LRG-47对日本血吸虫感染抗性调节的研究

发布时间：2018-05-24 13:02

本文选题：日本血吸虫 + LRG-47　；参考：《南京医科大学》2010年硕士论文

【摘要】：_p47 GTP酶家族是目前在脊椎动物中发现的分子量最小也是数量最多的干扰素(IFN)反应性GTP酶家族,已经克隆了小鼠_p47 GTP酶家族中的六个成员,分别是LRG-47、GTPI、IGTP、IRG-47、IIGP和TGTP/Mg21。研究发现,_p47 GTP酶能够特异地参与抵抗胞内细菌和原虫的感染,对病毒的清除能力较弱~[1-7]。虽然同属于_p47 GTP酶家族,但不同成员所表达的抗力具有病原体和时相特异性的特点。_P47 GTP酶家族因其成员所具有特异性防御病原体的能力而引起学者的广泛关注。然而,_p47 GTP酶在更为复杂的多细胞生物,例如重要的胞外寄生蠕虫—血吸虫感染中的作用知之甚少。本实验室前期研究发现~[8],_p47 GTP酶随日本血吸虫感染进程,信号强度呈下调趋势,且家族中各成员的表达强度存在明显差异。我们的前期研究还发现~[9],在日本血吸虫感染6周(w)后,IGTP KO小鼠和IRG-47 KO小鼠在感染/致病结局、整体/局部的免疫应答等方面均存在较大的差异。这提示不同_p47 GTP酶不仅参与胞内生物的感染,亦参与胞外多细胞生物的感染,而且各成员在多细胞生物感染中的作用可能不尽相同。日本血吸虫属吸虫纲的多细胞无脊椎动物,能引起宿主复杂的感染免疫应答,可作为多细胞生物研究的重要的生物模型之一。因此,对日本血吸虫感染/致病机制的深入研究,既能为我国今后血吸虫病的防治提供理论基础,更能扩展感染免疫学的基本知识。大量文献已报道p47 GTP酶家族成员LRG-47在多数胞内感染中具有明确的抗感染作用,但是对胞外血吸虫感染的作用却不明确。本研究采用lrg47基因敲除小鼠建立日本血吸虫急性感染模型,较为系统地观察LRG-47在日本血吸虫急性感染/致病阶段所诱导的宿主免疫应答特点,以进一步深化血吸虫的感染免疫学,为血吸虫病疫苗研制提供理论基础。本研究采用lrg47基因敲除(LRG-47 KO)与野生型C57BL/6J(wild-type,WT)小鼠,建立日本血吸虫自然感染的小鼠模型。为观察感染/致病结果,在感染后6w通过胸主动脉灌注法计数成虫数、消化肝脏计数虫卵数、以及计算每克肝卵数和每对成虫产卵数;以HE染色观察肝脏的病理变化。为观察感染后宿主的免疫应答特征,以间接ELISA法检测小鼠血清中日本血吸虫特异性IgG抗体水平;以双抗夹心ELISA法检测脾脏单个核细胞培养上清中Th1/Th2型细胞因子的表达水平;以流式细胞术检测脾脏中主要免疫细胞的比例变化;采用基因芯片技术,比较分析感染后6w小鼠脾脏单个核细胞中表达基因的转录水平,并对信号强度发生2倍及以上变化的差异基因进行GO分类及pathway的显著性分析。为观察基因敲除后对巨噬细胞功能的影响,以间接ELISA法检测脾巨噬细胞培养上清中TNF-α的表达水平;以硝酸还原酶法检测培养上清中NO的水平;以RT-PCR法检测脾巨噬细胞中tnfα和inos的转录水平。最后,采用虫源性抗原SEA免疫小鼠,以间接ELISA法检测小鼠血清中日本血吸虫特异性IgG抗体,脾脏单个核细胞培养上清中Th1/Th2型细胞因子的表达水平,以及流式技术检测脾脏中主要免疫细胞的比例变化。本研究获得如下主要结果: 1、日本血吸虫感染后6w,LRG-47 KO小鼠卵荷显著少于WT小鼠,且肝脏肉芽肿反应较轻。日本血吸虫感染后6w, LRG-47 KO组小鼠体内的成虫数与正常野生型小鼠相当,而LRG-47 KO小鼠的肝脏虫卵数、每克肝卵量以及每对成虫产卵数显著低于WT组。肝脏中的虫卵肉芽肿大多为急性期肉芽肿,可见在虫卵周围,大量炎症细胞浸润,LRG-47 KO组平均肉芽肿面积和组成细胞数显著小/少于WT组。 2、日本血吸虫感染后3w和6w,就抗原特异性IgG抗体而言,LRG-47 KO小鼠血清中SEA特异性IgG抗体水平显著低于WT小鼠。随着日本血吸虫感染的进行,小鼠血清中可溶性成虫抗原(SWAP)和可溶性虫卵抗原(SEA)诱导的特异性IgG抗体持续升高。但在感染后3w和6w,LRG-47 KO组与WT组SWAP特异的IgG抗体水平均无显著性差异。而LRG-47 KO组小鼠血清中SEA特异性IgG抗体水平均显著低于WT组。 3、日本血吸虫感染后6w,LRG-47 KO小鼠的CD4+T细胞的功能被削弱。脾脏单个核细胞经虫源性抗原SWAP、SEA和有丝分裂原ConA刺激后,培养上清中细胞因子水平显示: ConA与SEA刺激下,LRG-47 KO小鼠的IFN-γ水平显著低于WT小鼠;在SWAP刺激下,LRG-47 KO小鼠的IL-4水平显著低于WT小鼠;但在SEA刺激下,LRG-47 KO小鼠的TNF-α和IL-10水平显著高于WT小鼠。感染后6w脾中主要免疫细胞的比例为:脾中B细胞、NK细胞和巨噬细胞的比例在LRG-47 KO小鼠和WT小鼠两组之间无统计学差异;主要行使特异性细胞免疫应答功能的T细胞亚群:Tc1和Treg细胞的比例在LRG-47 KO小鼠显著高于WT小鼠;Th1细胞、Th2细胞和Tc2细胞的比例在LRG-47 KO小鼠显著低于WT小鼠。 4、采用高通量基因芯片技术分析比较日本血吸虫感染后6w脾脏单个核细胞基因转录水平的差异,发现LRG-47 KO小鼠中与免疫杀伤功能相关的基因被显著上调。与WT小鼠相比,在感染后6w的LRG-47 KO小鼠脾细胞中,有780个基因信号强度增强2倍以上,866个基因信号减弱2倍以上。对差异基因的GO分类及pathway的显著性分析发现,差异基因及参与的通路主要涉及免疫杀伤相关基因及通路。 5、日本血吸虫感染后6w的巨噬细胞应答:lrg47基因缺失后,不影响巨噬细胞中炎症因子NO和TNF-α的产生潜能。在日本血吸虫感染后6w,不论是LPS刺激,还是SEA刺激,LRG-47 KO小鼠巨噬细胞产生的TNF-α和NO水平与WT小鼠均无显著性差异;tnf-α和inos基因的转录水平在两组小鼠脾巨噬细胞亦无显著性差异。 6、SEA免疫后宿主免疫应答状况:LRG-47 KO小鼠血清SEA特异性IgG抗体水平明显低于WT小鼠;在SEA刺激下脾单个核细胞分泌TNF-α、IL-4和IL-10水平显著增高。日本血吸虫可溶性虫卵抗原(SEA)免疫小鼠后,小鼠血清中SEA特异的IgG抗体持续升高;在免疫后3w,LRG-47 KO组小鼠血清中SEA特异性IgG抗体水平显著低于WT组。脾脏单个核细胞经虫源性抗原SEA或有丝分裂原ConA刺激后,培养上清中细胞因子水平显示:在SEA刺激下,LRG-47 KO小鼠与WT小鼠的IFN-γ水平无显著的统计学差异,TNF-α、IL-4和IL-10水平显著高于WT小鼠;在ConA刺激下,LRG-47 KO小鼠的IFN-γ水平显著低于WT小鼠,TNF-α水平两组之间无显著性差异,IL-4的水平显著高于WT小鼠,IL-10的水平虽高于WT小鼠,但无统计学差异。 7、SEA免疫小鼠后脾脏中主要免疫细胞的比例:SEA免疫后3w,LRG-47 KO 小鼠的B细胞、Th1细胞和Tc2细胞的比例显著低于WT小鼠;LRG-47 KO 小鼠的Th2细胞和Tc1细胞比例略低于WT小鼠,但无统计学差异。综上,日本血吸虫感染后,与野生型小鼠相比,LRG-47信号缺失导致肝脏沉积的虫卵及每对成虫产卵量显著下降,肝脏肉芽肿反应较轻;LRG-47信号缺失增强TNF-α和IL-10的表达能力,显著上调免疫杀伤效应分子颗粒酶家族和TNF家族,增强杀伤细胞的免疫杀伤功能,这可能是导致LRG-47信号缺失小鼠低卵荷的重要因素。本研究结果提示LRG-47不仅参与了胞内生物感染,也参与胞外多细胞生物(如日本血吸虫)感染的抗性形成与调节。LRG-47在日本血吸虫感染过程中起负性调节作用。
[Abstract]:The _p47 GTP enzyme family is the least molecular weight and the largest number of interferon (IFN) reactive GTP enzyme family found in vertebrates, and has cloned six members of the _p47 GTP family of mice, namely, LRG-47, GTPI, IGTP, IRG-47, IIGP and TGTP/Mg21.. The infection of protozoa, the weakly scavenging ability to the virus ~[1-7]., although the same belongs to the _p47 GTP enzyme family, but the resistance expressed by the different members is characterized by pathogen and temporal specificity. The._P47 GTP family has attracted wide attention from scholars because of its members' ability to defend the pathogen. However, the _p47 GTP enzyme is more complex. There is little knowledge about the role of multicellular organisms, such as the important exoparasitic worms - Schistosoma infection. Earlier studies in our laboratory found that ~[8], _p47 GTP enzymes were down downward with the process of Schistosoma japonicum infection, and the expression intensity of each member in the family was clearly different. Our previous study also found that ~[9], in Japan, was also found in Japan. After 6 weeks of schistosomiasis infection (W), IGTP KO mice and IRG-47 KO mice have great differences in infection / pathogenetic outcome and overall / local immune response. This suggests that different _p47 GTP enzymes not only participate in intracellular infection, but also participate in the infection of extracellular multicellular organisms, and the role of each member in multicellular biological infection may be possible. It's not the same.
The multicellular invertebrates of the genus Schistosoma of the genus Schistosoma can cause complex host immune responses and can be one of the most important biological models for multicellular organisms. Therefore, the in-depth study of the infection / pathogenesis of Schistosoma japonicum can not only provide a theoretical basis for the prevention and cure of blood sucking disease in the future, but also extend the infection more. Basic knowledge of immunology. A large number of documents have reported that the P47 GTP family member LRG-47 has a clear anti infection role in most intracellular infections, but the effect on the infection of extracellular Schistosoma is not clear. This study used lrg47 knockout mice to establish acute infection model of Schistosoma japonicum, and systematically observed the blood of LRG-47 in Japanese blood. In order to further deepen the immunology of schistosomiasis and provide a theoretical basis for the development of schistosomiasis vaccine, the characteristics of the host immune response induced by the acute infection / pathogenic stage of the parasite are further deepened.
In this study, lrg47 gene knockout (LRG-47 KO) and wild type C57BL/6J (wild-type, WT) mice were used to establish a mouse model of natural infection of Schistosoma japonicum. In order to observe the infection / pathogenic results, the number of adult worms were counted by the thoracic aorta perfusion method in 6W after infection, the number of eggs was counted in the digestive liver, and the number of eggs per gram of liver and the number of eggs per pair of adults were calculated. The pathological changes of the liver were observed by HE staining. In order to observe the immune response characteristics of the infected host, the specific IgG antibody level of Schistosoma japonicum in the serum of mice was detected by indirect ELISA, and the expression level of Th1/Th2 cytokine in the culture supernatant of the spleen cells was detected by the double anti sandwich ELISA method, and the spleens were detected by flow cytometry. The change in the proportion of the main immune cells, the gene chip technology was used to compare the transcriptional level of the expression genes in the spleen mononuclear cells of 6W mice after infection, and the GO classification and the significant analysis of the difference genes of 2 times and more of the signal intensity were carried out, and the effect of the gene knockout on the function of macrophages after knockout was observed. The expression level of TNF- alpha in the supernatant of spleen macrophage culture was detected by indirect ELISA method, the level of NO in the culture supernatant was detected by the nitrate reductase method, and the transcription level of TNF alpha and iNOS in the spleen macrophages was detected by RT-PCR method. Finally, the worm derived antigen SEA was used to immunize mice, and the specific IgG in the mice serum was detected by indirect ELISA method. Antibodies, expression levels of Th1/Th2 cytokines in culture supernatants of splenic mononuclear cells, and flow cytometry were used to detect the proportion of major immune cells in spleen.
The main results of this study are as follows:
1, after Schistosoma japonicum infection 6W, LRG-47 KO mice were significantly less than WT mice, and the liver granuloma reaction was lighter. The number of adult worms in 6W, LRG-47 KO group after Schistosoma japonicum infection was equivalent to that of normal wild type mice. The number of eggs of liver eggs per gram of LRG-47 KO mice, the number of eggs per gram of liver and the number of eggs per pair of adults were significantly lower than that of the WT group. Most of the egg granuloma in the dirty is acute stage granuloma, which can be seen around the eggs, and a large number of inflammatory cells infiltrate. The average granulomatous area and the number of the constituent cells in the LRG-47 KO group are significantly smaller / less than that of the WT group.
2, 3W and 6W after Schistosoma japonicum infection, the level of SEA specific IgG antibody in the serum of LRG-47 KO mice was significantly lower than that of WT mice in terms of the antigen specific IgG antibody. With the infection of Schistosoma japonicum, the specific IgG antibody induced by the soluble adult antigen (SWAP) and soluble egg antigen (SEA) in the serum of the mice continued to rise. There was no significant difference in the level of specific IgG antibody between 3W and 6W, LRG-47 KO and WT group SWAP, but the level of SEA specific IgG antibody in the serum of LRG-47 KO mice was significantly lower than that in the WT group.
3, the function of CD4+T cells in 6W, LRG-47 KO mice after Schistosoma japonicum infection was weakened. The level of cytokines in the culture supernatant of spleen mononuclear cells stimulated by insect derived antigen SWAP, SEA and mitogen ConA showed that the level of IFN- gamma in LRG-47 KO mice was significantly lower than that of LRG-47 KO mice under the stimulus of ConA and SEA. The IL-4 level of rats was significantly lower than that of WT mice, but the level of TNF- alpha and IL-10 in LRG-47 KO mice was significantly higher than that of WT mice under SEA stimulation. The proportion of the main immune cells in the spleen of the spleen was: the proportion of B cells in the spleen and the proportion of NK cells and macrophages in the two groups of LRG-47 mice and mice. The T cell subsets of the pestilence function: the proportion of Tc1 and Treg cells in LRG-47 KO mice was significantly higher than that of WT mice; the proportion of Th1, Th2 and Tc2 cells in LRG-47 KO mice was significantly lower than that of WT mice.
4, using high throughput gene chip technique to analyze the difference in gene transcription level of 6W splenic mononuclear cells after Schistosoma japonicum infection, it was found that the genes related to immune killing function in LRG-47 KO mice were significantly up-regulated. Compared with WT mice, 780 gene signal intensities were increased by 2 times in 6W LRG-47 KO mice splenocytes after infection. Above, the 866 gene signals weakened by more than 2 times. The GO classification of the differential genes and the significant analysis of pathway found that the differential genes and the pathways involved were mainly involved in the immune killer related genes and pathways.
5, 6W macrophage response after Schistosoma japonicum infection: lrg47 gene deletion, does not affect the production potential of inflammatory factors NO and TNF- alpha in macrophages. 6W, no matter LPS stimulation or SEA stimulation, no significant difference in TNF- A and NO levels produced by macrophages from LRG-47 KO mice after the infection of Schistosoma japonicum, and there is no significant difference between LRG-47 KO mouse macrophages and WT mice. There was no significant difference in gene transcription level between the two groups of mice spleen macrophages.
6, the immune response status of SEA after immunization: the level of SEA specific IgG antibody in the serum of LRG-47 KO mice was significantly lower than that of WT mice; the secretion of TNF- a in the spleen mononuclear cells under SEA stimulated the level of IL-4 and IL-10 significantly increased. The level of SEA specific IgG antibody in the serum of 3W and LRG-47 KO mice was significantly lower than that in the WT group. The level of cytokines in the culture supernatant of the spleen mononuclear cells stimulated by the insect derived antigen SEA or the mitogen ConA showed that there was no significant difference between the LRG-47 KO mice and the WT mice. The level of LRG-47 KO mice was significantly higher than that of WT mice, and the level of IFN- gamma in LRG-47 KO mice was significantly lower than that of WT mice, and there was no significant difference between the two groups of TNF- a level. The level of IL-4 was significantly higher than that of the WT mice. Although the level of IL-10 was higher than that of the WT mice, there was no statistical difference.
7, the proportion of major immune cells in spleen after SEA immunization was 3W, LRG-47 KO after SEA immunization.
The proportion of B cells, Th1 cells and Tc2 cells in mice was significantly lower than that in WT mice; LRG-47 KO
The proportion of Th2 cells and Tc1 cells in mice was slightly lower than that of WT mice, but there was no statistical difference. In conclusion, after Schistosoma japonicum infection, the absence of LRG-47 signals resulted in a significant decrease in the egg of the liver and the amount of eggs per pair of adults, the light of the liver granuloma, and the lack of LRG-47 signal to enhance the expression of TNF- A and IL-10. A significant up-regulated immuno killing molecular granzyme family and TNF family enhanced the immune killing function of killer cells. This may be an important factor leading to low egg load in mice with LRG-47 signal deletion. The results of this study suggest that LRG-47 not only participates in intracellular biological infection, but also participates in the resistance of extracellular multicellular organisms (such as Schistosoma japonicum). The formation and regulation of.LRG-47 play a negative role in the regulation of Schistosoma japonicum infection.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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