本文選題：避孕 + mZP3蛋白　； 參考：《新疆大學(xué)》2007年博士論文

【摘要】： 哺乳動(dòng)物卵子周圍包裹的卵透明帶(ZP:Zona pellucida),主要由糖蛋白組成。小鼠的卵透明帶是有三種糖蛋白組成的,分別為ZP1、ZP2和ZP3,其中ZP3作為精子的初級(jí)受體介導(dǎo)了精-卵結(jié)合,并引發(fā)了頂體反應(yīng)。作為精子初級(jí)受體的ZP3誘導(dǎo)產(chǎn)生的自身抗體能夠阻止卵子受精并且已經(jīng)被作為人類不孕疫苗的候選靶抗原。由于引發(fā)的自身反應(yīng)性T細(xì)胞炎癥反應(yīng)可導(dǎo)致卵巢功能的破壞,去除T細(xì)胞表位的ZP3多肽疫苗成為了優(yōu)先選擇的避孕方法。但由于多肽疫苗的免疫原性較差,只能誘導(dǎo)短期的、較低水平的體液免疫反應(yīng)。我們?cè)诒狙芯恐刑接懥艘环N新的免疫避孕策略即:共免疫鼠卵透明帶3(mZP3) DNA和蛋白疫苗在保留高水平抗體的同時(shí),不僅能夠消除T細(xì)胞炎癥反應(yīng),而且造成小鼠生育率的顯著下降。共免疫不育疫苗的抗生育率達(dá)到了70%(從naive小鼠的100%下降到了30%),其它各組抗生育率在40%-60%之間。抗體分析顯示,共免疫組的抗體水平達(dá)到了最高。在共免疫組,不孕小鼠和生育小鼠抗體水平的差異是顯著的,高水平的抗體反應(yīng)和生育率的下降是呈正相關(guān)的;然而,其它各組生育率下降和抗體水平之間沒有相關(guān)性。 組織學(xué)分析顯示,在共免疫組的不孕小鼠卵巢中呈現(xiàn)出正常的卵泡發(fā)育,而其它各免疫組的大部分不孕小鼠卵巢缺少成熟的卵泡,這可能是造成這些小鼠不孕的原因。在共免疫小鼠中,正常卵泡發(fā)育和T細(xì)胞反應(yīng)的抑制之間具有明顯的相關(guān)性;同時(shí),共免疫可減少炎癥細(xì)胞因子IFN-γ的表達(dá),增加IL-10和FoxP3的表達(dá),揭示這種抗炎癥反應(yīng)可能是調(diào)節(jié)性T細(xì)胞介導(dǎo)的。 由于調(diào)節(jié)性T細(xì)胞能夠抑制免疫反應(yīng)和維持對(duì)自身抗原的免疫耐受,DNA疫苗和蛋白疫苗共免疫誘導(dǎo)的調(diào)節(jié)性T細(xì)胞能夠調(diào)控病理性T細(xì)胞反應(yīng),使得共免疫小鼠避免了異常的卵泡發(fā)育,我們以卵巢自主免疫疾病(autoimmune ovarian disease: AOD)作為研究模型來探討這類調(diào)節(jié)性T細(xì)胞的特征及作用機(jī)理。ZP3蛋白抗原引起的自主免疫反應(yīng)是AOD的重要誘因原因之一。用mZP3蛋白和完全弗氏佐劑誘導(dǎo)的AOD小鼠模型,我們探討了采用共免疫的方法來治療AOD的可行性。研究結(jié)果顯示,共免疫mZP3 DNA和蛋白疫苗顯著減輕了AOD的發(fā)病程度,并且抑制了抗原特異性的T細(xì)胞反應(yīng),降低了炎癥因子IL-12和INF-γ的表達(dá)水平,這些作用可能是由于共免疫誘導(dǎo)出了一群特異性的調(diào)節(jié)性T細(xì)胞來完成的。這群調(diào)節(jié)性T細(xì)胞高表達(dá)IL-10和FoxP3。在第一次共免疫的同時(shí),將抗CD25的抗體同時(shí)注射到小鼠體內(nèi),抗VP1的抗體作為無關(guān)抗體對(duì)照。結(jié)果顯示,抗CD25的抗體沒有影響共免疫的治療效果;同時(shí),與其它各組相比,共免疫小鼠脾臟和淋巴結(jié)的CD4~+T細(xì)胞CD25的表達(dá)沒有發(fā)生變化,這群調(diào)節(jié)性T細(xì)胞可能不屬于CD25~+。將CD4~+CD25~-和CD4~+CD25~+細(xì)胞從共免疫和錯(cuò)配免疫的小鼠中分離純化出來,通過尾靜脈過繼轉(zhuǎn)移到發(fā)病小鼠體內(nèi),發(fā)現(xiàn)只有從共免疫來源的CD4~+CD25~-細(xì)胞才能治療AOD。將純化的上述細(xì)胞進(jìn)行細(xì)胞因子表達(dá)檢測,結(jié)果顯示,只有CD4~+CD25~-細(xì)胞的IL-10和FoxP3的表達(dá)顯著增加。這些結(jié)果說明,共免疫誘導(dǎo)了調(diào)節(jié)性T細(xì)胞,并且這群調(diào)節(jié)性T細(xì)胞的特征是CD4~+/CD25~-/FoxP3~+,并能表達(dá)IL-10。我們嘗試了用共免疫DNA和蛋白疫苗的方法來治療自主免疫疾病。 總之,本研究證明了mZP3 DNA疫苗和蛋白疫苗共免疫顯著降低了小鼠的生育率。這種方法不僅誘導(dǎo)了最高水平的抗體反應(yīng),也抑制T細(xì)胞反應(yīng),避免了病理性T細(xì)胞反應(yīng)對(duì)卵巢造成的傷害。病理性T細(xì)胞反應(yīng)的抑制與共免疫誘導(dǎo)的CD4+CD25-調(diào)節(jié)性T細(xì)胞是密切相關(guān)的,這類調(diào)節(jié)性T細(xì)胞表達(dá)IL-10。 這些研究結(jié)果揭示了共免疫mZP3 DNA和蛋白疫苗能夠顯著提高抗生育效果,并且不影響卵泡的正常發(fā)育。這不僅克服了T細(xì)胞反應(yīng)造成的卵巢炎,也克服了只含有B細(xì)胞表位的多肽疫苗免疫原性的不足,為開發(fā)人用避孕疫苗提供了理論基礎(chǔ)。
[Abstract]:The zona pellucida (ZP:Zona pellucida), wrapped around the egg of a mammal, consists mainly of glycoproteins. The mouse oval zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3, in which ZP3 acts as a primary receptor for spermatozoa, which mediates sperm oval binding and triggering the acrosome reaction. It is induced by the ZP3 induced by the primary sperm receptor. Body antibody can prevent ovum fertilization and have been used as a candidate target antigen for the human infertility vaccine. The ZP3 polypeptide vaccine that removes the epitopes of T cells has become a preferred contraceptive method because of the inflammation of the autoreactive T cell caused by the self reactive cells. But the immunogenicity of the peptide vaccine is poor, only induced by the peptide vaccine. In this study, we have discussed a new strategy for immune contraception in this study: CO immunized zona pellucida 3 (mZP3) DNA and protein vaccine, while retaining high level of antibody, not only can eliminate the inflammatory response of T cells, but also make a significant decrease in the fertility rate of mice. Co immuno sterile vaccine. The anti fertility rate was 70% (from 100% of naive mice to 30%), and the other groups were between 40%-60%. Antibody analysis showed that the antibody level of the co immunization group reached the highest level. In the co immunization group, the difference in the antibody level between the infertile mice and the bearing mice was significant, and the high level of antibody response and the decline of fertility rate were present. Positive correlation; however, there was no correlation between the decline in fertility and antibody levels in other groups.
Histologic analysis showed normal follicle development in the ovaries of the infertile mice of the co immunization group, and the lack of mature follicles in most of the other groups of infertile mice, which may be the cause of infertility in these mice. In CO immunized mice, there is a significant difference between normal follicle development and the inhibition of T cell response in the co immunized mice. At the same time, CO immunization can reduce the expression of inflammatory cytokine IFN- gamma, increase the expression of IL-10 and FoxP3, and reveal that this anti-inflammatory response may be mediated by regulatory T cells.
Because the regulatory T cells can inhibit the immune response and maintain the immune tolerance to their own antigens, the DNA vaccine and the protein vaccine co immunized with regulatory T cells can regulate the pathological T cell response, making the co immunized mice avoid abnormal follicle development. We use the ovarian self main immune disease (autoimmune ovarian disease: AOD). As a study model to explore the characteristics and mechanism of this kind of regulatory T cells, the mechanism of.ZP3 protein antigen induced autonomic response is one of the important causes of AOD. The AOD mouse model induced by mZP3 protein and complete Freund's adjuvant has been used to explore the feasibility of using CO immunization to treat AOD. The pestilence mZP3 DNA and protein vaccine significantly reduce the incidence of AOD, inhibit the antigen specific T cell response, and reduce the expression level of the inflammatory factors IL-12 and INF- gamma, which may be due to a group of specific regulatory T cells induced by CO immunization. This group of regulatory T cells highly expresses IL-10 and FoxP3.. In the first co immunization, the anti CD25 antibody was injected into the mice and the anti VP1 antibody was used as the independent antibody control. The results showed that the anti CD25 antibody did not affect the therapeutic effect of CO immunization; at the same time, compared with other groups, the expression of CD25 in the spleen and lymph nodes of the mice was not changed, and the group of CD4~+T cells of the spleen and lymph nodes were not changed. The regulatory T cells may not belong to CD25~+. to separate CD4~+CD25~- and CD4~+CD25~+ cells from the co immunized and mismatched mice, and then pass through the tail vein to the infected mice. It is found that only the CD4~+CD25~- cells from the co immunized source can be treated by AOD. to detect the expression of the cytokine expression in the purified cells. The results showed that only the expression of IL-10 and FoxP3 in CD4~+CD25~- cells increased significantly. These results suggest that CO immunological induction of regulatory T cells, and this group of regulatory T cells is characterized by CD4~+/CD25~-/FoxP3~+, and can express IL-10.. We have tried to treat autoimmune disease by using CO immunized DNA and egg white vaccine.
In conclusion, the study demonstrated that the co immunization of mZP3 DNA vaccine and protein vaccine significantly reduced the fertility rate of mice. This method not only induces the highest level of antibody response, but also inhibits the reaction of T cells, avoids the injury caused by the pathological T cell response to the ovary. The inhibition of pathological T cell reaction and the CD4+CD25- regulation induced by CO immunization Sex T cells are closely related, and these regulatory T cells express IL-10..
These findings reveal that CO immunization of mZP3 DNA and protein vaccines can significantly improve the anti fertility effect and do not affect the normal development of follicles. This not only overcomes the ovarian inflammation caused by T cell response, but also overcomes the deficiency of the peptide vaccine containing the epitopes of only B cells, which provides a theoretical basis for developing a human contraceptive vaccine.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392;R711.6

【引證文獻(xiàn)】

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本文編號(hào)：2117389