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  本文選題：日本血吸蟲 + Sj22.6kDa抗原��； 參考：《南京醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 【目的】研究日本血吸蟲22.6kDa(Sj22.6)抗原基因序列中是否存在免疫抑制性片段。 【方法】依據(jù)已經(jīng)報道的免疫抑制性序列的結(jié)構(gòu)特征,從編碼Sj22.6kDa抗原的基因序列中選取若干長度均為20～30bp的寡脫氧核苷酸(oligodeoxynucleotide,ODN),人工合成這些短的DNA序列,以目前國際上公認的對小鼠具有很強免疫刺激作用的ODN CpG1826作為刺激劑,用于后續(xù)實驗,并設(shè)立對照。分離正常小鼠脾臟單個核細胞進行體外培養(yǎng),將不同ODN分別與CpG 1826共同加入細胞培養(yǎng)體系,同位素~3H-TdR摻入法測淋巴細胞增殖實驗觀察各組細胞增殖水平的變化,以篩選出抑制性O(shè)DN并觀察抑制性O(shè)DN作用的量效關(guān)系和時間效應(yīng)關(guān)系。以刀豆蛋白A(Concanavalin A,ConA)做刺激物,~3H-TdR摻入法測淋巴細胞增殖實驗觀察抑制性O(shè)DN對ConA誘導(dǎo)的細胞增殖的影響。采用酶聯(lián)免疫吸附試驗(ELISA)觀察抑制性O(shè)DN在體內(nèi)和體外分別對CpG 1826引起的Th1型細胞因子IFN-γ和IL-12分泌的影響。采用流式細胞術(shù)檢測抑制性O(shè)DN對細胞結(jié)合和攝取CpG 1826的影響。最后采用半定量PCR的方法檢測抑制性O(shè)DN對CpG 1826的特異性受體Toll樣受體9(TLR9)mRNA表達水平的影響。 【結(jié)果】(1)淋巴細胞增殖試驗結(jié)果顯示,Sj22.6kDa抗原基因序列中的ODN F546和F311能抑制CpG 1826誘導(dǎo)的小鼠脾臟淋巴細胞體外增殖,在各ODN終濃度均為1μM時,ODN F546和F311抑制率分別為87%和11%。(2)淋巴細胞增殖試驗結(jié)果顯示,當CpG 1826的量一定時,ODN F546和F311的抑制作用隨著它們量的增多而增強。當與CpG 1826的摩爾比為1∶1時,ODN F546或F311的抑制作用在同時或先于CpG 1826加入時最強。(3)酶聯(lián)免疫吸附實驗結(jié)果顯示,在體外和體內(nèi)實驗中,ODN F546均顯著降低CpG 1826誘導(dǎo)的小鼠脾細胞產(chǎn)生的Th1型細胞因子IFN-γ和IL-12的分泌量。(4)流式細胞術(shù)檢測結(jié)果表明,ODN F546使結(jié)合CpG 1826的細胞占總細胞的百分比由9.7%減少為4%(P＜0.05),使攝取CpG 1826的細胞占總細胞的百分比由36%減少為13%(P＜0.01)。(5)半定量RT-PCR結(jié)果顯示,體外培養(yǎng)時,CpG 1826刺激后小鼠脾細胞TLR9mRNA大量表達,ODN F546顯著降低CpG 1826引起的TLR9 mRNA表達(P＜0.01)。 【結(jié)論】Sj22.6抗原基因序列中存在具有明顯免疫抑制作用的ODNF546和F311,它們特異性抑制刺激性CpG誘導(dǎo)的淋巴細胞增殖,抑制作用呈現(xiàn)一定的劑量效應(yīng)關(guān)系和時間效應(yīng)關(guān)系。ODN F546能抑制刺激性CpG誘導(dǎo)的細胞因子IFN-γ和IL-12的分泌。減少細胞對刺激性CpG的結(jié)合及攝取、降低刺激性CpG特異性受體TLR9的基因表達可能與其作用機制相關(guān)。研究結(jié)果提示存在免疫抑制性O(shè)DN可能與編碼Sj22.6kDa抗原的核酸疫苗不能誘導(dǎo)產(chǎn)生有效保護力有關(guān)。
[Abstract]:[Objective] to study whether there are immunosuppressive fragments in the sequence of 22.6kDa (Sj22.6) antigen of Schistosoma japonicum.
[Methods] according to the structural features of the reported immunosuppressive sequences, the oligodeoxynucleotides (oligodeoxynucleotide, ODN), with a number of 20 to 30bp, were selected from the sequence of the gene encoding Sj22.6kDa antigen, and these short DNA sequences were synthesized artificially, with the internationally recognized ODN of strong immune stimulation to mice. CpG1826 was used as a stimulant in the follow-up experiment, and the control was set up. The spleen mononuclear cells of normal mice were isolated and cultured in vitro. The different ODN and CpG 1826 were added to the cell culture system. The proliferation of lymphocytes was observed by the isotope ~3H-TdR incorporation test, and the proliferation level of each group was observed in order to screen the inhibitory ODN. The relationship between the dose effect and the time effect of inhibitory ODN. Using A (Concanavalin A, ConA) as a stimulator. The effect of the inhibitory ODN on the proliferation of ConA induced cell proliferation was observed by ~3H-TdR incorporation assay. The inhibitory ODN was observed in vivo and in vitro by enzyme linked immunosorbent assay (ELISA) for CpG 1826, respectively. The effects of Th1 type cytokine IFN- gamma and IL-12 secretion were induced. Flow cytometry was used to detect the effects of inhibitory ODN on cell binding and uptake of CpG 1826. Finally, semi quantitative PCR was used to detect the effect of inhibitory ODN on the expression of Toll like receptor 9 (TLR9) mRNA expression of CpG 1826.
[results] (1) the lymphocyte proliferation test showed that ODN F546 and F311 in the Sj22.6kDa antigen gene sequence could inhibit the proliferation of spleen lymphocytes induced by CpG 1826 in vitro. The inhibition rates of ODN F546 and F311 were 87% and 11%. (2) lymphocyte proliferation test, respectively, when the ODN terminal concentration was 1 Mu M, respectively, when the amount of CpG 1826 was measured. The inhibitory effects of ODN F546 and F311 were enhanced as their amount increased. When the molar ratio of CpG 1826 was 1 to 1, the inhibitory effects of ODN F546 or F311 were strongest at the same time or before CpG 1826. (3) enzyme linked immunosorbent assay showed that in vitro and in vivo, ODN F546 significantly reduced the CpG 1826 induced mouse spleen The secretion of Th1 type cytokine IFN- gamma and IL-12 produced by the cells. (4) flow cytometry results showed that ODN F546 reduced the percentage of cells with CpG 1826 to total cells from 9.7% to 4% (P < 0.05), and reduced the percentage of CpG 1826 to total cells from 36% to 13% (P < 0.01). (5) semi quantitative RT-PCR results showed that body When cultured in vitro, CpG 1826 stimulated TLR9mRNA expression in splenocytes of mice, and ODN F546 significantly decreased TLR9 mRNA expression induced by CpG 1826 (P < 0.01).
[Conclusion] there is an obvious immunosuppressive effect of ODNF546 and F311 in the Sj22.6 antigen gene sequence. They specifically inhibit the proliferation of lymphocytes induced by stimulating CpG. The inhibitory effect presents a certain dose effect relationship and time effect relationship..ODN F546 can inhibit the secretion of IFN- gamma and IL-12 induced by stimulating CpG induced cytokines. The gene expression of CpG specific receptor TLR9 may be related to the binding and uptake of small cells to stimulating CpG, and the results suggest that the existence of immunosuppressive ODN may be related to the inability of the nucleic acid vaccine to encode the Sj22.6kDa antigen to induce effective protection.
【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】：2008
【分類號】：R392

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本文編號：1996372