Tim-3阻斷對H.pylori感染免疫致病和疫苗免疫保護作用影響及機制的研究
發(fā)布時間：2018-08-15 17:13
【摘要】： 背景和目的: 機體的免疫反應在幽門螺桿菌(Helicobacter pylori, H.pylori)致病和H.pylori疫苗的免疫保護中均起重要作用,因此,闡明H.pylori感染的免疫致病機制和H.pylori疫苗的免疫保護機制,對于H.pylori相關性疾病的防治具有十分重要的意義。T細胞免疫球蛋白黏蛋白結構域相關分子(T-cell immunoglobulin and mucin-domain-containing molecule,Tim)是一新近發(fā)現(xiàn)的T細胞表面的跨膜蛋白家族,它對CD4+T細胞分化成Th1和Th2細胞,調控效應T細胞(Th1和Th2)的應答起著重要作用。Tim-3是Tim家族中的重要成員,它表達在分化成熟的Th1細胞,調節(jié)不同CD4+T細胞亞群的功能,并影響Toll樣受體(Toll like receptor, TLR)信號通路和調節(jié)性T細胞(Regulatory T cell, Treg)的功能。但Tim-3在H. pylori感染免疫致病和H. pylori疫苗免疫保護中是否起作用,目前尚不清楚。為此,本文研究了Tim-3阻斷對H. pylori感染和不同佐劑的H. pylori疫苗接種小鼠胃黏膜內H. pylori定植、炎癥反應、TLR信號通路和Treg的影響,從一新的角度探討H. pylori致病機制和疫苗的免疫保護機制。 方法: 1.H. pylori全菌蛋白抗原殼聚糖微球的制備及體外釋放特性研究 采用Berthold沉淀法制備殼聚糖微球,并根據(jù)不同的殼聚糖、不同的沉淀劑、不同冰乙酸濃度、不同pH值和是否進行超聲處理來優(yōu)化殼聚糖微球制備條件;使用掃描電鏡及粒徑分析儀觀察殼聚糖微球的形態(tài)及粒徑分布;以不同量比的微球與抗原,進行殼聚糖微球包裹H. pylori全菌蛋白抗原;采用BCA蛋白定量法測量分析微球的抗原包裹率、包裹量及釋放率。 2. Tim-3阻斷對H. pylori感染免疫致病和疫苗免疫保護作用影響及機制的研究 (1)Tim-3阻斷對H. pylori疫苗免疫保護作用影響及機制的研究:6~8周齡的SPF級BALB/C小鼠隨機分為以下5組:①正常對照組:PBS溶液;②H. pylori抗原+CT;③抗Tim-3單抗預處理+H. pylori抗原+CT;④殼聚糖微球-H. pylori抗原;⑤抗Tim-3單抗預處理+殼聚糖微球-H. pylori抗原;各組于第0、7、14、21天灌胃各免疫1次,免疫后4周后4組小鼠給予1×109活菌/ml的SS1 H. pylori菌液0.5ml/只進行攻擊,隔日一次,共4次。未次攻擊后4周,處死小鼠,取標本待測。 (2)Tim-3阻斷對H. pylori感染免疫致病作用影響及機制的研究:6~8周齡的SPF級BALB/C小鼠隨機分為以下2組:①直接建立H. pylori感染模型;②Tim-3單抗預處理后建立H. pylori感染模型。模型的建立:給予含109/ml的SS1 H. pylori菌液,0.5ml/只灌胃,隔日1次,共5次。末次灌胃12周后,處死小鼠,取標本待測。 (3)各項指標檢測:①胃黏膜內H. pylori的檢測:采用改良Giemsa染色檢測;②胃黏膜炎癥程度檢測:采用HE染色,并按Sakagami法評分;③胃黏膜內TLR4、MyD88、NF-κB p65、Foxp3蛋白檢測:采用免疫組織化學染色法檢測;④胃黏膜內TLR4和MyD88 mRNA檢測:采用RT-PCR方法檢測;⑤血清抗H. pylori IgG含量的檢測:采用間接ELISA法檢測。 結果: 1. H. pylori全菌蛋白抗原殼聚糖微球的制備及體外釋放特性研究 從32種殼聚糖微球制備方案中篩選出了以海得貝殼聚糖為原料、冰乙酸的濃度為1%、硫酸鈉為沉淀劑、pH值為5.0、不進行超聲處理的最佳制備方案;經掃描電鏡(SEM)示微球光滑圓整、致密,粒徑分布在1.0-5.0μm;抗原與微球的量比為1:5時包裹率最大,包裹時間為3h時包裹率可達最大,抗原包裹率為79.92%,包裹量為16.47%;體外釋放實驗表明,總抗原釋放率為20.39%,呈緩慢釋放狀態(tài)。 2. Tim-3阻斷對H. pylori感染免疫致病和疫苗免疫保護作用影響及機制的研究 (1)兩種佐劑的H. pylori疫苗組預先給予Tim-3單抗阻斷小鼠胃黏膜內H. pylori定植密度均低于未阻斷小鼠(P0.05),與正常對照組無差別(P0.05);不同佐劑的H. pylori疫苗接種小鼠的H. pylori定植密度無差別(P0.05)。 (2)H. pylori活菌接種后胃黏膜內H. pylori定植密度顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷對H. pylori定植密度無影響(P0.05)。 (3)兩種佐劑的H. pylori疫苗接種小鼠胃粘膜炎癥程度均高于正常對照組(P0.05,0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜炎癥程度均高于未阻斷小鼠(P0.05,0.01),不同佐劑的H. pylori疫苗接種小鼠胃黏膜炎癥程度無差別(P0.05)。 (4)H.pylori活菌接種后胃黏膜炎癥程度顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜炎癥程度顯著高于未阻斷小鼠(P0.05)。 (5)兩種佐劑的H. pylori疫苗接種小鼠胃黏膜內TLR4 mRNA的表達和TLR4陽性細胞積分均顯著高于正常對照組(P0.05,0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜內TLR4陽性細胞積分均顯著高于未阻斷小鼠(P0.05,0.01,0.001),在以殼聚糖微球為佐劑的H. pylori疫苗組TLR4 mRNA的表達在Tim-3單抗阻斷組顯著高于未阻斷組(P0.001),而在以CT為佐劑小鼠Tim-3單抗阻斷組雖高于未阻斷組,但無統(tǒng)計學差別(P0.05)。不同佐劑的H. pylori疫苗接種小鼠胃黏膜內TLR4陽性細胞積分無差別(P0.05),TLR4 mRNA表達在無Tim-3單抗阻斷小鼠,在以CT為佐劑組顯著高于以殼聚糖微球為佐劑組(P0.05),但在Tim-3單抗阻斷小鼠二組之間無差別(P0.05)。 (6)H. pylori活菌接種后胃黏膜內TLR4 mRNA的表達和TLR4陽性細胞積分均顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜TLR4 mRNA的表達和TLR4陽性細胞積分顯著高于未阻斷小鼠(P0.05,0.001)。 (7)兩種佐劑的H. pylori疫苗接種小鼠胃黏膜內MyD88 mRNA的表達和MyD88陽性細胞積分均顯著高于正常對照組(P0.05,0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜內MyD88 mRNA的表達和MyD88陽性細胞積分均顯著高于未阻斷小鼠(P0.05,0.001)。不同佐劑的H. pylori疫苗接種小鼠胃黏膜內MyD88陽性細胞積分無差別(P0.05),MyD88 mRNA表達在無Tim-3單抗阻斷小鼠,在以CT為佐劑組顯著高于以殼聚糖微球為佐劑組(P0.05),但在Tim-3單抗阻斷小鼠二組之間無差別(P0.05)。 (8)H. pylori活菌接種后胃黏膜內MyD88 mRNA的表達和MyD88陽性細胞積分均顯著高于正常對照組(P0.001,0.01),預先給予Tim-3單抗阻斷小鼠胃黏膜MyD88 mRNA的表達和MyD88陽性細胞積分顯著高于未阻斷小鼠(P0.01,0.001)。 (9)兩種佐劑的H. pylori疫苗接種小鼠胃黏膜內Foxp3陽性細胞百分比均顯著高于正常對照組(P0.05,0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜內Foxp3陽性細胞百分比顯著低于未阻斷小鼠(P0.001)。不同佐劑的H. pylori疫苗接種小鼠胃黏膜內Foxp3陽性細胞百分比無差別(P0.05)。 (10)H. pylori活菌接種后胃黏膜內Foxp3陽性細胞百分比顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜內Foxp3陽性細胞百分比顯著低于未阻斷小鼠(P0.001)。 (11)兩種佐劑的H. pylori疫苗接種小鼠胃黏膜內NF-κBp65陽性細胞積分均顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷小鼠胃黏膜內NF-κBp65陽性細胞積分高于未阻斷小鼠(P0.01)。不同佐劑的H. pylori疫苗接種小鼠胃黏膜內NF-κBp65陽性細胞積分無差別(P0.05)。 (12)H. pylori活菌接種后胃黏膜內NF-κBp65陽性細胞積分均顯著高于正常對照組(P0.001,0.01),預先給予Tim-3單抗阻斷小鼠胃黏膜內NF-κBp65陽性細胞積分高于未阻斷小鼠(P0.001)。 (13)兩種佐劑的H. pylori疫苗接種小鼠血清抗H. pylori IgG含量均顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷對小鼠血清抗H. pylori IgG含量無影響(P0.05)。進一步分析不同佐劑的H. pylori疫苗接種小鼠血清抗H. pyloriIgG含量無差別(P0.05)。 (14) H. pylori活菌接種小鼠血清抗H. pylori IgG含量均顯著高于正常對照組(P0.001),預先給予Tim-3單抗阻斷對小鼠血清抗H. pylori IgG含量無影響(P0.05)。進一步分析H. pylori疫苗接種小鼠血清抗H. pylori IgG含量均顯著高于H. pylori感染小鼠(P0.05)。 結論: 1.本研究篩選的殼聚糖微球制備條件所制備的殼聚糖微球對H. pylori全菌蛋白抗原具有較高的包裹率和較好的控釋效果。 2. Tim-3阻斷可提高H. pylori疫苗的免疫保護率,但并不降低H. pylori自然感染小鼠胃黏膜內H. pylori定植密度。 3. Tim-3阻斷可加劇H. pylori疫苗接種小鼠和H. pylori感染小鼠胃黏膜炎癥程度。 4. Tim-3阻斷可上調TLR4、MyD88的表達和促進NF-κB活化,降低CD4+CD25+Foxp3+Treg的數(shù)量,這可能是它增強H .pylori疫苗的免疫保護作用的機制,但并不影響H. pylori自然感染小鼠胃黏膜內H. pylori定植密度。 5.以殼聚糖微球為佐劑的H. pylori疫苗與傳統(tǒng)的佐劑CT為佐劑的H. pylori疫苗對H. pylori感染具有同樣的免疫保護作用。
[Abstract]:Background and purpose:
The immune response plays an important role in the pathogenesis of Helicobacter pylori (H. pylori) and the immune protection of H. pylori vaccine. Therefore, it is very important to clarify the immunopathogenic mechanism of H. pylori infection and the immune protection mechanism of H. pylori vaccine for the prevention and treatment of H. pylori-related diseases. T-cell immunoglobulin and mucin-domain-containing molecule (Tim) is a newly discovered transmembrane protein family on the surface of T cells. Tim-3 plays an important role in the differentiation of CD4+T cells into Th1 and Th2 cells and in regulating the response of effector T cells (Th1 and Th2). Tim-3 is an important member of the Tim family. It is not clear whether Tim-3 plays a role in the immunopathogenesis of H.pylori infection and the immune protection of H.pylori vaccine. The effects of Tim-3 blockade on H.pylori colonization, inflammation, TLR signaling pathway and Treg in gastric mucosa of mice inoculated with H.pylori infection and H.pylori vaccine with different adjuvants were studied. The pathogenesis of H.pylori and the immune protection mechanism of the vaccine were discussed from a new perspective.
Method:
Preparation and in vitro release characteristics of 1.H. pylori whole protein antigen chitosan microspheres
The preparation conditions of chitosan microspheres were optimized by Berthold precipitation method according to different chitosan, different precipitating agent, different concentration of glacial acetic acid, different pH value and whether or not ultrasonic treatment was carried out. Chitosan microspheres were used to encapsulate H.pylori whole bacterial protein antigen, and BCA protein quantitative method was used to measure and analyze the antigen encapsulation rate, encapsulation amount and release rate of the microspheres.
Effects of 2. Tim-3 blockade on immune pathogenesis and vaccine protection of H. pylori infection and its mechanism
(1) Effect and mechanism of Tim-3 blockade on immune protection of H.pylori vaccine: SPF BALB/C mice aged 6-8 weeks were randomly divided into five groups: normal control group: PBS solution; H.pylori antigen + CT; anti-Tim-3 monoclonal antibody preconditioning + H.pylori antigen + CT; chitosan microsphere-H.pylori antigen; _anti-Tim-3 monoclonal antibody preconditioning + chitosan micro-antigen Spherical H.pylori antigen was administered intragastrically once on day 0, 7, 14 and 21 in each group. Four weeks after immunization, the mice in each group were given SSH.pylori solution of 1 *109 living bacteria/ml once every other day for 4 times.
(2) Effect of Tim-3 blockade on immunopathogenesis of H.pylori infection and its mechanism: 6-8 weeks old SPF BALB/C mice were randomly divided into the following two groups: 1) H.pylori infection model was established directly; 2) H.pylori infection model was established after pretreatment with Tim-3 monoclonal antibody. A total of 5 times. 12 weeks after the last gavage, the mice were sacrificed and the specimens were taken for testing.
(3) Indicators: detection of H.pylori in gastric mucosa by modified Giemsa staining; detection of gastric mucosal inflammation by HE staining and Sakagami score; detection of TLR4, MyD88, NF-kappa B p65, Foxp3 protein in gastric mucosa by immunohistochemical staining; detection of TLR4 and MyD88 mRNA in gastric mucosa by RT -PCR method was used to detect the content of anti H. pylori IgG in serum. Indirect ELISA was used to detect the content.
Result:
Preparation and in vitro release characteristics of 1. H. pylori total bacterial protein antigen chitosan microspheres
From 32 preparation schemes of chitosan microspheres, the optimum preparation scheme was selected, in which chitosan was used as raw material, acetic acid concentration was 1%, sodium sulfate as precipitator, pH value was 5.0, without ultrasonic treatment; scanning electron microscopy (SEM) showed that the microspheres were smooth, compact, and the particle size distribution was 1.0-5.0 micron; the encapsulation rate of antigen and microspheres was 1:5. The highest encapsulation rate was obtained when the encapsulation time was 3 hours. The encapsulation rate of antigen was 79.92% and the encapsulation amount was 16.47%. The release rate of total antigen was 20.39% in vitro, showing a slow release state.
Effects of 2. Tim-3 blockade on immune pathogenesis and vaccine protection of H. pylori infection and its mechanism
(1) The density of H.pylori colonization in gastric mucosa of the mice pretreated with Tim-3 monoclonal antibody was lower than that of the normal control group (P 0.05), and the density of H.pylori colonization in the mice inoculated with different adjuvants was not different (P 0.05).
(2) The colonization density of H.pylori in gastric mucosa was significantly higher than that in normal control group (P 0.001) after H.pylori inoculation. Pre-administration of Tim-3 monoclonal antibody had no effect on the colonization density of H.pylori (P 0.05).
(3) The degree of gastric mucosal inflammation in mice inoculated with H.pylori vaccine of two adjuvants was higher than that in normal control group (P 0.05,0.001). The degree of gastric mucosal inflammation in mice inoculated with Tim-3 monoclonal antibody was higher than that in mice inoculated with H.pylori vaccine of no adjuvant (P 0.05).
(4) The degree of gastric mucosal inflammation after H.pylori inoculation was significantly higher than that of the normal control group (P 0.001). The degree of gastric mucosal inflammation was significantly higher in pre-administration of Tim-3 monoclonal antibody in blocked mice than in non-blocked mice (P 0.05).
(5) The expression of TLR4 mRNA and the score of TLR4 positive cells in gastric mucosa of mice inoculated with two adjuvants of H.pylori vaccine were significantly higher than those of normal control group (P 0.05,0.001). The score of TLR4 positive cells in gastric mucosa of mice inoculated with Tim-3 monoclonal antibody was significantly higher than that of mice inoculated with non-blocking H.pylori vaccine (P 0.05,0.01,0.001). The expression of TLR4 mRNA in the Tim-3 monoclonal antibody blocked group was significantly higher than that in the non-blocked group (P 0.001), while the expression of TLR4 mRNA in the T-3 monoclonal antibody blocked group was significantly higher than that in the non-blocked group (P 0.05). Tim-3 monoclonal antibody blocked mice significantly higher in the CT adjuvant group than in the chitosan microsphere adjuvant group (P 0.05), but there was no difference between the Tim-3 monoclonal antibody blocked mice in the two groups (P 0.05).
(6) The expression of TLR4 mRNA and the score of TLR4 positive cells in gastric mucosa after H.pylori inoculation were significantly higher than those in normal control group (P 0.001). The expression of TLR4 mRNA and the score of TLR4 positive cells in gastric mucosa of mice blocked by Tim-3 monoclonal antibody in advance were significantly higher than those in non-blocked mice (P 0.05, 0.001).
(7) The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were significantly higher than those of normal control group (P 0.05,0.001). The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were significantly higher than those of mice inoculated without H.pylori vaccine (P 0.05,0.001). The score of MyD88 positive cells in gastric mucosa of mice inoculated with pylori vaccine had no difference (P 0.05). The expression of MyD88 mRNA in mice without Tim-3 monoclonal antibody blocking was significantly higher in the CT adjuvant group than in the chitosan microsphere adjuvant group (P 0.05), but there was no difference between the two groups (P 0.05).
(8) The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of H.pylori inoculated mice were significantly higher than those of normal control group (P 0.001,0.01). The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of mice blocked by Tim-3 monoclonal antibody in advance were significantly higher than those of non-blocked mice (P 0.01,0.001).
(9) The percentage of Foxp3 positive cells in gastric mucosa of mice inoculated with H. pylori vaccine of two adjuvants was significantly higher than that of normal control group (P 0.05, 0.001). The percentage of Foxp3 positive cells in gastric mucosa of mice inoculated with H. pylori vaccine of different adjuvants was significantly lower than that of non-blocked mice (P 0.001). The percentage of sex cells was not different (P0.05).
(10) The percentage of Foxp3 positive cells in gastric mucosa after H. pylori inoculation was significantly higher than that in normal control group (P 0.001). The percentage of Foxp3 positive cells in gastric mucosa of mice blocked by Tim-3 monoclonal antibody in advance was significantly lower than that of non-blocked mice (P 0.001).
(11) The scores of NF-kappa Bp65 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were significantly higher than those of normal control group (P 0.001). The scores of NF-kappa Bp65 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were higher than those of mice inoculated with non-blocking H.pylori vaccine (P 0.01). There was no difference in cell integration (P0.05).
(12) The scores of NF-kappa Bp65 positive cells in gastric mucosa after H.pylori inoculation were significantly higher than those in normal control group (P 0.001,0.01). The scores of NF-kappa Bp65 positive cells in gastric mucosa of pre-treated Tim-3 monoclonal antibody-blocked mice were higher than those of non-blocked mice (P 0.001).
(13) The levels of anti-H.pylori IgG in serum of mice inoculated with H.pylori adjuvant vaccine were significantly higher than those of normal control group (P 0.001). Pre-administration of Tim-3 monoclonal antibody had no effect on the level of anti-H.pylori IgG in serum of mice inoculated with H.pylori adjuvant vaccine (P 0.05).
(14) The serum anti-H.pylori IgG levels of mice inoculated with H.pylori live bacteria were significantly higher than those of normal control group (P 0.001). Pre-administration of Tim-3 monoclonal antibody had no effect on the serum anti-H.pylori IgG levels of mice inoculated with H.pylori vaccine (P 0.05).
Conclusion:
1. The chitosan microspheres prepared by this study have higher encapsulation rate and better controlled release effect on H. pylori whole bacterial protein antigen.
2. Tim-3 blockade increased the immune protection rate of H. pylori vaccine, but did not decrease the colonization density of H. pylori in gastric mucosa of H. pylori infected mice.
3. Tim-3 blockade could aggravate the degree of gastric mucosal inflammation in mice inoculated with H. pylori vaccine and H. pylori.
4. Tim-3 blockade can up-regulate the expression of TLR4, MyD88 and promote the activation of NF-kappa B, and decrease the number of CD4+CD25+Foxp3+Treg. This may be the mechanism of enhancing the immune protection of H. pylori vaccine, but it does not affect the colonization density of H. pylori in gastric mucosa of H. pylori infected mice.
5. H. pylori vaccine with chitosan microspheres as adjuvant and traditional adjuvant CT as adjuvant has the same immune protective effect on H. pylori infection.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：2184875
[Abstract]:Background and purpose:
The immune response plays an important role in the pathogenesis of Helicobacter pylori (H. pylori) and the immune protection of H. pylori vaccine. Therefore, it is very important to clarify the immunopathogenic mechanism of H. pylori infection and the immune protection mechanism of H. pylori vaccine for the prevention and treatment of H. pylori-related diseases. T-cell immunoglobulin and mucin-domain-containing molecule (Tim) is a newly discovered transmembrane protein family on the surface of T cells. Tim-3 plays an important role in the differentiation of CD4+T cells into Th1 and Th2 cells and in regulating the response of effector T cells (Th1 and Th2). Tim-3 is an important member of the Tim family. It is not clear whether Tim-3 plays a role in the immunopathogenesis of H.pylori infection and the immune protection of H.pylori vaccine. The effects of Tim-3 blockade on H.pylori colonization, inflammation, TLR signaling pathway and Treg in gastric mucosa of mice inoculated with H.pylori infection and H.pylori vaccine with different adjuvants were studied. The pathogenesis of H.pylori and the immune protection mechanism of the vaccine were discussed from a new perspective.
Method:
Preparation and in vitro release characteristics of 1.H. pylori whole protein antigen chitosan microspheres
The preparation conditions of chitosan microspheres were optimized by Berthold precipitation method according to different chitosan, different precipitating agent, different concentration of glacial acetic acid, different pH value and whether or not ultrasonic treatment was carried out. Chitosan microspheres were used to encapsulate H.pylori whole bacterial protein antigen, and BCA protein quantitative method was used to measure and analyze the antigen encapsulation rate, encapsulation amount and release rate of the microspheres.
Effects of 2. Tim-3 blockade on immune pathogenesis and vaccine protection of H. pylori infection and its mechanism
(1) Effect and mechanism of Tim-3 blockade on immune protection of H.pylori vaccine: SPF BALB/C mice aged 6-8 weeks were randomly divided into five groups: normal control group: PBS solution; H.pylori antigen + CT; anti-Tim-3 monoclonal antibody preconditioning + H.pylori antigen + CT; chitosan microsphere-H.pylori antigen; _anti-Tim-3 monoclonal antibody preconditioning + chitosan micro-antigen Spherical H.pylori antigen was administered intragastrically once on day 0, 7, 14 and 21 in each group. Four weeks after immunization, the mice in each group were given SSH.pylori solution of 1 *109 living bacteria/ml once every other day for 4 times.
(2) Effect of Tim-3 blockade on immunopathogenesis of H.pylori infection and its mechanism: 6-8 weeks old SPF BALB/C mice were randomly divided into the following two groups: 1) H.pylori infection model was established directly; 2) H.pylori infection model was established after pretreatment with Tim-3 monoclonal antibody. A total of 5 times. 12 weeks after the last gavage, the mice were sacrificed and the specimens were taken for testing.
(3) Indicators: detection of H.pylori in gastric mucosa by modified Giemsa staining; detection of gastric mucosal inflammation by HE staining and Sakagami score; detection of TLR4, MyD88, NF-kappa B p65, Foxp3 protein in gastric mucosa by immunohistochemical staining; detection of TLR4 and MyD88 mRNA in gastric mucosa by RT -PCR method was used to detect the content of anti H. pylori IgG in serum. Indirect ELISA was used to detect the content.
Result:
Preparation and in vitro release characteristics of 1. H. pylori total bacterial protein antigen chitosan microspheres
From 32 preparation schemes of chitosan microspheres, the optimum preparation scheme was selected, in which chitosan was used as raw material, acetic acid concentration was 1%, sodium sulfate as precipitator, pH value was 5.0, without ultrasonic treatment; scanning electron microscopy (SEM) showed that the microspheres were smooth, compact, and the particle size distribution was 1.0-5.0 micron; the encapsulation rate of antigen and microspheres was 1:5. The highest encapsulation rate was obtained when the encapsulation time was 3 hours. The encapsulation rate of antigen was 79.92% and the encapsulation amount was 16.47%. The release rate of total antigen was 20.39% in vitro, showing a slow release state.
Effects of 2. Tim-3 blockade on immune pathogenesis and vaccine protection of H. pylori infection and its mechanism
(1) The density of H.pylori colonization in gastric mucosa of the mice pretreated with Tim-3 monoclonal antibody was lower than that of the normal control group (P 0.05), and the density of H.pylori colonization in the mice inoculated with different adjuvants was not different (P 0.05).
(2) The colonization density of H.pylori in gastric mucosa was significantly higher than that in normal control group (P 0.001) after H.pylori inoculation. Pre-administration of Tim-3 monoclonal antibody had no effect on the colonization density of H.pylori (P 0.05).
(3) The degree of gastric mucosal inflammation in mice inoculated with H.pylori vaccine of two adjuvants was higher than that in normal control group (P 0.05,0.001). The degree of gastric mucosal inflammation in mice inoculated with Tim-3 monoclonal antibody was higher than that in mice inoculated with H.pylori vaccine of no adjuvant (P 0.05).
(4) The degree of gastric mucosal inflammation after H.pylori inoculation was significantly higher than that of the normal control group (P 0.001). The degree of gastric mucosal inflammation was significantly higher in pre-administration of Tim-3 monoclonal antibody in blocked mice than in non-blocked mice (P 0.05).
(5) The expression of TLR4 mRNA and the score of TLR4 positive cells in gastric mucosa of mice inoculated with two adjuvants of H.pylori vaccine were significantly higher than those of normal control group (P 0.05,0.001). The score of TLR4 positive cells in gastric mucosa of mice inoculated with Tim-3 monoclonal antibody was significantly higher than that of mice inoculated with non-blocking H.pylori vaccine (P 0.05,0.01,0.001). The expression of TLR4 mRNA in the Tim-3 monoclonal antibody blocked group was significantly higher than that in the non-blocked group (P 0.001), while the expression of TLR4 mRNA in the T-3 monoclonal antibody blocked group was significantly higher than that in the non-blocked group (P 0.05). Tim-3 monoclonal antibody blocked mice significantly higher in the CT adjuvant group than in the chitosan microsphere adjuvant group (P 0.05), but there was no difference between the Tim-3 monoclonal antibody blocked mice in the two groups (P 0.05).
(6) The expression of TLR4 mRNA and the score of TLR4 positive cells in gastric mucosa after H.pylori inoculation were significantly higher than those in normal control group (P 0.001). The expression of TLR4 mRNA and the score of TLR4 positive cells in gastric mucosa of mice blocked by Tim-3 monoclonal antibody in advance were significantly higher than those in non-blocked mice (P 0.05, 0.001).
(7) The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were significantly higher than those of normal control group (P 0.05,0.001). The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were significantly higher than those of mice inoculated without H.pylori vaccine (P 0.05,0.001). The score of MyD88 positive cells in gastric mucosa of mice inoculated with pylori vaccine had no difference (P 0.05). The expression of MyD88 mRNA in mice without Tim-3 monoclonal antibody blocking was significantly higher in the CT adjuvant group than in the chitosan microsphere adjuvant group (P 0.05), but there was no difference between the two groups (P 0.05).
(8) The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of H.pylori inoculated mice were significantly higher than those of normal control group (P 0.001,0.01). The expression of MyD88 mRNA and the score of MyD88 positive cells in gastric mucosa of mice blocked by Tim-3 monoclonal antibody in advance were significantly higher than those of non-blocked mice (P 0.01,0.001).
(9) The percentage of Foxp3 positive cells in gastric mucosa of mice inoculated with H. pylori vaccine of two adjuvants was significantly higher than that of normal control group (P 0.05, 0.001). The percentage of Foxp3 positive cells in gastric mucosa of mice inoculated with H. pylori vaccine of different adjuvants was significantly lower than that of non-blocked mice (P 0.001). The percentage of sex cells was not different (P0.05).
(10) The percentage of Foxp3 positive cells in gastric mucosa after H. pylori inoculation was significantly higher than that in normal control group (P 0.001). The percentage of Foxp3 positive cells in gastric mucosa of mice blocked by Tim-3 monoclonal antibody in advance was significantly lower than that of non-blocked mice (P 0.001).
(11) The scores of NF-kappa Bp65 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were significantly higher than those of normal control group (P 0.001). The scores of NF-kappa Bp65 positive cells in gastric mucosa of mice inoculated with H.pylori vaccine and Tim-3 monoclonal antibody were higher than those of mice inoculated with non-blocking H.pylori vaccine (P 0.01). There was no difference in cell integration (P0.05).
(12) The scores of NF-kappa Bp65 positive cells in gastric mucosa after H.pylori inoculation were significantly higher than those in normal control group (P 0.001,0.01). The scores of NF-kappa Bp65 positive cells in gastric mucosa of pre-treated Tim-3 monoclonal antibody-blocked mice were higher than those of non-blocked mice (P 0.001).
(13) The levels of anti-H.pylori IgG in serum of mice inoculated with H.pylori adjuvant vaccine were significantly higher than those of normal control group (P 0.001). Pre-administration of Tim-3 monoclonal antibody had no effect on the level of anti-H.pylori IgG in serum of mice inoculated with H.pylori adjuvant vaccine (P 0.05).
(14) The serum anti-H.pylori IgG levels of mice inoculated with H.pylori live bacteria were significantly higher than those of normal control group (P 0.001). Pre-administration of Tim-3 monoclonal antibody had no effect on the serum anti-H.pylori IgG levels of mice inoculated with H.pylori vaccine (P 0.05).
Conclusion:
1. The chitosan microspheres prepared by this study have higher encapsulation rate and better controlled release effect on H. pylori whole bacterial protein antigen.
2. Tim-3 blockade increased the immune protection rate of H. pylori vaccine, but did not decrease the colonization density of H. pylori in gastric mucosa of H. pylori infected mice.
3. Tim-3 blockade could aggravate the degree of gastric mucosal inflammation in mice inoculated with H. pylori vaccine and H. pylori.
4. Tim-3 blockade can up-regulate the expression of TLR4, MyD88 and promote the activation of NF-kappa B, and decrease the number of CD4+CD25+Foxp3+Treg. This may be the mechanism of enhancing the immune protection of H. pylori vaccine, but it does not affect the colonization density of H. pylori in gastric mucosa of H. pylori infected mice.
5. H. pylori vaccine with chitosan microspheres as adjuvant and traditional adjuvant CT as adjuvant has the same immune protective effect on H. pylori infection.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
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