滋養(yǎng)層細(xì)胞促融合表位肽疫苗的體液免疫效應(yīng)及其抗生育潛能的研究
發(fā)布時(shí)間：2018-09-19 07:29
【摘要】： 目的 以人滋養(yǎng)層細(xì)胞促融合表位模擬肽(P09-04)為基礎(chǔ)設(shè)計(jì)、合成多肽免疫原,免疫C57BL/6型小鼠,觀察體液免疫反應(yīng)的特點(diǎn),評(píng)價(jià)其免疫原性,探討如何將表位模擬肽構(gòu)建成有效的表位肽疫苗;通過(guò)分析表位多肽所誘導(dǎo)的抗血清與天然抗原的交叉反應(yīng)性,及其體外抑制人絨毛膜癌細(xì)胞(BeWo)融合的能力,評(píng)估其抗生育的潛能。 方法 1.以人滋養(yǎng)層細(xì)胞促融合表位模擬肽與一種通用輔助T細(xì)胞表位(PADRE)作為骨架,設(shè)計(jì)出八分枝多價(jià)抗原肽(PADRE-P09-04)8-MAP和線性嵌合肽(PADRE-P09-04)兩種結(jié)構(gòu)免疫原。在固相自動(dòng)肽合成儀上進(jìn)行合成,高效液相色譜儀上進(jìn)行純化及純度分析,純化后的多肽行質(zhì)譜鑒定。 2.純化后的MAP結(jié)構(gòu)肽(P1)、線性結(jié)構(gòu)肽(P2)和表位模擬肽(P3),分別與等量弗氏佐劑混懸后皮下免疫雌性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周取樣,應(yīng)用ELISA間接法測(cè)定血清IgG及子宮粘膜沖洗液中IgA抗體生成情況,比較不同合成肽的免疫原性。 3.取正常妊娠早期(6～8周)人工流產(chǎn)的新鮮絨毛組織,以效價(jià)較高的小鼠免疫血清為一抗,以未免疫的正常小鼠血清作為陰性對(duì)照,通過(guò)免疫組化法檢測(cè)免疫血清中特異性抗體識(shí)別人滋養(yǎng)層細(xì)胞相關(guān)抗原表位的能力。 4.應(yīng)用forskolin誘導(dǎo)的BeWo細(xì)胞融合來(lái)模擬體內(nèi)滋養(yǎng)細(xì)胞的分化融合過(guò)程。首先通過(guò)MTT法檢測(cè)小鼠血清存在下BeWo細(xì)胞增殖及活性情況,然后將細(xì)胞分為四組:Ⅰ組:應(yīng)用HAM’s F-12培養(yǎng)基培養(yǎng)細(xì)胞;Ⅱ組:含有100μM Forskolin的培養(yǎng)基培養(yǎng)細(xì)胞;Ⅲ組:含100μM Forskolin、10%未免疫鼠血清的培養(yǎng)基培養(yǎng)細(xì)胞;Ⅳ組:含100μM Forskolin、10%鼠抗血清培養(yǎng)基培養(yǎng)細(xì)胞。各組細(xì)胞培養(yǎng)48小時(shí)后,應(yīng)用細(xì)胞免疫熒光法分別標(biāo)記BeWo細(xì)胞膜與胞核,統(tǒng)計(jì)各組細(xì)胞的融合率,分析抗血清抑制BeWo細(xì)胞融合的能力。 結(jié)果 1.純化后的合成肽行HPLC均顯示為單峰,經(jīng)積分計(jì)算,純度均達(dá)到95%以上,質(zhì)譜分析結(jié)果顯示分子量分別為23372、2826.3和1721.8,與理論分子量相吻合。 2.小鼠血清中抗表位模擬肽IgG抗體的檢測(cè):合成肽P1組第5周出現(xiàn)陽(yáng)性,抗體水平隨免疫次數(shù)和時(shí)間而升高,至第10周達(dá)到最高(1:1200);合成多肽P2組第8周檢測(cè)到低水平的針對(duì)模擬表位的IgG抗體(1:200),抗體水平隨時(shí)間變化不大;合成多肽P3在在測(cè)定的時(shí)間未檢測(cè)到抗體的產(chǎn)生,與陰性對(duì)照組不具有顯著性差異。且五次血清抗體測(cè)量,都呈現(xiàn)出了各組多肽誘導(dǎo)抗體滴度的趨勢(shì)為:P1P2P3。 3.小鼠免疫后子宮粘膜沖洗液中抗表位模擬肽IgA抗體的檢測(cè):合成肽P1組第8周檢測(cè)到了低水平的抗表位模擬肽IgA抗體,第10周達(dá)到最高,而合成肽P2與P3組在觀測(cè)時(shí)間內(nèi)未檢測(cè)出特異性抗體,并且與陰性對(duì)照組無(wú)顯著性差異。且五次子宮粘膜沖洗液的抗體測(cè)量,都呈現(xiàn)出了各組多肽誘導(dǎo)抗體滴度的趨勢(shì)為:P1P2/P3。 4.應(yīng)用P1組免疫血清作為一抗,經(jīng)免疫組化法顯示抗血清與早期人流產(chǎn)絨毛組織表面的合體滋養(yǎng)層細(xì)胞及內(nèi)側(cè)的細(xì)胞滋養(yǎng)層細(xì)胞呈現(xiàn)特異的結(jié)合,主要分布在細(xì)胞胞膜上和胞漿中,但與絨毛中其他組織細(xì)胞無(wú)反應(yīng)。未免疫鼠正常血清與人絨毛組織間無(wú)交叉反應(yīng)。 5.應(yīng)用MTT法檢測(cè)結(jié)果提示鼠血清對(duì)BeWo細(xì)胞的增值及活性無(wú)明顯影響,為下一步比較各組細(xì)胞融合率提供了前提條件。應(yīng)用BeWo細(xì)胞融合來(lái)模擬體內(nèi)滋養(yǎng)細(xì)胞的分化融合的實(shí)驗(yàn)顯示:在forskolin誘導(dǎo)下,BeWo細(xì)胞間融合率由1.59%(Ⅰ組)升高到11.45%(Ⅱ組),在10%抗血清存在下,細(xì)胞間融合率明顯下降至6.97%(Ⅳ組),且Ⅱ組細(xì)胞融合率與Ⅳ組之間具有非常顯著性差異(P0.01),而含有10%未免疫鼠血清的Ⅲ組細(xì)胞融合率為11.1%,與Ⅱ組無(wú)明顯差異,但與Ⅳ組細(xì)胞融合率之間具有非常顯著性差異(P0.01)。 結(jié)論 以人滋養(yǎng)層細(xì)胞促融合表位模擬肽與一種通用輔助T細(xì)胞表位(PADRE)作為基礎(chǔ),所設(shè)計(jì)、合成的MAP結(jié)構(gòu)表位多肽具有較好的免疫原性,能夠在雌性C57BL/6小鼠體內(nèi)誘導(dǎo)出較強(qiáng)的體液免疫反應(yīng)。并且其抗血清可與人滋養(yǎng)層細(xì)胞相關(guān)抗原結(jié)合,并在體外對(duì)forskolin誘導(dǎo)的人絨癌細(xì)胞(BeWo)間融合具有一定的抑制效果,初步表明所設(shè)計(jì)的包含人滋養(yǎng)層細(xì)胞促融合模擬表位的MAP結(jié)構(gòu)肽可能具有一定抗生育潛力,為進(jìn)一步研究滋養(yǎng)層細(xì)胞抗原表位為基礎(chǔ)的抗生育疫苗提供了實(shí)驗(yàn)依據(jù)和理論參考。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
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