滋养层细胞促融合表位肽疫苗的体液免疫效应及其抗生育潜能的研究
发布时间：2018-09-19 07:29
【摘要】： 目的 以人滋养层细胞促融合表位模拟肽(P09-04)为基础设计、合成多肽免疫原,免疫C57BL/6型小鼠,观察体液免疫反应的特点,评价其免疫原性,探讨如何将表位模拟肽构建成有效的表位肽疫苗;通过分析表位多肽所诱导的抗血清与天然抗原的交叉反应性,及其体外抑制人绒毛膜癌细胞(BeWo)融合的能力,评估其抗生育的潜能。 方法 1.以人滋养层细胞促融合表位模拟肽与一种通用辅助T细胞表位(PADRE)作为骨架,设计出八分枝多价抗原肽(PADRE-P09-04)8-MAP和线性嵌合肽(PADRE-P09-04)两种结构免疫原。在固相自动肽合成仪上进行合成,高效液相色谱仪上进行纯化及纯度分析,纯化后的多肽行质谱鉴定。 2.纯化后的MAP结构肽(P1)、线性结构肽(P2)和表位模拟肽(P3),分别与等量弗氏佐剂混悬后皮下免疫雌性C57BL/6小鼠,采用第0、3、6周三次免疫程序,100μg/只,首次免疫后第2、5、8、10、12周取样,应用ELISA间接法测定血清IgG及子宫粘膜冲洗液中IgA抗体生成情况,比较不同合成肽的免疫原性。 3.取正常妊娠早期(6～8周)人工流产的新鲜绒毛组织,以效价较高的小鼠免疫血清为一抗,以未免疫的正常小鼠血清作为阴性对照,通过免疫组化法检测免疫血清中特异性抗体识别人滋养层细胞相关抗原表位的能力。 4.应用forskolin诱导的BeWo细胞融合来模拟体内滋养细胞的分化融合过程。首先通过MTT法检测小鼠血清存在下BeWo细胞增殖及活性情况,然后将细胞分为四组:Ⅰ组:应用HAM’s F-12培养基培养细胞;Ⅱ组:含有100μM Forskolin的培养基培养细胞;Ⅲ组:含100μM Forskolin、10%未免疫鼠血清的培养基培养细胞;Ⅳ组:含100μM Forskolin、10%鼠抗血清培养基培养细胞。各组细胞培养48小时后,应用细胞免疫荧光法分别标记BeWo细胞膜与胞核,统计各组细胞的融合率,分析抗血清抑制BeWo细胞融合的能力。 结果 1.纯化后的合成肽行HPLC均显示为单峰,经积分计算,纯度均达到95%以上,质谱分析结果显示分子量分别为23372、2826.3和1721.8,与理论分子量相吻合。 2.小鼠血清中抗表位模拟肽IgG抗体的检测:合成肽P1组第5周出现阳性,抗体水平随免疫次数和时间而升高,至第10周达到最高(1:1200);合成多肽P2组第8周检测到低水平的针对模拟表位的IgG抗体(1:200),抗体水平随时间变化不大;合成多肽P3在在测定的时间未检测到抗体的产生,与阴性对照组不具有显著性差异。且五次血清抗体测量,都呈现出了各组多肽诱导抗体滴度的趋势为:P1P2P3。 3.小鼠免疫后子宫粘膜冲洗液中抗表位模拟肽IgA抗体的检测:合成肽P1组第8周检测到了低水平的抗表位模拟肽IgA抗体,第10周达到最高,而合成肽P2与P3组在观测时间内未检测出特异性抗体,并且与阴性对照组无显著性差异。且五次子宫粘膜冲洗液的抗体测量,都呈现出了各组多肽诱导抗体滴度的趋势为:P1P2/P3。 4.应用P1组免疫血清作为一抗,经免疫组化法显示抗血清与早期人流产绒毛组织表面的合体滋养层细胞及内侧的细胞滋养层细胞呈现特异的结合,主要分布在细胞胞膜上和胞浆中,但与绒毛中其他组织细胞无反应。未免疫鼠正常血清与人绒毛组织间无交叉反应。 5.应用MTT法检测结果提示鼠血清对BeWo细胞的增值及活性无明显影响,为下一步比较各组细胞融合率提供了前提条件。应用BeWo细胞融合来模拟体内滋养细胞的分化融合的实验显示:在forskolin诱导下,BeWo细胞间融合率由1.59%(Ⅰ组)升高到11.45%(Ⅱ组),在10%抗血清存在下,细胞间融合率明显下降至6.97%(Ⅳ组),且Ⅱ组细胞融合率与Ⅳ组之间具有非常显著性差异(P0.01),而含有10%未免疫鼠血清的Ⅲ组细胞融合率为11.1%,与Ⅱ组无明显差异,但与Ⅳ组细胞融合率之间具有非常显著性差异(P0.01)。 结论 以人滋养层细胞促融合表位模拟肽与一种通用辅助T细胞表位(PADRE)作为基础,所设计、合成的MAP结构表位多肽具有较好的免疫原性,能够在雌性C57BL/6小鼠体内诱导出较强的体液免疫反应。并且其抗血清可与人滋养层细胞相关抗原结合,并在体外对forskolin诱导的人绒癌细胞(BeWo)间融合具有一定的抑制效果,初步表明所设计的包含人滋养层细胞促融合模拟表位的MAP结构肽可能具有一定抗生育潜力,为进一步研究滋养层细胞抗原表位为基础的抗生育疫苗提供了实验依据和理论参考。
[Abstract]:objective
Based on human trophoblastic epitope mimic peptide (P09-04), peptide immunogen was synthesized to immunize C57BL/6 mice. The characteristics of humoral immune response were observed, and the immunogenicity of C57BL/6 mice was evaluated. Cross-reactivity and its ability to inhibit fusion of human choriocarcinoma cells (BeWo) in vitro were evaluated to evaluate their antifertility potential.
Method
1. Using human trophoblast fusion epitope mimic peptide and a universal helper T cell epitope (PADRE) as the skeleton, we designed eight-branched polyvalent antigen peptide (PADRE-P09-04) 8-MAP and linear chimeric peptide (PADRE-P09-04). The purified peptides were identified by mass spectrometry.
2. Purified MAP structural peptide (P1), linear structural peptide (P2) and epitope mimic peptide (P3) were suspended with Freund's adjuvant and subcutaneously immunized female C57BL/6 mice. Three immunization procedures were used at weeks 0, 3 and 6, 100 ug/mouse. Samples were taken at weeks 2, 5, 8, 10 and 12 after the first immunization. Serum IgG and IgA antibodies in uterine mucosal lavage fluid were determined by ELISA indirectly. The immunogenicity of different synthetic peptides was compared.
3. The ability of specific antibodies in the immune serum to recognize the epitopes of trophoblast-associated antigens was detected by immunohistochemical method.
4. Forskolin-induced fusion of BeWo cells was used to simulate the process of differentiation and fusion of trophoblasts in vivo. Firstly, the proliferation and activity of BeWo cells in the presence of mouse serum were detected by MTT, and then the cells were divided into four groups: group I: cells were cultured in HAM's F-12 medium; group II: cells were cultured in the medium containing 100 mu Forskolin; Group III: cells cultured on medium containing 100 mu Forskolin and 10% unimmunized mouse serum; Group IV: cells cultured on medium containing 100 mu Forskolin and 10% mouse antiserum. After 48 hours of culture, the cell membrane and nucleus of BeWo cells were labeled with immunofluorescence method, and the fusion rate of cells in each group was counted, and the inhibition of BeWo cell fusion by antiserum was analyzed. Ability.
Result
1. The purity of the purified peptides was over 95% by integral calculation. The molecular weights of the purified peptides were 23372, 2826.3 and 1721.8, respectively, which were consistent with the theoretical molecular weights.
2. Detection of anti-epitope mimic peptide IgG antibody in serum of mice: Synthetic peptide P1 group appeared positive at the 5th week, and the antibody level increased with the number and time of immunization, and reached the highest at the 10th week (1:1200); Synthetic peptide P2 group detected low level of anti-epitope mimic peptide IgG antibody (1:200) at the 8th week, the antibody level changed little with time; Synthetic peptide P3 No antibody was detected in the assay time, and there was no significant difference between the negative control group and the negative control group.
3. Detection of anti-epitope mimic peptide IgA antibody in the uterine mucosal lavage fluid of mice after immunization: Low level of anti-epitope mimic peptide IgA antibody was detected in the synthetic peptide P1 group at the 8th week, and reached the highest level at the 10th week. No specific antibody was detected in the synthetic peptide P2 and P3 groups during the observation period, and there was no significant difference between the synthetic peptide P2 and the negative control group. The antibody measurement of mucosal washings showed a trend of polypeptide induced antibody titer in each group: P1P2/P3.
4. Using P1 immunoserum as an antibody, immunohistochemistry showed that the antiserum had specific binding with syncytiotrophoblast cells on the surface and medial cytotrophoblast cells on the villi of early abortion, mainly distributed on the cell membrane and cytoplasm, but did not react with other tissue cells in the villi. There was no cross reaction between human villi.
5. MTT assay showed that there was no significant effect of rat serum on the proliferation and activity of BeWo cells, which provided a precondition for further comparing the fusion rate of each group. 1.45% (group II), in the presence of 10% antiserum, the cell fusion rate decreased significantly to 6.97% (group IV), and there was a very significant difference between group II and group IV (P 0.01), while the cell fusion rate of group III with 10% unimmunized mouse serum was 11.1%, which had no significant difference with group II, but there was a very significant difference between group IV and group II. Sex differences (P0.01).
conclusion
Based on the human trophoblast fusion epitope mimic peptide and a universal helper T cell epitope (PADRE), the synthesized MAP epitope peptide has good immunogenicity and can induce strong humoral immune response in female C57BL/6 mice. The results showed that the designed MAP peptide containing human trophoblastic cell fusion mimic epitope might have certain antifertility potential, which provided experimental basis for further study of trophoblastic epitope-based antifertility vaccine. According to the theory and reference.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2249447
[Abstract]:objective
Based on human trophoblastic epitope mimic peptide (P09-04), peptide immunogen was synthesized to immunize C57BL/6 mice. The characteristics of humoral immune response were observed, and the immunogenicity of C57BL/6 mice was evaluated. Cross-reactivity and its ability to inhibit fusion of human choriocarcinoma cells (BeWo) in vitro were evaluated to evaluate their antifertility potential.
Method
1. Using human trophoblast fusion epitope mimic peptide and a universal helper T cell epitope (PADRE) as the skeleton, we designed eight-branched polyvalent antigen peptide (PADRE-P09-04) 8-MAP and linear chimeric peptide (PADRE-P09-04). The purified peptides were identified by mass spectrometry.
2. Purified MAP structural peptide (P1), linear structural peptide (P2) and epitope mimic peptide (P3) were suspended with Freund's adjuvant and subcutaneously immunized female C57BL/6 mice. Three immunization procedures were used at weeks 0, 3 and 6, 100 ug/mouse. Samples were taken at weeks 2, 5, 8, 10 and 12 after the first immunization. Serum IgG and IgA antibodies in uterine mucosal lavage fluid were determined by ELISA indirectly. The immunogenicity of different synthetic peptides was compared.
3. The ability of specific antibodies in the immune serum to recognize the epitopes of trophoblast-associated antigens was detected by immunohistochemical method.
4. Forskolin-induced fusion of BeWo cells was used to simulate the process of differentiation and fusion of trophoblasts in vivo. Firstly, the proliferation and activity of BeWo cells in the presence of mouse serum were detected by MTT, and then the cells were divided into four groups: group I: cells were cultured in HAM's F-12 medium; group II: cells were cultured in the medium containing 100 mu Forskolin; Group III: cells cultured on medium containing 100 mu Forskolin and 10% unimmunized mouse serum; Group IV: cells cultured on medium containing 100 mu Forskolin and 10% mouse antiserum. After 48 hours of culture, the cell membrane and nucleus of BeWo cells were labeled with immunofluorescence method, and the fusion rate of cells in each group was counted, and the inhibition of BeWo cell fusion by antiserum was analyzed. Ability.
Result
1. The purity of the purified peptides was over 95% by integral calculation. The molecular weights of the purified peptides were 23372, 2826.3 and 1721.8, respectively, which were consistent with the theoretical molecular weights.
2. Detection of anti-epitope mimic peptide IgG antibody in serum of mice: Synthetic peptide P1 group appeared positive at the 5th week, and the antibody level increased with the number and time of immunization, and reached the highest at the 10th week (1:1200); Synthetic peptide P2 group detected low level of anti-epitope mimic peptide IgG antibody (1:200) at the 8th week, the antibody level changed little with time; Synthetic peptide P3 No antibody was detected in the assay time, and there was no significant difference between the negative control group and the negative control group.
3. Detection of anti-epitope mimic peptide IgA antibody in the uterine mucosal lavage fluid of mice after immunization: Low level of anti-epitope mimic peptide IgA antibody was detected in the synthetic peptide P1 group at the 8th week, and reached the highest level at the 10th week. No specific antibody was detected in the synthetic peptide P2 and P3 groups during the observation period, and there was no significant difference between the synthetic peptide P2 and the negative control group. The antibody measurement of mucosal washings showed a trend of polypeptide induced antibody titer in each group: P1P2/P3.
4. Using P1 immunoserum as an antibody, immunohistochemistry showed that the antiserum had specific binding with syncytiotrophoblast cells on the surface and medial cytotrophoblast cells on the villi of early abortion, mainly distributed on the cell membrane and cytoplasm, but did not react with other tissue cells in the villi. There was no cross reaction between human villi.
5. MTT assay showed that there was no significant effect of rat serum on the proliferation and activity of BeWo cells, which provided a precondition for further comparing the fusion rate of each group. 1.45% (group II), in the presence of 10% antiserum, the cell fusion rate decreased significantly to 6.97% (group IV), and there was a very significant difference between group II and group IV (P 0.01), while the cell fusion rate of group III with 10% unimmunized mouse serum was 11.1%, which had no significant difference with group II, but there was a very significant difference between group IV and group II. Sex differences (P0.01).
conclusion
Based on the human trophoblast fusion epitope mimic peptide and a universal helper T cell epitope (PADRE), the synthesized MAP epitope peptide has good immunogenicity and can induce strong humoral immune response in female C57BL/6 mice. The results showed that the designed MAP peptide containing human trophoblastic cell fusion mimic epitope might have certain antifertility potential, which provided experimental basis for further study of trophoblastic epitope-based antifertility vaccine. According to the theory and reference.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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