通过共免疫鼠卵透明带3 DNA和蛋白疫苗技术增强免疫不孕效果及降低卵巢炎症反应的研究

发布时间：2018-07-12 14:02

本文选题：避孕 + mZP3蛋白　；参考：《新疆大学》2007年博士论文

【摘要】： 哺乳动物卵子周围包裹的卵透明带(ZP:Zona pellucida),主要由糖蛋白组成。小鼠的卵透明带是有三种糖蛋白组成的,分别为ZP1、ZP2和ZP3,其中ZP3作为精子的初级受体介导了精-卵结合,并引发了顶体反应。作为精子初级受体的ZP3诱导产生的自身抗体能够阻止卵子受精并且已经被作为人类不孕疫苗的候选靶抗原。由于引发的自身反应性T细胞炎症反应可导致卵巢功能的破坏,去除T细胞表位的ZP3多肽疫苗成为了优先选择的避孕方法。但由于多肽疫苗的免疫原性较差,只能诱导短期的、较低水平的体液免疫反应。我们在本研究中探讨了一种新的免疫避孕策略即:共免疫鼠卵透明带3(mZP3) DNA和蛋白疫苗在保留高水平抗体的同时,不仅能够消除T细胞炎症反应,而且造成小鼠生育率的显著下降。共免疫不育疫苗的抗生育率达到了70%(从naive小鼠的100%下降到了30%),其它各组抗生育率在40%-60%之间。抗体分析显示,共免疫组的抗体水平达到了最高。在共免疫组,不孕小鼠和生育小鼠抗体水平的差异是显著的,高水平的抗体反应和生育率的下降是呈正相关的;然而,其它各组生育率下降和抗体水平之间没有相关性。组织学分析显示,在共免疫组的不孕小鼠卵巢中呈现出正常的卵泡发育,而其它各免疫组的大部分不孕小鼠卵巢缺少成熟的卵泡,这可能是造成这些小鼠不孕的原因。在共免疫小鼠中,正常卵泡发育和T细胞反应的抑制之间具有明显的相关性;同时,共免疫可减少炎症细胞因子IFN-γ的表达,增加IL-10和FoxP3的表达,揭示这种抗炎症反应可能是调节性T细胞介导的。由于调节性T细胞能够抑制免疫反应和维持对自身抗原的免疫耐受,DNA疫苗和蛋白疫苗共免疫诱导的调节性T细胞能够调控病理性T细胞反应,使得共免疫小鼠避免了异常的卵泡发育,我们以卵巢自主免疫疾病(autoimmune ovarian disease: AOD)作为研究模型来探讨这类调节性T细胞的特征及作用机理。ZP3蛋白抗原引起的自主免疫反应是AOD的重要诱因原因之一。用mZP3蛋白和完全弗氏佐剂诱导的AOD小鼠模型,我们探讨了采用共免疫的方法来治疗AOD的可行性。研究结果显示,共免疫mZP3 DNA和蛋白疫苗显著减轻了AOD的发病程度,并且抑制了抗原特异性的T细胞反应,降低了炎症因子IL-12和INF-γ的表达水平,这些作用可能是由于共免疫诱导出了一群特异性的调节性T细胞来完成的。这群调节性T细胞高表达IL-10和FoxP3。在第一次共免疫的同时,将抗CD25的抗体同时注射到小鼠体内,抗VP1的抗体作为无关抗体对照。结果显示,抗CD25的抗体没有影响共免疫的治疗效果;同时,与其它各组相比,共免疫小鼠脾脏和淋巴结的CD4~+T细胞CD25的表达没有发生变化,这群调节性T细胞可能不属于CD25~+。将CD4~+CD25~-和CD4~+CD25~+细胞从共免疫和错配免疫的小鼠中分离纯化出来,通过尾静脉过继转移到发病小鼠体内,发现只有从共免疫来源的CD4~+CD25~-细胞才能治疗AOD。将纯化的上述细胞进行细胞因子表达检测,结果显示,只有CD4~+CD25~-细胞的IL-10和FoxP3的表达显著增加。这些结果说明,共免疫诱导了调节性T细胞,并且这群调节性T细胞的特征是CD4~+/CD25~-/FoxP3~+,并能表达IL-10。我们尝试了用共免疫DNA和蛋白疫苗的方法来治疗自主免疫疾病。总之,本研究证明了mZP3 DNA疫苗和蛋白疫苗共免疫显著降低了小鼠的生育率。这种方法不仅诱导了最高水平的抗体反应,也抑制T细胞反应,避免了病理性T细胞反应对卵巢造成的伤害。病理性T细胞反应的抑制与共免疫诱导的CD4+CD25-调节性T细胞是密切相关的,这类调节性T细胞表达IL-10。这些研究结果揭示了共免疫mZP3 DNA和蛋白疫苗能够显著提高抗生育效果,并且不影响卵泡的正常发育。这不仅克服了T细胞反应造成的卵巢炎,也克服了只含有B细胞表位的多肽疫苗免疫原性的不足,为开发人用避孕疫苗提供了理论基础。
[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.
【学位授予单位】：新疆大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392;R711.6

【引证文献】