本文選題：日本血吸蟲 + LRG-47�。� 參考：《南京醫(yī)科大學(xué)》2010年碩士論文

【摘要】：_p47 GTP酶家族是目前在脊椎動物中發(fā)現(xiàn)的分子量最小也是數(shù)量最多的干擾素(IFN)反應(yīng)性GTP酶家族,已經(jīng)克隆了小鼠_p47 GTP酶家族中的六個成員,分別是LRG-47、GTPI、IGTP、IRG-47、IIGP和TGTP/Mg21。研究發(fā)現(xiàn),_p47 GTP酶能夠特異地參與抵抗胞內(nèi)細(xì)菌和原蟲的感染,對病毒的清除能力較弱~[1-7]。雖然同屬于_p47 GTP酶家族,但不同成員所表達(dá)的抗力具有病原體和時相特異性的特點。_P47 GTP酶家族因其成員所具有特異性防御病原體的能力而引起學(xué)者的廣泛關(guān)注。然而,_p47 GTP酶在更為復(fù)雜的多細(xì)胞生物,例如重要的胞外寄生蠕蟲—血吸蟲感染中的作用知之甚少。本實驗室前期研究發(fā)現(xiàn)~[8],_p47 GTP酶隨日本血吸蟲感染進程,信號強度呈下調(diào)趨勢,且家族中各成員的表達(dá)強度存在明顯差異。我們的前期研究還發(fā)現(xiàn)~[9],在日本血吸蟲感染6周(w)后,IGTP KO小鼠和IRG-47 KO小鼠在感染/致病結(jié)局、整體/局部的免疫應(yīng)答等方面均存在較大的差異。這提示不同_p47 GTP酶不僅參與胞內(nèi)生物的感染,亦參與胞外多細(xì)胞生物的感染,而且各成員在多細(xì)胞生物感染中的作用可能不盡相同。 日本血吸蟲屬吸蟲綱的多細(xì)胞無脊椎動物,能引起宿主復(fù)雜的感染免疫應(yīng)答,可作為多細(xì)胞生物研究的重要的生物模型之一。因此,對日本血吸蟲感染/致病機制的深入研究,既能為我國今后血吸蟲病的防治提供理論基礎(chǔ),更能擴展感染免疫學(xué)的基本知識。大量文獻(xiàn)已報道p47 GTP酶家族成員LRG-47在多數(shù)胞內(nèi)感染中具有明確的抗感染作用,但是對胞外血吸蟲感染的作用卻不明確。本研究采用lrg47基因敲除小鼠建立日本血吸蟲急性感染模型,較為系統(tǒng)地觀察LRG-47在日本血吸蟲急性感染/致病階段所誘導(dǎo)的宿主免疫應(yīng)答特點,以進一步深化血吸蟲的感染免疫學(xué),為血吸蟲病疫苗研制提供理論基礎(chǔ)。 本研究采用lrg47基因敲除(LRG-47 KO)與野生型C57BL/6J(wild-type,WT)小鼠,建立日本血吸蟲自然感染的小鼠模型。為觀察感染/致病結(jié)果,在感染后6w通過胸主動脈灌注法計數(shù)成蟲數(shù)、消化肝臟計數(shù)蟲卵數(shù)、以及計算每克肝卵數(shù)和每對成蟲產(chǎn)卵數(shù);以HE染色觀察肝臟的病理變化。為觀察感染后宿主的免疫應(yīng)答特征,以間接ELISA法檢測小鼠血清中日本血吸蟲特異性IgG抗體水平;以雙抗夾心ELISA法檢測脾臟單個核細(xì)胞培養(yǎng)上清中Th1/Th2型細(xì)胞因子的表達(dá)水平;以流式細(xì)胞術(shù)檢測脾臟中主要免疫細(xì)胞的比例變化;采用基因芯片技術(shù),比較分析感染后6w小鼠脾臟單個核細(xì)胞中表達(dá)基因的轉(zhuǎn)錄水平,并對信號強度發(fā)生2倍及以上變化的差異基因進行GO分類及pathway的顯著性分析。為觀察基因敲除后對巨噬細(xì)胞功能的影響,以間接ELISA法檢測脾巨噬細(xì)胞培養(yǎng)上清中TNF-α的表達(dá)水平;以硝酸還原酶法檢測培養(yǎng)上清中NO的水平;以RT-PCR法檢測脾巨噬細(xì)胞中tnfα和inos的轉(zhuǎn)錄水平。最后,采用蟲源性抗原SEA免疫小鼠,以間接ELISA法檢測小鼠血清中日本血吸蟲特異性IgG抗體,脾臟單個核細(xì)胞培養(yǎng)上清中Th1/Th2型細(xì)胞因子的表達(dá)水平,以及流式技術(shù)檢測脾臟中主要免疫細(xì)胞的比例變化。 本研究獲得如下主要結(jié)果: 1、日本血吸蟲感染后6w,LRG-47 KO小鼠卵荷顯著少于WT小鼠,且肝臟肉芽腫反應(yīng)較輕。日本血吸蟲感染后6w, LRG-47 KO組小鼠體內(nèi)的成蟲數(shù)與正常野生型小鼠相當(dāng),而LRG-47 KO小鼠的肝臟蟲卵數(shù)、每克肝卵量以及每對成蟲產(chǎn)卵數(shù)顯著低于WT組。肝臟中的蟲卵肉芽腫大多為急性期肉芽腫,可見在蟲卵周圍,大量炎癥細(xì)胞浸潤,LRG-47 KO組平均肉芽腫面積和組成細(xì)胞數(shù)顯著小/少于WT組。 2、日本血吸蟲感染后3w和6w,就抗原特異性IgG抗體而言,LRG-47 KO小鼠血清中SEA特異性IgG抗體水平顯著低于WT小鼠。隨著日本血吸蟲感染的進行,小鼠血清中可溶性成蟲抗原(SWAP)和可溶性蟲卵抗原(SEA)誘導(dǎo)的特異性IgG抗體持續(xù)升高。但在感染后3w和6w,LRG-47 KO組與WT組SWAP特異的IgG抗體水平均無顯著性差異。而LRG-47 KO組小鼠血清中SEA特異性IgG抗體水平均顯著低于WT組。 3、日本血吸蟲感染后6w,LRG-47 KO小鼠的CD4+T細(xì)胞的功能被削弱。脾臟單個核細(xì)胞經(jīng)蟲源性抗原SWAP、SEA和有絲分裂原ConA刺激后,培養(yǎng)上清中細(xì)胞因子水平顯示: ConA與SEA刺激下,LRG-47 KO小鼠的IFN-γ水平顯著低于WT小鼠;在SWAP刺激下,LRG-47 KO小鼠的IL-4水平顯著低于WT小鼠;但在SEA刺激下,LRG-47 KO小鼠的TNF-α和IL-10水平顯著高于WT小鼠。感染后6w脾中主要免疫細(xì)胞的比例為:脾中B細(xì)胞、NK細(xì)胞和巨噬細(xì)胞的比例在LRG-47 KO小鼠和WT小鼠兩組之間無統(tǒng)計學(xué)差異;主要行使特異性細(xì)胞免疫應(yīng)答功能的T細(xì)胞亞群:Tc1和Treg細(xì)胞的比例在LRG-47 KO小鼠顯著高于WT小鼠;Th1細(xì)胞、Th2細(xì)胞和Tc2細(xì)胞的比例在LRG-47 KO小鼠顯著低于WT小鼠。 4、采用高通量基因芯片技術(shù)分析比較日本血吸蟲感染后6w脾臟單個核細(xì)胞基因轉(zhuǎn)錄水平的差異,發(fā)現(xiàn)LRG-47 KO小鼠中與免疫殺傷功能相關(guān)的基因被顯著上調(diào)。與WT小鼠相比,在感染后6w的LRG-47 KO小鼠脾細(xì)胞中,有780個基因信號強度增強2倍以上,866個基因信號減弱2倍以上。對差異基因的GO分類及pathway的顯著性分析發(fā)現(xiàn),差異基因及參與的通路主要涉及免疫殺傷相關(guān)基因及通路。 5、日本血吸蟲感染后6w的巨噬細(xì)胞應(yīng)答:lrg47基因缺失后,不影響巨噬細(xì)胞中炎癥因子NO和TNF-α的產(chǎn)生潛能。在日本血吸蟲感染后6w,不論是LPS刺激,還是SEA刺激,LRG-47 KO小鼠巨噬細(xì)胞產(chǎn)生的TNF-α和NO水平與WT小鼠均無顯著性差異;tnf-α和inos基因的轉(zhuǎn)錄水平在兩組小鼠脾巨噬細(xì)胞亦無顯著性差異。 6、SEA免疫后宿主免疫應(yīng)答狀況:LRG-47 KO小鼠血清SEA特異性IgG抗體水平明顯低于WT小鼠;在SEA刺激下脾單個核細(xì)胞分泌TNF-α、IL-4和IL-10水平顯著增高。日本血吸蟲可溶性蟲卵抗原(SEA)免疫小鼠后,小鼠血清中SEA特異的IgG抗體持續(xù)升高;在免疫后3w,LRG-47 KO組小鼠血清中SEA特異性IgG抗體水平顯著低于WT組。脾臟單個核細(xì)胞經(jīng)蟲源性抗原SEA或有絲分裂原ConA刺激后,培養(yǎng)上清中細(xì)胞因子水平顯示:在SEA刺激下,LRG-47 KO小鼠與WT小鼠的IFN-γ水平無顯著的統(tǒng)計學(xué)差異,TNF-α、IL-4和IL-10水平顯著高于WT小鼠;在ConA刺激下,LRG-47 KO小鼠的IFN-γ水平顯著低于WT小鼠,TNF-α水平兩組之間無顯著性差異,IL-4的水平顯著高于WT小鼠,IL-10的水平雖高于WT小鼠,但無統(tǒng)計學(xué)差異。 7、SEA免疫小鼠后脾臟中主要免疫細(xì)胞的比例:SEA免疫后3w,LRG-47 KO 小鼠的B細(xì)胞、Th1細(xì)胞和Tc2細(xì)胞的比例顯著低于WT小鼠;LRG-47 KO 小鼠的Th2細(xì)胞和Tc1細(xì)胞比例略低于WT小鼠,但無統(tǒng)計學(xué)差異。綜上,日本血吸蟲感染后,與野生型小鼠相比,LRG-47信號缺失導(dǎo)致肝臟沉積的蟲卵及每對成蟲產(chǎn)卵量顯著下降,肝臟肉芽腫反應(yīng)較輕;LRG-47信號缺失增強TNF-α和IL-10的表達(dá)能力,顯著上調(diào)免疫殺傷效應(yīng)分子顆粒酶家族和TNF家族,增強殺傷細(xì)胞的免疫殺傷功能,這可能是導(dǎo)致LRG-47信號缺失小鼠低卵荷的重要因素。本研究結(jié)果提示LRG-47不僅參與了胞內(nèi)生物感染,也參與胞外多細(xì)胞生物(如日本血吸蟲)感染的抗性形成與調(diào)節(jié)。LRG-47在日本血吸蟲感染過程中起負(fù)性調(diào)節(jié)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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