【摘要】： 近年的研究表明,具有特定序列的單鏈脫氧寡核苷酸(ODN)具有免疫負(fù)調(diào)節(jié)作用,可能通過(guò)干預(yù)免疫系統(tǒng)或免疫細(xì)胞的異常活化,成為治療自身免疫病、超敏反應(yīng)和Toll樣受體活化相關(guān)性疾病的藥物。這類ODN被稱為抑制性O(shè)DN,為人王合成的、長(zhǎng)約幾十個(gè)核苷酸的單鏈DNA分子。有研究表明,抑制性O(shè)DN可以抑制含CpG基序ODN(CpG ODN)的免疫刺激作用,也可能抑制LPS誘導(dǎo)的免疫反應(yīng)等,且多為富含鳥(niǎo)嘌吟(G)的ODN。 根據(jù)哺乳動(dòng)物線粒體DNA的序列特點(diǎn)以及對(duì)可能具有免疫負(fù)調(diào)節(jié)活性O(shè)DN的結(jié)構(gòu)特性認(rèn)識(shí),我們?cè)O(shè)計(jì)了13條單鏈脫氧寡核苷酸,分別命名為MT00～12。然后,利用生物學(xué)活性方法對(duì)MT00～12是否能抑制免疫激活劑誘導(dǎo)免疫細(xì)胞產(chǎn)生抗病毒物質(zhì)進(jìn)行了篩選,發(fā)現(xiàn)富含胞嘧啶的MT01表現(xiàn)出較強(qiáng)的抑制活性。進(jìn)一步研究顯示:1)MT01不僅能抑制TLR9激動(dòng)劑CpG ODN誘導(dǎo)的免疫反應(yīng),也能抑制滅活的DNA病毒、TLR7激動(dòng)劑咪喹莫特或滅活的RNA病毒誘導(dǎo)的免疫反應(yīng),但對(duì)TLR4誘導(dǎo)的免疫反應(yīng)則無(wú)抑制作用;2)MT01對(duì)SLE病人血清刺激的免疫反應(yīng)也具有明顯的抑制作用:3)MT01的結(jié)構(gòu),尤其富含胞嘧啶區(qū)域與其生物學(xué)活性直接相關(guān),構(gòu)效關(guān)系研究顯示其免疫負(fù)調(diào)節(jié)活性可能是序列依賴性的:4)MT01在小鼠體內(nèi)能明顯抑制CpG ODN對(duì)乙肝表面抗原特異性抗體誘生的增強(qiáng)作用,但對(duì)乙肝表面抗原刺激抗體產(chǎn)生無(wú)抑制作用,說(shuō)明MT01可能只對(duì)過(guò)強(qiáng)的免疫反應(yīng)具有抑制作用;5)MT01可抑制小鼠B細(xì)胞活化,這提示其對(duì)抗原特異性抗體產(chǎn)生的抑制作用可能是通過(guò)抑制B細(xì)胞活化而實(shí)現(xiàn)的。 綜上,一種富含胞嘧啶的單鏈脫氧寡核苷酸(MT01)具有選擇性免疫負(fù)調(diào)節(jié)活性,可能具有治療自身免疫病或TLR7/9活化相關(guān)性疾病的潛在應(yīng)用價(jià)值。
[Abstract]:Recent studies have shown that single-stranded oligonucleotides (ODN) with specific sequences have negative immunomodulatory effects, which may be used to treat autoimmune diseases by interfering with the abnormal activation of immune system or immune cells. Drugs associated with hypersensitivity and Toll-like receptor activation-associated diseases. This type of ODN is known as a mono-stranded DNA molecule of about dozens of nucleotides synthesized by inhibitory ODN,. Some studies have shown that inhibitory ODN can inhibit the immunostimulatory effect of ODN (CpG ODN) containing CpG motif, and may also inhibit the immune response induced by LPS, and most of them are ODN. rich in CpG (G). According to the sequence characteristics of mammalian mitochondrial DNA and the recognition of the structural characteristics of ODN with possible immuno-negative regulatory activity, we designed 13 single-stranded oligodeoxynucleotides named MT00~12.. Then, the biological activity method was used to screen whether MT00~12 could inhibit the production of antiviral substances in immune cells induced by immunoactivator. It was found that MT01 rich in cytosine showed strong inhibitory activity. Further studies showed that: 1) MT01 could not only inhibit the immune response induced by TLR9 agonist CpG ODN, but also inhibit the immune response induced by inactivated DNA virus, TLR7 agonist Imiquimod or inactivated RNA virus. But the immune response induced by TLR4 was not inhibited. 2) MT01 also inhibited the immune response to serum stimulation in patients with SLE. 3) the structure of MT01, especially cytosine rich region, was directly related to its biological activity. The study of structure-activity relationship showed that its negative immunomodulatory activity might be sequence-dependent: 4) MT01 could significantly inhibit the enhancement of CpG ODN induced by HBsAg specific antibody in mice. However, there was no inhibitory effect on the antibody stimulated by hepatitis B surface antigen, indicating that MT01 may only inhibit the excessive immune response. 5) MT01 can inhibit the activation of B cells in mice, which suggests that the inhibition of antigen-specific antibodies may be achieved by inhibiting the activation of B cells. In conclusion, a cytosine rich single-strand deoxyoligodeoxynucleotide (MT01) has selective negative immunomodulatory activity, which may have potential application value in the treatment of autoimmune diseases or TLR7/9 activation-related diseases.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.11

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