本文選題：粘膜免疫 + HMGB1�。� 參考：《蘇州大學(xué)》2012年碩士論文

【摘要】：病毒性心肌炎（viral myocarditis，VMC）是一種臨床常見的心血管系統(tǒng)疾病。由柯薩奇病毒、埃可(ECHO)、脊髓灰質(zhì)炎、腺病毒40、41，流感病毒等多種病毒感染引起的心肌局限性或彌漫性的急性或慢性炎癥病變，屬于感染性心肌疾病。其中以腸道病毒，特別是柯薩奇病毒B3型感染最為常見。目前尚無根治病毒感染的有效治療方法，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主要經(jīng)胃腸道粘膜入侵機(jī)體，誘導(dǎo)有效的粘膜免疫應(yīng)答對于有效控制病毒感染及后續(xù)心肌炎發(fā)病具有重要意義。考慮到粘膜疫苗是誘導(dǎo)粘膜免疫應(yīng)答的最有效方式，因此制備有效的CVB3特異性粘膜疫苗成為病毒性心肌炎防治的重要策略。 我們實(shí)驗(yàn)室前期制備了編碼CVB3優(yōu)勢抗原VP1的真核表達(dá)質(zhì)粒pVP1，通過滴鼻免疫小鼠,誘生了免疫應(yīng)答，保護(hù)了30%多的感染CVB3的小鼠免于死亡。然而，其免疫效果尚需進(jìn)一步加強(qiáng)，如何提高該疫苗的保護(hù)效應(yīng)成為我們后續(xù)研究的重點(diǎn)。HMGB1存在于真核細(xì)胞核內(nèi)的非組蛋白染色體結(jié)合蛋白，因其在聚丙烯酞胺凝膠電泳(PAGE)中遷移速度快而得名。作為新型的粘膜免疫佐劑，與編碼CVB3重要保護(hù)性抗原VP1質(zhì)粒相混合然后用chitosan包裹進(jìn)行滴鼻免疫小鼠。為了檢驗(yàn)疫苗引發(fā)的全身免疫反應(yīng)。收集免疫小鼠的血清行ELISA檢測。結(jié)果顯示：與單獨(dú)免疫組相比，，HMGB1共免疫組誘生了全身性特異性IgG，同時為了驗(yàn)證免疫反應(yīng)是Th1還是Th2反應(yīng)，我們檢測了IgG亞型IgG2a和IgG1，結(jié)果發(fā)現(xiàn)HMGB1共免疫組誘生的全身免疫反應(yīng)傾向于Th1反應(yīng)。提示HMGB1作為粘膜佐劑可以促進(jìn)免疫反應(yīng)向Th1進(jìn)行，這樣更有利于機(jī)體抵御病毒的感染。更重要的是HMGB1增加了腸道分泌IgA的能力，因CVB3是腸道病毒，所以這對病毒的清除有重要的意義。 細(xì)胞免疫對病毒的清除有著關(guān)鍵的作用，尤其是T細(xì)胞。為此我們先是檢驗(yàn)了T細(xì)胞的增殖，Brdu增殖結(jié)果表明： HMGB1共免疫組小鼠脾臟淋巴細(xì)胞（OD370nm處的吸光度為1.24）比其他組小鼠脾臟淋巴細(xì)胞（VP1組小鼠OD370nm處的吸光度為0.75；HMGB1組小鼠OD370nm處的吸光度為0.36；空載組小鼠OD370nm處的吸光度為0.25）增殖幅度明顯增加。同時腸系膜淋巴結(jié)淋巴細(xì)胞（OD370nm處的吸光度為0.72）的增殖幅度也顯著大于其他組小鼠（VP1組小鼠OD370nm處的吸光度為0.36；HMGB1組小鼠OD370nm處的吸光度為0.16；空載組小鼠OD370nm處的吸光度為0.10）腸系膜淋巴細(xì)胞的增殖。HMGB1共免疫組較其他組更能增加脾臟和腸系膜淋巴結(jié)中T細(xì)胞的增殖，為發(fā)揮免疫效應(yīng)打下了基礎(chǔ)。 T細(xì)胞免疫作用主要是CTL，而CTL對于抗病毒的作用又十分關(guān)鍵。前面結(jié)果已經(jīng)表明HMGB1能增加T細(xì)胞的增殖，那么為了檢測增殖后的作用，我們做了脾臟和淋巴結(jié)細(xì)胞的CTL，結(jié)果表明HMGB1共免疫組小鼠無論是脾臟細(xì)胞還是腸系膜淋巴結(jié)細(xì)胞的CTL作用都高于單免疫組小鼠和空載體小鼠。提示了HMGB1能有效的增強(qiáng)T細(xì)胞的CTL作用，對于增強(qiáng)機(jī)體抵御病毒的能力有重要作用。 淋巴細(xì)胞增殖后發(fā)揮效應(yīng)作用，IFN-γ是效應(yīng)細(xì)胞分泌的一種清除病毒的最重要的細(xì)胞因子。為了驗(yàn)證HMGB1作為新型的粘膜佐劑對于T細(xì)胞的IFN-γ分泌量的影響，我們檢測了脾臟細(xì)胞和腸系膜淋巴結(jié)細(xì)胞的IFN-γ的分泌量。ELISPOT結(jié)果顯示HGMB1共免疫組小鼠不僅脾臟細(xì)胞IFN-γ+T細(xì)胞的頻率（304SFC/106）高于其他組小鼠脾臟細(xì)胞IFN-γ+T細(xì)胞的頻率（VP1組小鼠188SFC/106；HMGB1組小鼠46SFC/106；空載體組小鼠9SFC/106），而且腸系膜淋巴結(jié)細(xì)胞HMGB1共免疫組IFN-γ+T細(xì)胞的頻率（128SFC/106）也顯著高于其他組（VP1組小鼠70SFC/106；HMGB1組小鼠31SFC/106；空載體組小鼠8SFC/106）IFN-γ+T細(xì)胞的頻率，提示HMGB1能顯著增加淋巴細(xì)胞分泌IFN-γ的能力，加強(qiáng)疫苗更好的發(fā)揮作用。 疫苗的主要作用就是預(yù)防作用，因此為了評估疫苗的保護(hù)效果我們將一批小鼠分成四組分別進(jìn)行滴鼻免疫小鼠，末次免疫2周后以致死劑量的CVB3腹腔注射小鼠，觀察28天生存率。結(jié)果是HMGB1共免疫組小鼠的生存率80%，而VP1組小鼠的生存率為60%左右，但是空載體組小鼠在攻毒后不到10天全部死亡。說明了HMGB1作為粘膜佐劑能有效增強(qiáng)機(jī)體抵抗病毒的免疫反應(yīng)，強(qiáng)化了疫苗的保護(hù)效果。同時心臟病理切片也可以看到HMGB1共免疫組小鼠幾乎無炎癥細(xì)胞侵潤，壞死的細(xì)胞也比較少，心肌結(jié)構(gòu)幾乎是完整的。而其他組心臟病理切片可以看到有大了炎癥細(xì)胞侵潤，還有壞死的細(xì)胞，心肌結(jié)構(gòu)的輪轂已經(jīng)不清晰了。說明HMGB1作為粘膜佐劑加速了體內(nèi)清除病毒的能力，有效地預(yù)防了小鼠嚴(yán)重心肌炎疾病的發(fā)生。 為了進(jìn)一步了解HMGB1做為粘膜佐劑所起的作用機(jī)制探討,我們?nèi)∶庖吆笮∈蟮钠⑴K和腸系膜淋巴結(jié)細(xì)胞進(jìn)行CD80和CD11c雙染進(jìn)行流式細(xì)胞儀檢測。結(jié)果顯示共免疫組小鼠無論是脾臟細(xì)胞還是腸系膜淋巴結(jié)細(xì)胞的DC的成熟度及數(shù)量都高于其他組。說明HMGB1可以促進(jìn)DC的成熟，進(jìn)而可以增加對于病毒的清除能力。 本研究中，我們探討了HMGB1作為粘膜免疫佐劑對CVB3VP1疫苗發(fā)揮了重要的作用。從HMGB1能增強(qiáng)全身免疫應(yīng)答到增強(qiáng)粘膜局部免疫應(yīng)答，提示HMGB1對免疫應(yīng)答很重要。那么就檢測了HMGB1對于淋巴細(xì)胞的增殖作用，結(jié)果HMGB1在脾臟和腸系膜淋巴結(jié)處的淋巴細(xì)胞都發(fā)生了增殖作用，同時HMGB1又促進(jìn)了CD8+T細(xì)胞的CTL作用和增加效應(yīng)細(xì)胞分泌IFN-γ的量。這些結(jié)果提示HMGB1為新型的疫苗研發(fā)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳至善!200052上海,熊顯華!200052上海;腎癌細(xì)胞增殖活性與預(yù)后的關(guān)系[J];臨床腫瘤學(xué)雜志;1998年02期

2 張秀榮,孟如松

本文編號：2107869