Tim-3阻断对H.pylori感染免疫致病和疫苗免疫保护作用影响及机制的研究
发布时间：2018-08-15 17:13
【摘要】： 背景和目的: 机体的免疫反应在幽门螺杆菌(Helicobacter pylori, H.pylori)致病和H.pylori疫苗的免疫保护中均起重要作用,因此,阐明H.pylori感染的免疫致病机制和H.pylori疫苗的免疫保护机制,对于H.pylori相关性疾病的防治具有十分重要的意义。T细胞免疫球蛋白黏蛋白结构域相关分子(T-cell immunoglobulin and mucin-domain-containing molecule,Tim)是一新近发现的T细胞表面的跨膜蛋白家族,它对CD4+T细胞分化成Th1和Th2细胞,调控效应T细胞(Th1和Th2)的应答起着重要作用。Tim-3是Tim家族中的重要成员,它表达在分化成熟的Th1细胞,调节不同CD4+T细胞亚群的功能,并影响Toll样受体(Toll like receptor, TLR)信号通路和调节性T细胞(Regulatory T cell, Treg)的功能。但Tim-3在H. pylori感染免疫致病和H. pylori疫苗免疫保护中是否起作用,目前尚不清楚。为此,本文研究了Tim-3阻断对H. pylori感染和不同佐剂的H. pylori疫苗接种小鼠胃黏膜内H. pylori定植、炎症反应、TLR信号通路和Treg的影响,从一新的角度探讨H. pylori致病机制和疫苗的免疫保护机制。 方法: 1.H. pylori全菌蛋白抗原壳聚糖微球的制备及体外释放特性研究 采用Berthold沉淀法制备壳聚糖微球,并根据不同的壳聚糖、不同的沉淀剂、不同冰乙酸浓度、不同pH值和是否进行超声处理来优化壳聚糖微球制备条件;使用扫描电镜及粒径分析仪观察壳聚糖微球的形态及粒径分布;以不同量比的微球与抗原,进行壳聚糖微球包裹H. pylori全菌蛋白抗原;采用BCA蛋白定量法测量分析微球的抗原包裹率、包裹量及释放率。 2. Tim-3阻断对H. pylori感染免疫致病和疫苗免疫保护作用影响及机制的研究 (1)Tim-3阻断对H. pylori疫苗免疫保护作用影响及机制的研究:6~8周龄的SPF级BALB/C小鼠随机分为以下5组:①正常对照组:PBS溶液;②H. pylori抗原+CT;③抗Tim-3单抗预处理+H. pylori抗原+CT;④壳聚糖微球-H. pylori抗原;⑤抗Tim-3单抗预处理+壳聚糖微球-H. pylori抗原;各组于第0、7、14、21天灌胃各免疫1次,免疫后4周后4组小鼠给予1×109活菌/ml的SS1 H. pylori菌液0.5ml/只进行攻击,隔日一次,共4次。未次攻击后4周,处死小鼠,取标本待测。 (2)Tim-3阻断对H. pylori感染免疫致病作用影响及机制的研究:6~8周龄的SPF级BALB/C小鼠随机分为以下2组:①直接建立H. pylori感染模型;②Tim-3单抗预处理后建立H. pylori感染模型。模型的建立:给予含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,处死小鼠,取标本待测。 (3)各项指标检测:①胃黏膜内H. pylori的检测:采用改良Giemsa染色检测;②胃黏膜炎症程度检测:采用HE染色,并按Sakagami法评分;③胃黏膜内TLR4、MyD88、NF-κB p65、Foxp3蛋白检测:采用免疫组织化学染色法检测;④胃黏膜内TLR4和MyD88 mRNA检测:采用RT-PCR方法检测;⑤血清抗H. pylori IgG含量的检测:采用间接ELISA法检测。 结果: 1. H. pylori全菌蛋白抗原壳聚糖微球的制备及体外释放特性研究 从32种壳聚糖微球制备方案中筛选出了以海得贝壳聚糖为原料、冰乙酸的浓度为1%、硫酸钠为沉淀剂、pH值为5.0、不进行超声处理的最佳制备方案;经扫描电镜(SEM)示微球光滑圆整、致密,粒径分布在1.0-5.0μm;抗原与微球的量比为1:5时包裹率最大,包裹时间为3h时包裹率可达最大,抗原包裹率为79.92%,包裹量为16.47%;体外释放实验表明,总抗原释放率为20.39%,呈缓慢释放状态。 2. Tim-3阻断对H. pylori感染免疫致病和疫苗免疫保护作用影响及机制的研究 (1)两种佐剂的H. pylori疫苗组预先给予Tim-3单抗阻断小鼠胃黏膜内H. pylori定植密度均低于未阻断小鼠(P0.05),与正常对照组无差别(P0.05);不同佐剂的H. pylori疫苗接种小鼠的H. pylori定植密度无差别(P0.05)。 (2)H. pylori活菌接种后胃黏膜内H. pylori定植密度显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断对H. pylori定植密度无影响(P0.05)。 (3)两种佐剂的H. pylori疫苗接种小鼠胃粘膜炎症程度均高于正常对照组(P0.05,0.001),预先给予Tim-3单抗阻断小鼠胃黏膜炎症程度均高于未阻断小鼠(P0.05,0.01),不同佐剂的H. pylori疫苗接种小鼠胃黏膜炎症程度无差别(P0.05)。 (4)H.pylori活菌接种后胃黏膜炎症程度显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断小鼠胃黏膜炎症程度显著高于未阻断小鼠(P0.05)。 (5)两种佐剂的H. pylori疫苗接种小鼠胃黏膜内TLR4 mRNA的表达和TLR4阳性细胞积分均显著高于正常对照组(P0.05,0.001),预先给予Tim-3单抗阻断小鼠胃黏膜内TLR4阳性细胞积分均显著高于未阻断小鼠(P0.05,0.01,0.001),在以壳聚糖微球为佐剂的H. pylori疫苗组TLR4 mRNA的表达在Tim-3单抗阻断组显著高于未阻断组(P0.001),而在以CT为佐剂小鼠Tim-3单抗阻断组虽高于未阻断组,但无统计学差别(P0.05)。不同佐剂的H. pylori疫苗接种小鼠胃黏膜内TLR4阳性细胞积分无差别(P0.05),TLR4 mRNA表达在无Tim-3单抗阻断小鼠,在以CT为佐剂组显著高于以壳聚糖微球为佐剂组(P0.05),但在Tim-3单抗阻断小鼠二组之间无差别(P0.05)。 (6)H. pylori活菌接种后胃黏膜内TLR4 mRNA的表达和TLR4阳性细胞积分均显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断小鼠胃黏膜TLR4 mRNA的表达和TLR4阳性细胞积分显著高于未阻断小鼠(P0.05,0.001)。 (7)两种佐剂的H. pylori疫苗接种小鼠胃黏膜内MyD88 mRNA的表达和MyD88阳性细胞积分均显著高于正常对照组(P0.05,0.001),预先给予Tim-3单抗阻断小鼠胃黏膜内MyD88 mRNA的表达和MyD88阳性细胞积分均显著高于未阻断小鼠(P0.05,0.001)。不同佐剂的H. pylori疫苗接种小鼠胃黏膜内MyD88阳性细胞积分无差别(P0.05),MyD88 mRNA表达在无Tim-3单抗阻断小鼠,在以CT为佐剂组显著高于以壳聚糖微球为佐剂组(P0.05),但在Tim-3单抗阻断小鼠二组之间无差别(P0.05)。 (8)H. pylori活菌接种后胃黏膜内MyD88 mRNA的表达和MyD88阳性细胞积分均显著高于正常对照组(P0.001,0.01),预先给予Tim-3单抗阻断小鼠胃黏膜MyD88 mRNA的表达和MyD88阳性细胞积分显著高于未阻断小鼠(P0.01,0.001)。 (9)两种佐剂的H. pylori疫苗接种小鼠胃黏膜内Foxp3阳性细胞百分比均显著高于正常对照组(P0.05,0.001),预先给予Tim-3单抗阻断小鼠胃黏膜内Foxp3阳性细胞百分比显著低于未阻断小鼠(P0.001)。不同佐剂的H. pylori疫苗接种小鼠胃黏膜内Foxp3阳性细胞百分比无差别(P0.05)。 (10)H. pylori活菌接种后胃黏膜内Foxp3阳性细胞百分比显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断小鼠胃黏膜内Foxp3阳性细胞百分比显著低于未阻断小鼠(P0.001)。 (11)两种佐剂的H. pylori疫苗接种小鼠胃黏膜内NF-κBp65阳性细胞积分均显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断小鼠胃黏膜内NF-κBp65阳性细胞积分高于未阻断小鼠(P0.01)。不同佐剂的H. pylori疫苗接种小鼠胃黏膜内NF-κBp65阳性细胞积分无差别(P0.05)。 (12)H. pylori活菌接种后胃黏膜内NF-κBp65阳性细胞积分均显著高于正常对照组(P0.001,0.01),预先给予Tim-3单抗阻断小鼠胃黏膜内NF-κBp65阳性细胞积分高于未阻断小鼠(P0.001)。 (13)两种佐剂的H. pylori疫苗接种小鼠血清抗H. pylori IgG含量均显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断对小鼠血清抗H. pylori IgG含量无影响(P0.05)。进一步分析不同佐剂的H. pylori疫苗接种小鼠血清抗H. pyloriIgG含量无差别(P0.05)。 (14) H. pylori活菌接种小鼠血清抗H. pylori IgG含量均显著高于正常对照组(P0.001),预先给予Tim-3单抗阻断对小鼠血清抗H. pylori IgG含量无影响(P0.05)。进一步分析H. pylori疫苗接种小鼠血清抗H. pylori IgG含量均显著高于H. pylori感染小鼠(P0.05)。 结论: 1.本研究筛选的壳聚糖微球制备条件所制备的壳聚糖微球对H. pylori全菌蛋白抗原具有较高的包裹率和较好的控释效果。 2. Tim-3阻断可提高H. pylori疫苗的免疫保护率,但并不降低H. pylori自然感染小鼠胃黏膜内H. pylori定植密度。 3. Tim-3阻断可加剧H. pylori疫苗接种小鼠和H. pylori感染小鼠胃黏膜炎症程度。 4. Tim-3阻断可上调TLR4、MyD88的表达和促进NF-κB活化,降低CD4+CD25+Foxp3+Treg的数量,这可能是它增强H .pylori疫苗的免疫保护作用的机制,但并不影响H. pylori自然感染小鼠胃黏膜内H. pylori定植密度。 5.以壳聚糖微球为佐剂的H. pylori疫苗与传统的佐剂CT为佐剂的H. pylori疫苗对H. pylori感染具有同样的免疫保护作用。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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