Shown in table 21, each organizes IL-2, IL-10 in the gastric mucosa, there were significant differences for IL-4 content (P＜0.001).Wherein IL-2 is significantly higher than matched group and simple Heliobacter pylori antigen group (P＜0.001-0.05) containing each group of adjuvant; IL-10 is significantly higher than matched group and simple Heliobacter pylori antigen group (P＜0.001-0.01) is being adjuvant group (P＜0.01) with the chitosan for the adjuvant group is significantly higher than with CT simultaneously containing each group of adjuvant; IL-4 is being significantly higher than matched group, simple Heliobacter pylori antigen group with the chitosan for the adjuvant group and is being adjuvant group (P＜0.001-0.05), be significantly higher than matched group (P＜0.05) with CT for the adjuvant group simultaneously with CT.In sum, the present invention confirms through zoopery: (1), be that the helicobacter pylori vaccine of adjuvant is consistent with CT with the immune clearance rate to the protective rate of the infection of helicobacter pylori with the chitosan, while discovery chitosan in the detection of helicobacter pylori plantation density also can be worked in coordination with the adjuvant effect of enhanced CT, and the prompting chitosan can be used as the mucosa adjuvant of helicobacter pylori vaccine; (2), be that the helicobacter pylori vaccine of adjuvant can induce gastric mucosa to produce specificity anti-helicobacter pylori IgA and non-specific secretory IgA with the chitosan, and share synergism with CT, simultaneously simple chitosan also can promote the non-specific secretory IgA secretion of gastric mucosa, illustrates that chitosan can promote local mucosa-immune reaction effectively; (3), be that the helicobacter pylori vaccine of adjuvant is consistent with CT with the chitosan, can promote the secretion of cytokine IL-2, and synergism be arranged that the immunoreation that it can promote TH1 is described, this may participate in its immanoprotection action with CT; (4), with the chitosan be the helicobacter pylori vaccine of adjuvant can obviously promote TH2 cytokine IL-4, IL-10 before attack secretion, can reverse simultaneously the inhibition of attacking back TH2, TH1/TH2 due to the recovery helicobacter pylori infections is unbalance, and this may play an important role in its immunoprotection; (5), with the chitosan be the helicobacter pylori vaccine of adjuvant can alleviate immunity back gastritis when producing immunoprotection and therapeutical effect the order of severity; (6), chitosan solution or chitosan particle are that adjuvant can be used as the protection mammal and comprises that the people avoids the preventative vaccine of helicobacter pylori or comprises people's therapeutic vaccine as the mammal that has infected helicobacter pylori.

Description of drawings

Fig. 1 is each group helicobacter pylori quantitative culture clump count scatterplot;

Fig. 2 respectively organizes serum anti-helicobacter pylori IgG, helicobacter pylori IgG1, helicobacter pylori IgG2a comparison diagram for before and after attacking;

Fig. 3 is anti-helicobacter pylori IgA content comparison diagram in gastric mucosa before and after attacking and the saliva;

Fig. 4 is the positive body of gland percent of sIgA comparison diagram in the gastric mucosa before and after attacking;

Fig. 5 is IL-2, IL-10 and IL-4 content comparison diagram in the gastric mucosa before and after attacking;

The specific embodiment

Be described in further detail the present invention below in conjunction with embodiment, but should understand the scope that scope of the present invention is not limited only to these embodiment.

Embodiment 1:

Use method well-known in the art, with 88.5% deacetylation chitosan is raw material, with 88.5% deacetylation chitosan in 3% ratio be dissolved in the 0.8% acetic acid saline the chitosan acid solution, be the mucosa adjuvant with the chitosan acid solution again, be prepared into vaccine with Heliobacter pylori antigen, be used for the prevention and the treatment of helicobacter pylori infections.

Embodiment 2:

Use method well-known in the art, with 88.5% deacetylation chitosan is raw material, 88.5% deacetylation chitosan and normal saline are made into the solution of 10mg/ml, handle 2 times through ultrasonic (output 80HZ) again, each 5 minutes, 1 minute at interval, get its supernatant with 50g after centrifugal 10 minutes, and filter with 400 order mesh screens, collecting its precipitate after centrifugal 10 minutes with 1400g again, be the chitosan granule, is the mucosa adjuvant with the chitosan granule again, be prepared into vaccine with Heliobacter pylori antigen, be used for the prevention and the treatment of helicobacter pylori infections.

Claims (6)

1, the application of chitosan solution in the preparation helicobacter pylorus vaccine adjuvant.

2, the application of chitosan particle in the preparation helicobacter pylorus vaccine adjuvant.

3, the application of chitosan solution as claimed in claim 1 in the preparation helicobacter pylorus vaccine adjuvant, chitosan solution is used to protect mammal to comprise that the people avoids the preventative vaccine of helicobacter pylori as adjuvant.

4, chitosan solution as claimed in claim 1 is in the application of preparation in the helicobacter pylorus vaccine adjuvant, and the mammal that chitosan solution has been used to infect helicobacter pylori as adjuvant comprises people's therapeutic vaccine.

5, the application of chitosan particle as claimed in claim 2 in the preparation helicobacter pylorus vaccine adjuvant, chitosan particle is used to protect mammal to comprise that the people avoids the preventative vaccine of helicobacter pylori as adjuvant.

6, chitosan particle as claimed in claim 2 is in the application of preparation in the helicobacter pylorus vaccine adjuvant, and the mammal that chitosan particle has been used to infect helicobacter pylori as adjuvant comprises people's therapeutic vaccine.

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