PHEV感染对树突状细胞的活化及其细胞疫苗免疫保护作用

发布时间：2018-07-14 14:14

【摘要】：猪血凝性脑脊髓炎病毒（Porcine hemagglutinating encephalomyelitis virus，PHEV）属于β冠状病毒属成员，其主要侵害3周龄以内的仔猪，临床上常常表现为两种类型，即呕吐、衰竭或/和明显的神经症状，死亡率20%～100%。临床上，PHEV多呈隐性感染，在猪群中不易察觉。尽管成年猪能够较好的抵抗PHEV，但感染后会出现消瘦和发育不良等症状，且易于继发其他病原的混合感染。目前，PHEV已在世界范围内广泛传播，本课题组调查结果显示，我国吉林、辽宁、山东等省份的猪群中PHEV感染率均为50%左右。研究表明，PHEV感染小鼠后通过神经传播，由于其发病急，机体的获得性免疫应答并未完全启动，感染小鼠即已死亡，死亡率几乎为100%。但在该病毒感染初期是否能被机体的免疫细胞所识别，目前还不清楚。树突状细胞（Dendritic cells，DC）是目前已知的处理呈递抗原能力最强的抗原提呈细胞（Antigen presenting cells，APCs）。对其他多种病毒感染的研究资料证实，DC能够识别并呈递病毒蛋白，分泌Ⅰ型干扰素及炎性细胞因子，以激活机体免疫系统清除并杀伤病毒感染细胞。但PHEV能否刺激DC活化，并且活化的DC对机体有无保护力未曾研究。为了解PHEV刺激对DC活化的影响，以及DC在PHEV感染过程中参与免疫应答的能力，本研究首先分离了Balb\c小鼠骨髓细胞，并通过条件培养基诱导培养8天，获得纯度约77.69%的DC。将PHEV接种DC后，观察到细胞体积变大、产生大量突起、贴壁能力强，说明细胞成熟、吞噬及黏附能力增强。PHEV刺激DC后可分泌大量的炎性因子，如TNF-α、IL-1β、IL-10和IL-12等，，其中IL-1β在24h分泌量达到对照组的12倍；TNF-α持续高水平分泌，约为800～1000pg/mL，为对照组的16倍，差异极显著；表明DC的免疫调节能力处于较高水平。FACS检测到DC的CD40、CD86、MHC Ⅱ等表达也均上调25%左右，说明DC呈递抗原、刺激活化T细胞的能力增强。qPCR和Western Blotting结果证明NF κB和炎性复合体通路活化；但IFN-α、β的表达被抑制，24h和48h的表达量不到对照组的10%，TLR7的表达也同样被抑制。上述研究结果说明PHEV在体外能有效的活化DC并促使其成熟为有效的抗原提呈细胞，具有潜在的激活淋巴细胞反应的能力，为机体提供有效免疫保护。但被激活的DC并不能通过Ⅰ型干扰素途径达到抗病毒反应。为了确定活化、成熟的DC对小鼠是否具有免疫保护作用，以负载PHEV的DC作为细胞疫苗免疫小鼠，检测到免疫小鼠血清中的细胞因子TNF-α、IL-6和IFN-γ上调1倍以上；血清PHEV抗体均呈阳性，说明小鼠体液免疫系统被激活。FACS分析免疫小鼠的脾脏细胞发现，无论是负载灭活病毒还是活病毒的DC疫苗经过两次免疫后，CD4/CD8均比第一次免疫后高接近1倍，IFN-γ分泌T细胞数量是对照组的1.5倍，说明小鼠的细胞免疫系统也同时被活化。两次加强免疫后，滴鼻接种致死剂量的PHEV，14天内负载PHEV的DC组死亡率为25%，而负载灭活PHEV的DC组死亡率仅为20%，对照组则几乎为100%，说明负载病毒的DC能够为小鼠提供一定的免疫保护力抵抗PHEV感染。由此可见，PHEV能够激活DC，促使其成熟、活化，表面蛋白表达，细胞因子分泌及NF κB和炎性复合体通路信号激活；负载病毒的DC疫苗能够在一定程度上对小鼠提供免疫保护力。这些研究成果不仅为DC参与PHEV感染过程中的免疫应答机制奠定基础，而且也为开发DC细胞疫苗抵抗PHEV感染提供了重要的参考依据。
[Abstract]:Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus beta coronavirus, which mainly infringe on the piglets within 3 weeks of age. In clinical, it often shows two types, namely, vomiting, failure or / and obvious neurologic symptoms. The death rate is 20% to 100%., and the PHEV is mostly recessive and in the pig group. It is not easy to detect. Although adult pigs are able to resist PHEV better, the symptoms of emaciation and dysplasia will occur after infection, and it is easy to secondary infection of other pathogens. At present, PHEV has been widely spread worldwide. The results of this research group showed that the rate of PHEV infection in the pigs of Jilin, Liaoning, Shandong and other provinces of our country was 50% The study showed that PHEV infected mice through the nerve, because of its acute onset, the body's acquired immune response was not fully activated, the infected mice were dead, the mortality rate was almost 100%., but it is not clear at the beginning of the virus infection by the immune cells of the body. The Dendritic cells (DC) is not clear. Antigen presenting cells (APCs) is the most potent antigen presenting antigen (cells, APCs). Research data on many other viral infections confirm that DC can identify and present viral proteins, secrete type I interferon and inflammatory cytokines to activate the body immune system to clear and kill virus infected cells. But PHEV Whether DC activation can be stimulated, and whether the activated DC has no protective effect on the organism has not been studied.
In order to understand the effect of PHEV stimulation on the activation of DC and the ability of DC to participate in the immune response during PHEV infection, the bone marrow cells of Balbc mice were first isolated and cultured for 8 days through the conditioned medium, and the purity of DC. was obtained by inoculating PHEV to DC, and observed that the cell volume became larger, a large number of protrusions were produced and the adhesion ability was strong, and the ability of adherence was strong. The maturation of the cells, phagocytosis and adhesion ability enhanced.PHEV stimulated DC to secrete a large number of inflammatory factors, such as TNF- alpha, IL-1 beta, IL-10 and IL-12, in which the secretion of IL-1 beta in 24h reached 12 times that of the control group; TNF- alpha sustained high level secretion of about 800 ~ 1000pg/mL, 16 times the control group, the difference was very significant; indicating DC immunoregulation The expression of CD40, CD86, MHC II and other expressions of DC were also up to 25%, indicating that DC presented antigen, and the ability to activate T cells stimulated.QPCR and Western Blotting to prove that NF kappa B and inflammatory complex pathway was activated, but the expression of alpha and beta was inhibited, and the expression of CD86 and CD86 was less than that of the control group. The results show that PHEV can activate DC effectively in vitro and promote its maturation as an effective antigen presenting cell, which has the potential to activate lymphocyte reaction and provide effective immune protection for the body. But the activated DC does not reach the antiviral response through the type I interferon pathway.
In order to determine whether the mature DC had immuno protective effect on mice and immunized mice with PHEV DC as a cell vaccine, the cytokine TNF- alpha in the serum of the immune mice was detected by 1 times more than that of IL-6 and IFN- gamma, and the serum PHEV antibody was positive, indicating that the mice's humoral immune system was activated by.FACS to immunize mice. The spleen cells found that, after two times of immunization, both the CD4/CD8 and the live virus DC vaccines were nearly 1 times higher than the first immunization, and the number of IFN- gamma secreted T cells was 1.5 times that of the control group, indicating that the cell immune system of the mice was also activated at the same time. After two strong immunizations, the nasal drops inoculated to the lethal dose PHEV, 14 days. The mortality of the DC group with internal load PHEV was 25%, while the DC group with the load inactivated PHEV was only 20%, and the control group was almost 100%, indicating that the DC of the loaded virus could provide certain immune protection against PHEV infection in mice.
Thus, PHEV can activate DC to stimulate its maturation, activation, surface protein expression, cytokine secretion and activation of NF kappa B and inflammatory complex pathway signals, and the DC vaccine of the loaded virus can provide immune protection to mice to a certain extent. These results not only lay the immune response mechanism of DC in the PHEV infection process. It also provides an important reference for developing DC cell vaccine against PHEV infection.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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