HMGB1对病毒性心肌炎DNA粘膜疫苗的免疫增强作用及其机制

发布时间：2018-07-08 15:26

本文选题：粘膜免疫 + HMGB1　；参考：《苏州大学》2012年硕士论文

【摘要】：病毒性心肌炎（viral myocarditis，VMC）是一种临床常见的心血管系统疾病。由柯萨奇病毒、埃可(ECHO)、脊髓灰质炎、腺病毒40、41，流感病毒等多种病毒感染引起的心肌局限性或弥漫性的急性或慢性炎症病变，属于感染性心肌疾病。其中以肠道病毒，特别是柯萨奇病毒B3型感染最为常见。目前尚无根治病毒感染的有效治疗方法，因此新型、高效的病毒性心肌炎疫苗的研制十分必要。由于CVB3主要经胃肠道粘膜入侵机体，诱导有效的粘膜免疫应答对于有效控制病毒感染及后续心肌炎发病具有重要意义。考虑到粘膜疫苗是诱导粘膜免疫应答的最有效方式，因此制备有效的CVB3特异性粘膜疫苗成为病毒性心肌炎防治的重要策略。我们实验室前期制备了编码CVB3优势抗原VP1的真核表达质粒pVP1，通过滴鼻免疫小鼠,诱生了免疫应答，保护了30%多的感染CVB3的小鼠免于死亡。然而，其免疫效果尚需进一步加强，如何提高该疫苗的保护效应成为我们后续研究的重点。HMGB1存在于真核细胞核内的非组蛋白染色体结合蛋白，因其在聚丙烯酞胺凝胶电泳(PAGE)中迁移速度快而得名。作为新型的粘膜免疫佐剂，与编码CVB3重要保护性抗原VP1质粒相混合然后用chitosan包裹进行滴鼻免疫小鼠。为了检验疫苗引发的全身免疫反应。收集免疫小鼠的血清行ELISA检测。结果显示：与单独免疫组相比，，HMGB1共免疫组诱生了全身性特异性IgG，同时为了验证免疫反应是Th1还是Th2反应，我们检测了IgG亚型IgG2a和IgG1，结果发现HMGB1共免疫组诱生的全身免疫反应倾向于Th1反应。提示HMGB1作为粘膜佐剂可以促进免疫反应向Th1进行，这样更有利于机体抵御病毒的感染。更重要的是HMGB1增加了肠道分泌IgA的能力，因CVB3是肠道病毒，所以这对病毒的清除有重要的意义。细胞免疫对病毒的清除有着关键的作用，尤其是T细胞。为此我们先是检验了T细胞的增殖，Brdu增殖结果表明： HMGB1共免疫组小鼠脾脏淋巴细胞（OD370nm处的吸光度为1.24）比其他组小鼠脾脏淋巴细胞（VP1组小鼠OD370nm处的吸光度为0.75；HMGB1组小鼠OD370nm处的吸光度为0.36；空载组小鼠OD370nm处的吸光度为0.25）增殖幅度明显增加。同时肠系膜淋巴结淋巴细胞（OD370nm处的吸光度为0.72）的增殖幅度也显著大于其他组小鼠（VP1组小鼠OD370nm处的吸光度为0.36；HMGB1组小鼠OD370nm处的吸光度为0.16；空载组小鼠OD370nm处的吸光度为0.10）肠系膜淋巴细胞的增殖。HMGB1共免疫组较其他组更能增加脾脏和肠系膜淋巴结中T细胞的增殖，为发挥免疫效应打下了基础。 T细胞免疫作用主要是CTL，而CTL对于抗病毒的作用又十分关键。前面结果已经表明HMGB1能增加T细胞的增殖，那么为了检测增殖后的作用，我们做了脾脏和淋巴结细胞的CTL，结果表明HMGB1共免疫组小鼠无论是脾脏细胞还是肠系膜淋巴结细胞的CTL作用都高于单免疫组小鼠和空载体小鼠。提示了HMGB1能有效的增强T细胞的CTL作用，对于增强机体抵御病毒的能力有重要作用。淋巴细胞增殖后发挥效应作用，IFN-γ是效应细胞分泌的一种清除病毒的最重要的细胞因子。为了验证HMGB1作为新型的粘膜佐剂对于T细胞的IFN-γ分泌量的影响，我们检测了脾脏细胞和肠系膜淋巴结细胞的IFN-γ的分泌量。ELISPOT结果显示HGMB1共免疫组小鼠不仅脾脏细胞IFN-γ+T细胞的频率（304SFC/106）高于其他组小鼠脾脏细胞IFN-γ+T细胞的频率（VP1组小鼠188SFC/106；HMGB1组小鼠46SFC/106；空载体组小鼠9SFC/106），而且肠系膜淋巴结细胞HMGB1共免疫组IFN-γ+T细胞的频率（128SFC/106）也显著高于其他组（VP1组小鼠70SFC/106；HMGB1组小鼠31SFC/106；空载体组小鼠8SFC/106）IFN-γ+T细胞的频率，提示HMGB1能显著增加淋巴细胞分泌IFN-γ的能力，加强疫苗更好的发挥作用。疫苗的主要作用就是预防作用，因此为了评估疫苗的保护效果我们将一批小鼠分成四组分别进行滴鼻免疫小鼠，末次免疫2周后以致死剂量的CVB3腹腔注射小鼠，观察28天生存率。结果是HMGB1共免疫组小鼠的生存率80%，而VP1组小鼠的生存率为60%左右，但是空载体组小鼠在攻毒后不到10天全部死亡。说明了HMGB1作为粘膜佐剂能有效增强机体抵抗病毒的免疫反应，强化了疫苗的保护效果。同时心脏病理切片也可以看到HMGB1共免疫组小鼠几乎无炎症细胞侵润，坏死的细胞也比较少，心肌结构几乎是完整的。而其他组心脏病理切片可以看到有大了炎症细胞侵润，还有坏死的细胞，心肌结构的轮毂已经不清晰了。说明HMGB1作为粘膜佐剂加速了体内清除病毒的能力，有效地预防了小鼠严重心肌炎疾病的发生。为了进一步了解HMGB1做为粘膜佐剂所起的作用机制探讨,我们取免疫后小鼠的脾脏和肠系膜淋巴结细胞进行CD80和CD11c双染进行流式细胞仪检测。结果显示共免疫组小鼠无论是脾脏细胞还是肠系膜淋巴结细胞的DC的成熟度及数量都高于其他组。说明HMGB1可以促进DC的成熟，进而可以增加对于病毒的清除能力。本研究中，我们探讨了HMGB1作为粘膜免疫佐剂对CVB3VP1疫苗发挥了重要的作用。从HMGB1能增强全身免疫应答到增强粘膜局部免疫应答，提示HMGB1对免疫应答很重要。那么就检测了HMGB1对于淋巴细胞的增殖作用，结果HMGB1在脾脏和肠系膜淋巴结处的淋巴细胞都发生了增殖作用，同时HMGB1又促进了CD8+T细胞的CTL作用和增加效应细胞分泌IFN-γ的量。这些结果提示HMGB1为新型的疫苗研发提供了很大可能。
[Abstract]:Viral myocarditis (VMC) is a common clinical disease of cardiovascular system. Myocardial limitation or diffuse acute or chronic inflammatory disease caused by Coxsackie virus (ECHO), poliomyelitis, adenovirus 40,41, influenza virus and other virus infections, belongs to infectious myocardial disease. In particular, Coxsackie virus type B3 infection is the most common. There is no effective treatment to cure the virus infection. Therefore, the development of a new and efficient viral myocarditis vaccine is necessary. The effective mucosal immunity should be induced to effectively control viral infection and subsequent myocarditis due to the invasion of CVB3 mainly through the gastric and intestinal mucosa. The pathogenesis is of great significance. Considering that the mucosal vaccine is the most effective way to induce the mucosal immune response, the preparation of an effective CVB3 specific mucosal vaccine has become an important strategy for the prevention and treatment of viral myocarditis.
In our laboratory, the eukaryotic expression plasmid pVP1 encoding CVB3 predominant antigen VP1 was prepared. The immune response was induced by intranasal immunity to mice, and more than 30% of the mice infected with CVB3 were protected from death. However, the immune effect needed to be further strengthened. How to improve the protective effect of the vaccine has become the focus of our follow-up study. The non histone binding protein, which exists in the eukaryotic cell nucleus, is named because of its rapid migration rate in polyacrylamide gel electrophoresis (PAGE). As a new mucosal immune adjuvant, it is mixed with the CVB3 important protective antigen VP1 plasmid and then enwrapped chitosan in the dripping nose to immunize mice. The body immunoreaction. The serum ELISA test of the immune mice was collected. The results showed that the HMGB1 co immunization group induced the systemic specific IgG compared with the individual immune group, and in order to verify the immune response was Th1 or Th2 reaction, we detected the IgG subtype IgG2a and IgG1, and found the tendency of systemic immune response induced by the HMGB1 co immunization group. As a mucosal adjuvant, it is suggested that HMGB1 as a mucosal adjuvant can promote the immune response to Th1, which is more beneficial to the body against virus infection. More importantly, HMGB1 increases the ability to secrete IgA in the intestinal tract, because CVB3 is an enterovirus, so it is of great significance for the clearance of the virus.
Cell immunity plays a key role in the clearance of the virus, especially T cells. For this reason, we first examined the proliferation of T cells, and the Brdu proliferation results showed that the spleen lymphocytes of the HMGB1 co immunized mice (the absorbance of OD370nm at 1.24) was more than the other group of spleen lymphocytes (the absorbance of OD370nm at VP1 mice was 0.75; HMGB1). The absorbance of the mice at OD370nm was 0.36, the absorbency of the OD370nm in the no-load group was 0.25) and the proliferation amplitude was significantly increased, and the proliferation of the mesenteric lymph node lymphocyte (the absorbance of OD370nm at 0.72) was significantly greater than that of the other groups (the absorbance of OD370nm at VP1 in group VP1 was 0.36, and the absorption of OD370nm in group HMGB1 mice). The luminosity was 0.16, the absorbance at OD370nm in the no-load group was 0.10) and the proliferation of the mesenteric lymphocyte proliferation.HMGB1 co immunization group increased the proliferation of T cells in the spleen and mesenteric lymph nodes, which laid the foundation for the immune effect.
The immunization of T cells is mainly CTL, and the effect of CTL on antiviral activity is crucial. The previous results have shown that HMGB1 can increase the proliferation of T cells. In order to detect the effect of proliferation, we have done CTL in the spleen and lymph node cells. The results showed that the HMGB1 co immunization group was the spleen cells and the mesenteric lymph node cells. The effect of CTL is higher than that of mice in the single immunization group and the empty carrier mice. It suggests that HMGB1 can effectively enhance the CTL effect of T cells and play an important role in enhancing the ability of the body to resist the virus.
After lymphocyte proliferation, IFN- gamma is the most important cytokine secreted by the effector cells. In order to verify the effect of HMGB1 as a new mucosal adjuvant on the IFN- gamma secretion of T cells, we detected the secretion of IFN- gamma in the spleen cells and mesenteric lymph node cells,.ELISPOT results showed HGM In B1 co immunization group, the frequency of IFN- gamma +T cells in spleen cells (304SFC/106) was higher than that of IFN- gamma +T cells in the spleen cells of other groups (VP1 group 188SFC/106, HMGB1 group 46SFC/106, no-load group mice 9SFC/106), and the frequency of the mesenteric lymph node cell HMGB1 co immunization group also showed the frequency. It is higher than other groups (group VP1 mice 70SFC/106, HMGB1 group 31SFC/106, 8SFC/106) IFN- gamma +T cells, suggesting that HMGB1 can significantly increase the ability of lymphocyte to secrete IFN- gamma and enhance the effect of the vaccine.
The main effect of the vaccine was the preventive effect, so in order to evaluate the protective effect of the vaccine, we divided a group of mice into four groups to immunize mice in four groups. After 2 weeks of last immunization, the mice were intraperitoneally injected with lethal dose of CVB3 to observe the natural survival rate of 28. The survival rate of the HMGB1 co immunization group was 80%, and the survival rate of the VP1 group. It was about 60%, but the mice in the empty body group died less than 10 days after the attack. It shows that HMGB1 as a mucosal adjuvant can effectively enhance the immune response of the body against the virus and strengthen the protective effect of the vaccine. At the same time, the pathological sections of the heart can also see that the mice of the HMGB1 co immunization group are not invaded by inflammatory cells, and the necrotic cells are also compared. Less, myocardial structure is almost complete. And other groups of heart pathological sections can see that there are large inflammatory cells embellish, and necrotic cells, and the hub of the myocardial structure is not clear. It shows that HMGB1 as a mucosal adjuvant accelerates the ability to clear the virus in the body, effectively preventing the occurrence of severe myocarditis in mice.
In order to further understand the mechanism of HMGB1 as a mucosal adjuvant, we examined the spleen and mesenteric lymph node cells of the spleen and mesenteric lymph nodes of the immunized mice by CD80 and CD11c double staining. The results showed that the maturity and number of DC in the co immunized mice was higher than that of the spleen cells or the mesenteric lymph node cells. In other groups, it indicates that HMGB1 can promote the maturation of DC and increase the ability of virus clearance.
In this study, we discussed that HMGB1 plays an important role in CVB3VP1 vaccine as a mucosal immune adjuvant. From HMGB1, it can enhance the systemic immune response to the enhancement of local mucosal immune response, suggesting that HMGB1 is important to the immune response. Then, the proliferation of HMGB1 to lymphocytes is detected, and HMGB1 is in the spleen and mesenteric lymph nodes. The lymphocytes in the junction were proliferating, and HMGB1 also promoted the CTL action of CD8+T cells and increased the amount of IFN- gamma secreted by the effector cells. These results suggest that HMGB1 provides a great possibility for the development of a new vaccine.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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