PHEV感染對樹突狀細胞的活化及其細胞疫苗免疫保護作用
發(fā)布時間：2018-07-14 14:14
【摘要】：豬血凝性腦脊髓炎病毒（Porcine hemagglutinating encephalomyelitis virus，PHEV）屬于β冠狀病毒屬成員，其主要侵害3周齡以內的仔豬，臨床上常常表現為兩種類型，即嘔吐、衰竭或/和明顯的神經癥狀，死亡率20%～100%。臨床上，PHEV多呈隱性感染，在豬群中不易察覺。盡管成年豬能夠較好的抵抗PHEV，但感染后會出現消瘦和發(fā)育不良等癥狀，且易于繼發(fā)其他病原的混合感染。目前，PHEV已在世界范圍內廣泛傳播，本課題組調查結果顯示，我國吉林、遼寧、山東等省份的豬群中PHEV感染率均為50%左右。研究表明，PHEV感染小鼠后通過神經傳播，由于其發(fā)病急，機體的獲得性免疫應答并未完全啟動，感染小鼠即已死亡，死亡率幾乎為100%。但在該病毒感染初期是否能被機體的免疫細胞所識別，目前還不清楚。樹突狀細胞（Dendritic cells，DC）是目前已知的處理呈遞抗原能力最強的抗原提呈細胞（Antigen presenting cells，APCs）。對其他多種病毒感染的研究資料證實，DC能夠識別并呈遞病毒蛋白，分泌Ⅰ型干擾素及炎性細胞因子，以激活機體免疫系統(tǒng)清除并殺傷病毒感染細胞。但PHEV能否刺激DC活化，并且活化的DC對機體有無保護力未曾研究。 為了解PHEV刺激對DC活化的影響，以及DC在PHEV感染過程中參與免疫應答的能力，本研究首先分離了Balb\c小鼠骨髓細胞，并通過條件培養(yǎng)基誘導培養(yǎng)8天，獲得純度約77.69%的DC。將PHEV接種DC后，觀察到細胞體積變大、產生大量突起、貼壁能力強，說明細胞成熟、吞噬及黏附能力增強。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分泌量達到對照組的12倍；TNF-α持續(xù)高水平分泌，約為800～1000pg/mL，為對照組的16倍，差異極顯著；表明DC的免疫調節(jié)能力處于較高水平。FACS檢測到DC的CD40、CD86、MHC Ⅱ等表達也均上調25%左右，說明DC呈遞抗原、刺激活化T細胞的能力增強。qPCR和Western Blotting結果證明NF κB和炎性復合體通路活化；但IFN-α、β的表達被抑制，24h和48h的表達量不到對照組的10%，TLR7的表達也同樣被抑制。上述研究結果說明PHEV在體外能有效的活化DC并促使其成熟為有效的抗原提呈細胞，具有潛在的激活淋巴細胞反應的能力，為機體提供有效免疫保護。但被激活的DC并不能通過Ⅰ型干擾素途徑達到抗病毒反應。 為了確定活化、成熟的DC對小鼠是否具有免疫保護作用，以負載PHEV的DC作為細胞疫苗免疫小鼠，檢測到免疫小鼠血清中的細胞因子TNF-α、IL-6和IFN-γ上調1倍以上；血清PHEV抗體均呈陽性，說明小鼠體液免疫系統(tǒng)被激活。FACS分析免疫小鼠的脾臟細胞發(fā)現，無論是負載滅活病毒還是活病毒的DC疫苗經過兩次免疫后，CD4/CD8均比第一次免疫后高接近1倍，IFN-γ分泌T細胞數量是對照組的1.5倍，說明小鼠的細胞免疫系統(tǒng)也同時被活化。兩次加強免疫后，滴鼻接種致死劑量的PHEV，14天內負載PHEV的DC組死亡率為25%，而負載滅活PHEV的DC組死亡率僅為20%，對照組則幾乎為100%，說明負載病毒的DC能夠為小鼠提供一定的免疫保護力抵抗PHEV感染。 由此可見，PHEV能夠激活DC，促使其成熟、活化，表面蛋白表達，細胞因子分泌及NF κB和炎性復合體通路信號激活；負載病毒的DC疫苗能夠在一定程度上對小鼠提供免疫保護力。這些研究成果不僅為DC參與PHEV感染過程中的免疫應答機制奠定基礎，而且也為開發(fā)DC細胞疫苗抵抗PHEV感染提供了重要的參考依據。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.3
本文編號：2121904
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.3
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本文編號：2121904
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