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
|
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:P1>P2>P3。
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:P1>P2/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(P<0.01),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(P<0.01)。
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。
Objective:
Based on fusogenic epitope mimic epeptid of trophoblastic cell(P09-04), antigenic peptides for immunocontraception were designed and synthesized to immune female C57BL/6 mice. Then we observed the antigenic peptides’humoral immune response and evaluate their immunogenicity to explore how to obtain an effective epitope polypeptide vaccine from epitope mimic peptide. Anti-fertile potential of the polypeptide vaccine was evaluated by studying the cross-reactivity between anti-serum induced by the epitope polypeptide and natural antigens, and anti-serum’s effect on inhibiting intercellular fusion of BeWo choriocarcinoma cells in vitro.
Methods:
1. Based on fusogenic epitope mimic peptide and synthetic non-natural Pan DR T Helper Epitope(PADRE), both Multiple Antigenic Peptide (PADRE-P09-04)8-MAP and linear structural peptide (PADRE-P09-04) were designed and subsequently synthesized on the peptide synthesizer. After the peptide was synthesized, they were purified in high performance liquid chromatography (HPLC) and identified in mass spectrometer.
2. Three antigens such as MAP (P1) ,linear structural peptide(P2), and epitope mimic peptide(P3) were combined with the equivalence Freund’s adjuvant to immunize the female C57BL/6 mice(100μg per mice) subcutaneouly at week 0, 2 and 4 respectively. To compare the immunogenicity of the three peptide antigens, the presence and reactivity of antibody (IgG and IgA) in serum and uterine mucous membrane washings were analyzed against epitope mimic peptide by ELISA at week 2, 5, 8, 10, and 12 after first immunization .
3. Fresh chorionic tissue was obtained from female patients undergoing induced abortion during their early timester of normal pregnancy. Murine anti-serum was applied as first antibody while normal murine serum without immunization was used as negative control. The potency of human trophoblastic cell related antigen epitope was identified by the specific antibody in immunized serum.
4. Forskolin-induced choriocarcinoma BeWo cells were applied to mimic the process of differentiation and fusion of villous cytotrophoblast in vivo. Firstly, we applied MTT assay to detect the proliferation and activity of BeWo cells under the influence of murine serum. Then, we divided the cells into four groups: GroupⅠ: BeWo cells were cultured in HAM’S F12 medium; GroupⅡ: BeWo cells were cultured in medium containing 100μM forskolin ; GroupⅢ: BeWo cells were cultured medium containing 100μM forskolin and 10% unimmunized murine serum; GroupⅣ: BeWo cells were cultured medium containing 100μM forskolin and 10% murine anti-serum. After 48 hours culture, the membrane and nuclear of BeWo cells were marked by immunofluorescence., then intercellular fusion was quantified to analyze the potency of anti-serum on inhibiting the fusion of BeWo cells.
Results:
1. Purified synthetic peptides were demonstrated as mono peak by HPL and their purity could reach 95% after integral calculation. The mass chromatographic analysis indicated molecular weight of synthetic peptides were 23372 and 2826.3 and 1727.8 respectively, which were identical with theorical molecular weight.
2. Detection of anti-epitope mimic polypeptide IgG in murie serum: synthetic peptide P1 became positive at week 5, the antibody levels were related with the numbers of immunization and time and reached maximum (1:1200) at week 10. Low levels of mimic eptipe IgG antibody (1:200)was detected at week 8 in synthetic peptide P2 group, but the levels of antibody did not show significant changes through time. In synthetic peptide P3 group, no antibody could be detected in our experiment in synthetic peptide P3 group and did not show significant difference when comparing with normal controls. In all, polypeptide induced antibody titer showed the trend to decrease from PI to P2 to P3 in five detections.
3. Detection of anti-epitope mimic polypeptide IgA in murie uterine mucous membrane washings: Low levels of mimic eptipe IgA antibody could be detected at week 8 and reached maximum at week 10 in synthetic peptide P1 group . But there were no specific antibody detected in synthetic peptide P2 and P3 groups, also there were no significant differences between normal controls , synthetic peptide P2 group, and synthetic peptide P3 group. In all, polypeptide induced antibody titer showed the trend to decrease from PI to P2 to P3 in five detection.
4. Immunized serum in P1 group served as first antibody. Then immunohistochemistry showed the specific binding between anit-serum, and syncytiotrophoblast as well as cytotrophoblast in chorionic tissue from female patients undergoing induced abortion during their early timester of normal pregnancy. The binding mainly happened on cell membrane and in Kytoplasma. But no reaction could be drawn between anti-serum and other tissue cells in chorionic villi. There is no reaction between normal murine serum and human chorionic villi.
5. MTT showed murine serum had no effect on proliferation and activity of BeWo cells, which permitted the next step of comparing the fusion rate in all groups. Choriocarcinoma BeWo cells were applied to mimic the differentiation and fusion of villous cytotrophoblast in vivo, and the results demonstrated that , under the induce of forskolin, the fusion rate between BeWo cells increased from 1.59% (groupⅠ) to 11.45%(groupⅡ); while under the influence of 10% anti-serum, the intercellular fusion rate decreased to 6.97 % ( groupⅣ) . There is significant difference betweenⅡgroup andⅣgroup(P<0.01) .The fusion rate in group III with normal murine serum was 11.1%. There was no significant difference between group III and groupⅡ, while the results between group III and group IV was contrary(P<0.01).
Conclusion:
MAP constructive epitope polypeptide based on fusogenic epitope mimic epeptid of trophoblastic cell and one general Th cells epitope(PADRE) was proved to possess good immunogenicity and induce strong humoral immune reaction in female C57BL/6 mice, furthermore its antirserum could identify natural antigen epitope of human trophoblastic cell and significantly inhibited the intercellular fusion of BeWo choriocarcinoma cells. Our study demonstrated that MAP constructive peptide with fusogenic epitope mimic epeptid of human trophoblastic cell was capable of antireproduction. It is feasible to design immunocontraception vaccine based on this epitope mimic peptide. Our study provided basic theory for immunocontraception vaccine acting on trophoblastic cell.
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:P1>P2>P3。
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:P1>P2/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(P<0.01),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(P<0.01)。
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。
Objective:
Based on fusogenic epitope mimic epeptid of trophoblastic cell(P09-04), antigenic peptides for immunocontraception were designed and synthesized to immune female C57BL/6 mice. Then we observed the antigenic peptides’humoral immune response and evaluate their immunogenicity to explore how to obtain an effective epitope polypeptide vaccine from epitope mimic peptide. Anti-fertile potential of the polypeptide vaccine was evaluated by studying the cross-reactivity between anti-serum induced by the epitope polypeptide and natural antigens, and anti-serum’s effect on inhibiting intercellular fusion of BeWo choriocarcinoma cells in vitro.
Methods:
1. Based on fusogenic epitope mimic peptide and synthetic non-natural Pan DR T Helper Epitope(PADRE), both Multiple Antigenic Peptide (PADRE-P09-04)8-MAP and linear structural peptide (PADRE-P09-04) were designed and subsequently synthesized on the peptide synthesizer. After the peptide was synthesized, they were purified in high performance liquid chromatography (HPLC) and identified in mass spectrometer.
2. Three antigens such as MAP (P1) ,linear structural peptide(P2), and epitope mimic peptide(P3) were combined with the equivalence Freund’s adjuvant to immunize the female C57BL/6 mice(100μg per mice) subcutaneouly at week 0, 2 and 4 respectively. To compare the immunogenicity of the three peptide antigens, the presence and reactivity of antibody (IgG and IgA) in serum and uterine mucous membrane washings were analyzed against epitope mimic peptide by ELISA at week 2, 5, 8, 10, and 12 after first immunization .
3. Fresh chorionic tissue was obtained from female patients undergoing induced abortion during their early timester of normal pregnancy. Murine anti-serum was applied as first antibody while normal murine serum without immunization was used as negative control. The potency of human trophoblastic cell related antigen epitope was identified by the specific antibody in immunized serum.
4. Forskolin-induced choriocarcinoma BeWo cells were applied to mimic the process of differentiation and fusion of villous cytotrophoblast in vivo. Firstly, we applied MTT assay to detect the proliferation and activity of BeWo cells under the influence of murine serum. Then, we divided the cells into four groups: GroupⅠ: BeWo cells were cultured in HAM’S F12 medium; GroupⅡ: BeWo cells were cultured in medium containing 100μM forskolin ; GroupⅢ: BeWo cells were cultured medium containing 100μM forskolin and 10% unimmunized murine serum; GroupⅣ: BeWo cells were cultured medium containing 100μM forskolin and 10% murine anti-serum. After 48 hours culture, the membrane and nuclear of BeWo cells were marked by immunofluorescence., then intercellular fusion was quantified to analyze the potency of anti-serum on inhibiting the fusion of BeWo cells.
Results:
1. Purified synthetic peptides were demonstrated as mono peak by HPL and their purity could reach 95% after integral calculation. The mass chromatographic analysis indicated molecular weight of synthetic peptides were 23372 and 2826.3 and 1727.8 respectively, which were identical with theorical molecular weight.
2. Detection of anti-epitope mimic polypeptide IgG in murie serum: synthetic peptide P1 became positive at week 5, the antibody levels were related with the numbers of immunization and time and reached maximum (1:1200) at week 10. Low levels of mimic eptipe IgG antibody (1:200)was detected at week 8 in synthetic peptide P2 group, but the levels of antibody did not show significant changes through time. In synthetic peptide P3 group, no antibody could be detected in our experiment in synthetic peptide P3 group and did not show significant difference when comparing with normal controls. In all, polypeptide induced antibody titer showed the trend to decrease from PI to P2 to P3 in five detections.
3. Detection of anti-epitope mimic polypeptide IgA in murie uterine mucous membrane washings: Low levels of mimic eptipe IgA antibody could be detected at week 8 and reached maximum at week 10 in synthetic peptide P1 group . But there were no specific antibody detected in synthetic peptide P2 and P3 groups, also there were no significant differences between normal controls , synthetic peptide P2 group, and synthetic peptide P3 group. In all, polypeptide induced antibody titer showed the trend to decrease from PI to P2 to P3 in five detection.
4. Immunized serum in P1 group served as first antibody. Then immunohistochemistry showed the specific binding between anit-serum, and syncytiotrophoblast as well as cytotrophoblast in chorionic tissue from female patients undergoing induced abortion during their early timester of normal pregnancy. The binding mainly happened on cell membrane and in Kytoplasma. But no reaction could be drawn between anti-serum and other tissue cells in chorionic villi. There is no reaction between normal murine serum and human chorionic villi.
5. MTT showed murine serum had no effect on proliferation and activity of BeWo cells, which permitted the next step of comparing the fusion rate in all groups. Choriocarcinoma BeWo cells were applied to mimic the differentiation and fusion of villous cytotrophoblast in vivo, and the results demonstrated that , under the induce of forskolin, the fusion rate between BeWo cells increased from 1.59% (groupⅠ) to 11.45%(groupⅡ); while under the influence of 10% anti-serum, the intercellular fusion rate decreased to 6.97 % ( groupⅣ) . There is significant difference betweenⅡgroup andⅣgroup(P<0.01) .The fusion rate in group III with normal murine serum was 11.1%. There was no significant difference between group III and groupⅡ, while the results between group III and group IV was contrary(P<0.01).
Conclusion:
MAP constructive epitope polypeptide based on fusogenic epitope mimic epeptid of trophoblastic cell and one general Th cells epitope(PADRE) was proved to possess good immunogenicity and induce strong humoral immune reaction in female C57BL/6 mice, furthermore its antirserum could identify natural antigen epitope of human trophoblastic cell and significantly inhibited the intercellular fusion of BeWo choriocarcinoma cells. Our study demonstrated that MAP constructive peptide with fusogenic epitope mimic epeptid of human trophoblastic cell was capable of antireproduction. It is feasible to design immunocontraception vaccine based on this epitope mimic peptide. Our study provided basic theory for immunocontraception vaccine acting on trophoblastic cell.
引文
1. Rajesh KN. Molecular and immunological characteristics of sperm antigens involved in egg binding[J]. J of Rep rod Immunol, 2002, 53:13-23.
2. Lea IA, Kurth B, O’Rand MG. Immune response to immunization with sperm antigens in the macaque oviduct[J]. Biol Rep rod, 1998, 58:794-800.
3. Tung KS, Primokoff P, Woolman GL, et al.Mechanism of inf ertility in male Guinea pigs immunised wit h sperm PH-20[J]. Biol Rep rod, 1997, 56:1133-1141.
4. Goldberg E, Herr JC. Rep roductive Immunology[J]. Narosa Press, 1999, pp:309-315.
5. Shetty J, Wolkowicz MJ, Digilio LC, et al. SAMP14,a novel,acrosomal membrane- associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokin- ase-type plasminogen activator receptor superfamilywith a role in sperm-egg interaction[J]. J Biol Chem, 2003, 278:30506-30515.
6. Miller LA, J ohnes B E, Killian GJ. Immunocont raception of white-tailed deer with GnR H vaccine[J]. Am J Rep rod Immunol, 2000, 44:266-274.
7. Peter J D, Torben L, Ivan MR. Antif ertility vaccines[J]. Trends in Immunol, 2002, 23: 213 - 219.
8. 王梦玖. 临床生殖免疫学(M). 第 1 版. 上海:上海科学技术出版社, 2000, 354-355
9. Bambra CS. Anti-trophectoderm vaccines:rationale and methods used for antigen identification and selection[J]. Scand J Immunol Suppl, 1992, 11: 131-136.
10. Frank HG, Bose P, Albieri-Borges A, Kaufmann P, et al. Evaluation of fusogenic tropho- blast surface epitopes as targets for immunecontraception[J]. Contraception, 2005, 71: 282-293.
11. Benirschke K, Kaufmann P. Pathology of the human placenta[M]. 4thed.New York: Springer Verlag, 2000.
12. Berger M, Shankar V, Vafai A, et al. Therapeutic applications of moloclonal antibodies [J]. AmJ Med Sci, 2002, 324:14-30.
13. AlexanderJ, SydneyJ, SouthwoodS, et al. Development of high potency universal DR- restricted helper epitopes by modification of high affinity DR-blocking peptides[J]. Immunity, 1994, 1:751-761.
14. 汪进,罗伟. 链霉亲和素-生物素-ELISA方法的建立及其在 HBsAg 测定中的应用[J].上海医学检验杂志, 1998, 13(4):213-214.
15. 贾秀娟, 谢晓雁, 张迪,等. 胰岛素瘤相关蛋白-2 自身抗体酶联免疫吸附测定方法的建立[J]. J Diagn concepts Pract, 2003, 2(2):110-112.
16. 吴玉章, 刘茂昌, 贾正才, 等. HBV 新型免疫原的设计、合成及免疫原性研究[J].第三军医大学学报, 2000, 22(10):919-923.
17. Scheibenbogen C, Sun Y, Keilholz U, et al. Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL2-based treatment[J]. Int J Cancer, 2002, 98(3): 409-414.
18. Panigada M, Sturniolo T, Besozzi G, et al .Identification of a promiscuous T-cell epitope in Mycobacteriumtuberculosis Mce proteins[J]. Infect Immun, 2002, 70(1): 79-85.
19. Hou Y, Gu XX. Development of peptide minotopes of lipooligosaccharide from nontypeable haemophilus influenzae as vaccine candidates[J]. J Immunol, 2003, 170(8): 4373- 4379.
20. Waiyan Candy NG, Mingchiu Fung. The Importance of Helper T cell Epitope in Vaccine Development[J]. 热带医学杂志, 2002, 2(3):207-213.
21. Del Guercio MF, Alexander J, Kubo RT, et al. Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper Epitopes(PADRE) for antibody responses in vivo[J]. vaccine, 1997, 15(4):441-448.
22. Nardin EH, Calvo-Calle JM, Oliveira GA, et al. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types[J]. J Immunol, 2001, 166(1): 481-489.
23. Jeff Alexander, Marie-France del Guercio, Barbara Frame, et al. Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE?)[J]. Vaccine, 2004, 22:2362-2367.
24. Partidos C, stanley C, steward M. The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides[J]. Eur J Immunol, 1992, 22(10):2675-2680.
25. Sette A, Fikes J. Epitope-based vaccines:an update on epitope identification, vaccine design and delivery[J]. Curr-Opin-Immunol, 2003, 15(4):461-470.
26. Alexander J, Oseroff C, Dahlberg C, et al. A decaepitope polypeptide primes for multiple CD8R IFN- and Th lymphocyte responses:evaluation of multiepitope poly- peptides as a mode for vaccine eelivery[J]. J Immunol, 2002, 168(12):6189-6198.
27. TamJP.Recent advances in multiple antigen peptides[J].Journal of immunological methods, 1996, 196(1):17-32.
28. Kawamura KS, Su R-C, Nguyen LT, et al. In vivo generation of cytotoxic T cells from epitopes displayed on peptide-based delivery vehicles[J]. J Immunol, 2002, 168(11): 5709-5715.
29. Del GG, Nardelli B, Huang W, et al. A multiple antigen peptide from the reptive sequenceof the plasmodiue malarise circumsporzoite protein induces a specific antibody response in mice of various H-2 haplotype[J]. Eur J Immunol, 1990, 20 (7):1619- 1622.
30. 曹胜利,菜孟深. 含两种不同肽段的血吸虫多抗原肽疫苗的合成与生物活性[J].药学学报, 1999, 34(10)751-754.
31. 汪海丹,丁淑香. 病毒 T 细胞表位和 T 细胞疫苗的研究进展[J]. 国外医学.预防诊断、治疗用生物制品分册, 1999, 22(4):152-156.
32. 潘勇, 顾正, 左嘉客. 局部避免和避孕疫苗[J]. 生殖与避孕, 1999, 19(6)328-334.
33. 何畏, 史常旭, 梁志清, 等. 多价精子抗原表位多肽-免疫刺激复合物的抗生育作用研究[J]. 重庆医学, 2002, 31(7)555-556.
34. Russell MW, Moldoveanu Z, White PL, et al. Salivary,nasal,genital,and systemic antibody responsesin monkeys immunized intranasally with a bacterial protein antigen and the cholera toxin B subunit[J]. Infect Immun, 1996, 64:1272 .
35. Borges M, Bose P, Frank HG, et al. A two-color fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines[J]. Placenta, 2003, 24(10): 959- 964.
36. Das.M, Xu.B, Rote.NS, et al. Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast[J]. Placenta, 2004, 25(5):396-407.
37. 成令忠. 组织学与胚胎学[M]. 第四版. 北京, 人民出版社, 1996, 265-267.
38. James C, Keith JD. Placental syncytin expression in normal and preeclampsia pregnancies[J]. AmJ Obstet Gynecol, 2002, 187(4):1122-1124.
39. Knerr I, Huppertz B, Weigel C, et al. Endogenous retroviral syncytin:compilation of experimental research on syncytin and its possible role in normal and disturbed human placentogenesis[J]. Mol Hum Reprod, 2004, 10(8):581-588.
40. Huppertz B, Tews DS, Kaufmann P. Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle[J]. Int Rev Cytol, 2001, 205:215-253.
41. Vignery A.Osteoclasts and giant cells:macrophage-macrophage fusion mechan- ism[J]. Int J Exp Pathol, 2000, 81(5):291-304.
42. Lee A, Morrow JS, Fowler VM. Caspase remodeling of the spectrin membrane skeleton during lens development and aging[J]. J Biol Chem, 2001, 276(23):20735 -20742.
43. Potgens AJ, Schmitz U, Bose P, et al.Mechanisms of syncytial fusion:a review[J]. Placenta Trophoblast Research, 2002, 23(6):S107-S113.
44. Gilpin BJ, Loechel F, Mattei MG, et al. A novel, secreted form of human ADAM
12(meltrin alpha)provokes myogenesis in vivo[J]. J-Biol-Chem, 1998, 273(1):157-166.
45. Frendo JL, Olivier D, Cheynet V, et al. Direct involvement of HERV-W Env glycol- protein in human trophoblast cell fusion and differiation[J]. Mol Cell Biol, 2003, 23(10):3566-3574.
46. Frendo JL, Cronier L, Bertin G, et al. Involvement of connexin 43 in human trophoblast cell fusion and differentiation[J]. J Cell Sci, 2003, 116(16):3413-3421.
47. Stoute JA, Ballou WR, Kolodny N, et al. Induction of humoral immune response against Plasmodiumfalciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library[J]. Infect Immun, 1995, 63:934-941.
48. Cortese R, Monaci P, Nicosia A, et al. Identification of biologically active peptides using random libraries displayed on phage[J]. Curr Opin Biotechnol, 1997, 6:73-80.
49. 王雁玲,庄临之.人胎盘滋养层细胞的功能分化及其调节[J].生殖与避孕, 1999, 19(6):335-341.
50. Sha M, Lee XH, Li XP, et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[J]. Nature, 2000, 403:785- 789.
1. Huppertz B, Tews DS, Kaufmann P. Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle[J]. Int Rev Cytol, 2001, 205: 215- 253.
2. Vignery A.Osteoclasts and giant cells:macrophage-macrophage fusion mecha- nism[J]. Int J Exp Pathol, 2000, 81(5):291-304.
3. Lee A, Morrow JS, Fowler VM. Caspase remodeling of the spectrin membrane skeleton during lens development and aging[J]. J Biol Chem, 2001, 276(23): 20735 -20742.
4. Potgens AJ, Schmitz U, Bose P, et al. Mechanisms of syncytial fusion:a review [J]. Placenta, 2002, 23(Suppl A):S107-S113.
5. Benirschke K, Kaufmann P. Pathology of the human placenta[M]. 4th ed.New York: Springer Verlag, 2000.
6. Daleke DL, Lyles JV. Identification and purification of aminophospholipid flippases[J]. Biochimica et Biophysica Acta, 2000, 1486(1):108-127.
7. Sims PJ, Wiedmer T. Unraveling the mysteries of phospholipid scrambling. Thrombosis and Haemostasis[J], 2001, 86(1):266-275.
8. Das.M,Xu.B, Rote. NS, et al. Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast[J]. Placenta, 2004, 25(5):396-407.
9. Adler RR, Ng AK, Rote NS. Monoclonal antiphosphatidylserine antibody inhib- its intercellular fusion of the choriocarcinoma line, JAR[J]. Biology of Reproduction, 1995, 53(4):905-910.
10. Black S, Kadyrov M, Huppertz B, et al. Syncytial fusion of human trophoblast depends on caspase8[J]. Cell-Death-Differ, 2004,11(1):90-98.
11. Blond JL, Beseme F, Duret L,et al. Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family[J]. J Virol, 1999, 73(2): 1175-1185.
12. Borges M, Bose P, Frank HG, et al. A two-color fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines[J]. Placenta, 2003, 24(10): 959-964.
13. Sha M, Lee XH, Li XP, et al. Syncytin is a captive retroviral envelope protein involvedin human placental morphogenesis[J]. Nature, 2000, 403(6765-6771):785-789.
14. Frendo JL, Olivier D, Cheynet V, et al. Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differiation[J]. Mol Cell Biol, 2003, 23(10):3566-3574.
15. Kudo Y, Boyd CA. Changs in expression and function of syncytin and receptors , amino acid transport system B(0)(ASCT2),in human placental choriocarcinoma BeWo cells during syncytialization[J]. Placenta, 2002, 23(7):536-541.
16. Gong R,Peng X,Kang S, et al. Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W[J]. Biochem- Biophys- Res-Commun. 2005, 331(4):1193-200.
17. Gilpin BJ, Loechel F, Mattei MG, et al. A novel, secreted form of human ADAM
12(meltrin alpha)provokes myogenesis in vivo[J]. J-Biol-Chem, 1998, 273(1): 157-166.
18. Cronier L, Defamie N, Malassine A, et al. Connexin expression and gap junctio- nal intercellular communication in human first trimester trophoblast[J]. Mol-Hum-Reprod, 2002, 8(11):1005-1013.
19. Cronier L, Frendo JL, Malassine A, et al. Requirement of gap junctional intercellular communication for human villous trophoblast differentiation [J]. Biol-Reprod, 2003, 69(5):1472-1480.
20. Frendo JL, Cronier L, Bertin G, et al. Involvement of connexin 43 in human trophoblast cell fusion and differentiation[J]. J Cell Sci, 2003, 116(16): 3413-3421.
21. Frank HG, Bose P, Kaufmann P, et al. Evaluation of fusogenic trophoblast surface epitopes as targets for immunecontraception [J]. Contraception, 2005, 71(4):282-293.
22. James C, Keith JD. Placental syncytin expression in normal and preeclampsia pregnancies[J]. AmJ Obstet Gynecol, 2002, 187(4):1122-1124.
23. Knerr I, Huppertz B, Weigel C, et al. Endogenous retroviral syncytin: compilation of experimental research on syncytin and its possible role in normal and disturbed human placentogenesis[J]. Mol Hum Rep rod, 2004, 10(8): 581-588.
24. Bose P, Kadyrov M, Goldin R, et al. Aberrations of early trophoblast differen- tiation predispose to pregnancy failure:lessons from the anti-phospholipid syndrome[J]. Placenta, 2006, 27(8):869-875.
1. Sette A, Fikes J. Epitope-based vaccines:an update on epitope identification, vaccine design and delivery[J]. Curr-Opin-Immunol. 2003, 15(4):461-470.
2. Hou Y, Gu XX. Development of peptide minotopes of lipooligosaccharide from nontypeable haemophilus influenzae as vaccine candidates[J]. J Immunol, 2003, 170(8):4373-4379.
3. Frank HG, Bose P, Albieri-Borges A, Kaufmann P, et al. Evaluation of fusogenic trophoblast surface epitopes as targets for immune contraception[J]. Contraception, 2005, 71(4):282-293.
4. Le Doussal JM, Piqueras B, DoganI, et al. Phage display of peptide/major histocompatibility complex[J]. J-Immunol-Methods, 2000, 241(2):147-158.
5. Scheibenbogen C, Sun Y, Keilholz U, et al. Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL2-based treatment[J]. Int J Cancer, 2002, 98 (3):409-414.
6. Panigada M, Sturniolo T,Besozzi G,et al. Identification of a promiscuous T-cell epitope in Mycobacteriumtuberculosis Mce proteins[J]. Infect Immun, 2002, 70(1):79-85.
7. Del Guercio MF, Alexander J, Kubo RT, et al. Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper Epitopes(PADRE) for antibody responses in vivo[J]. vaccine, 1997, 15(4): 441-448.
8. Nardin EH, Calvo-Calle JM, Oliveira GA, et al. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types [J]. J Immunol, 2001, 166(1): 481-489.
9. Livingston BD,Newman M,Crimi C, et al. Optimization of epitope processing enhances immunogenicity of DNA vaccines[J]. Vaccine, 2001, 19(32):4652-4660.
10. Livingston B, Crimi C, Newman M, et al. A rational strategy to design multiepitope immunogens-based multiple HTL epitopes[J]. J Immunol, 2002, 168 (11):5499-5506.
11. Velders MP,Weijzen S, Eiben GL, et al. Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNAvaccine[J]. J-Immunol, 2001, 166(9):5366-5373.
12. Partidos C, stanley C, steward M.The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides[J]. Eur J Immunol, 1992, 22(10):2675-2680.
13. Deliyannis G, Jackson DC, Ede NJ,et al. Induction of long-term memory CD8R T cells for recall of viral clearing responses against influenza virus[J]. J Virol, 2002, 76(9): 4212-4221.
14. BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines yesterday,today, and tomorrow[J]. Lancet Infec Dis, 2002, 2(7):425-431.
15. Kawamura KS, Su R-C, Nguyen LT, et al. In vivo generation of cytotoxic T cells from epitopes displayed on peptide-based delivery vehicles[J]. J Immunol, 2002, 168(11): 5709-5715.
16. 何畏, 史常旭, 梁志清, 等. 多价精子抗原表位多肽-免疫刺激复合物的抗生育作用研究[J].重庆医学, 2002, 31(7):555-556.
17. Alexander J, Oseroff C, Dahlberg C, et al. A decaepitope polypeptide primes for multiple CD8R IFN- and Th lymphocyte responses: evaluation of multi- epitope polypeptides as a mode for vaccine eelivery[J]. J Immunol, 2002, 168(12):6189-6198.
18. Zwaveling S, Ferreira Mota SC, Melief CJ, et al. Established human papill- omavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides[J]. J Immunol, 2002, 169(1):350-358.
19. Barton GM, Medzhitov R. Toll-like receptor signaling pathways[J]. Science, 2003, 300 (5625):1524-1525.
20. La Rosa C, Wang Z, Diamond DJ, et al. Preclinical development of an adjuvant free peptide vaccine with activity against CMV pp65 in HLA transgenic mice[J]. Blood, 2002, 100(10):3681-3689.
21. Chikh GG, Kong S, Bally MB, et al. Efficient delivery of antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8R T cells[J]. J Immunol, 2001, 167(11): 6462-6470.
22. Leifert JA, Harkins S, Whitton JL. Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity[J]. Gene Ther, 2002, 9(21):1422-1428.
2. Lea IA, Kurth B, O’Rand MG. Immune response to immunization with sperm antigens in the macaque oviduct[J]. Biol Rep rod, 1998, 58:794-800.
3. Tung KS, Primokoff P, Woolman GL, et al.Mechanism of inf ertility in male Guinea pigs immunised wit h sperm PH-20[J]. Biol Rep rod, 1997, 56:1133-1141.
4. Goldberg E, Herr JC. Rep roductive Immunology[J]. Narosa Press, 1999, pp:309-315.
5. Shetty J, Wolkowicz MJ, Digilio LC, et al. SAMP14,a novel,acrosomal membrane- associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokin- ase-type plasminogen activator receptor superfamilywith a role in sperm-egg interaction[J]. J Biol Chem, 2003, 278:30506-30515.
6. Miller LA, J ohnes B E, Killian GJ. Immunocont raception of white-tailed deer with GnR H vaccine[J]. Am J Rep rod Immunol, 2000, 44:266-274.
7. Peter J D, Torben L, Ivan MR. Antif ertility vaccines[J]. Trends in Immunol, 2002, 23: 213 - 219.
8. 王梦玖. 临床生殖免疫学(M). 第 1 版. 上海:上海科学技术出版社, 2000, 354-355
9. Bambra CS. Anti-trophectoderm vaccines:rationale and methods used for antigen identification and selection[J]. Scand J Immunol Suppl, 1992, 11: 131-136.
10. Frank HG, Bose P, Albieri-Borges A, Kaufmann P, et al. Evaluation of fusogenic tropho- blast surface epitopes as targets for immunecontraception[J]. Contraception, 2005, 71: 282-293.
11. Benirschke K, Kaufmann P. Pathology of the human placenta[M]. 4thed.New York: Springer Verlag, 2000.
12. Berger M, Shankar V, Vafai A, et al. Therapeutic applications of moloclonal antibodies [J]. AmJ Med Sci, 2002, 324:14-30.
13. AlexanderJ, SydneyJ, SouthwoodS, et al. Development of high potency universal DR- restricted helper epitopes by modification of high affinity DR-blocking peptides[J]. Immunity, 1994, 1:751-761.
14. 汪进,罗伟. 链霉亲和素-生物素-ELISA方法的建立及其在 HBsAg 测定中的应用[J].上海医学检验杂志, 1998, 13(4):213-214.
15. 贾秀娟, 谢晓雁, 张迪,等. 胰岛素瘤相关蛋白-2 自身抗体酶联免疫吸附测定方法的建立[J]. J Diagn concepts Pract, 2003, 2(2):110-112.
16. 吴玉章, 刘茂昌, 贾正才, 等. HBV 新型免疫原的设计、合成及免疫原性研究[J].第三军医大学学报, 2000, 22(10):919-923.
17. Scheibenbogen C, Sun Y, Keilholz U, et al. Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL2-based treatment[J]. Int J Cancer, 2002, 98(3): 409-414.
18. Panigada M, Sturniolo T, Besozzi G, et al .Identification of a promiscuous T-cell epitope in Mycobacteriumtuberculosis Mce proteins[J]. Infect Immun, 2002, 70(1): 79-85.
19. Hou Y, Gu XX. Development of peptide minotopes of lipooligosaccharide from nontypeable haemophilus influenzae as vaccine candidates[J]. J Immunol, 2003, 170(8): 4373- 4379.
20. Waiyan Candy NG, Mingchiu Fung. The Importance of Helper T cell Epitope in Vaccine Development[J]. 热带医学杂志, 2002, 2(3):207-213.
21. Del Guercio MF, Alexander J, Kubo RT, et al. Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper Epitopes(PADRE) for antibody responses in vivo[J]. vaccine, 1997, 15(4):441-448.
22. Nardin EH, Calvo-Calle JM, Oliveira GA, et al. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types[J]. J Immunol, 2001, 166(1): 481-489.
23. Jeff Alexander, Marie-France del Guercio, Barbara Frame, et al. Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE?)[J]. Vaccine, 2004, 22:2362-2367.
24. Partidos C, stanley C, steward M. The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides[J]. Eur J Immunol, 1992, 22(10):2675-2680.
25. Sette A, Fikes J. Epitope-based vaccines:an update on epitope identification, vaccine design and delivery[J]. Curr-Opin-Immunol, 2003, 15(4):461-470.
26. Alexander J, Oseroff C, Dahlberg C, et al. A decaepitope polypeptide primes for multiple CD8R IFN- and Th lymphocyte responses:evaluation of multiepitope poly- peptides as a mode for vaccine eelivery[J]. J Immunol, 2002, 168(12):6189-6198.
27. TamJP.Recent advances in multiple antigen peptides[J].Journal of immunological methods, 1996, 196(1):17-32.
28. Kawamura KS, Su R-C, Nguyen LT, et al. In vivo generation of cytotoxic T cells from epitopes displayed on peptide-based delivery vehicles[J]. J Immunol, 2002, 168(11): 5709-5715.
29. Del GG, Nardelli B, Huang W, et al. A multiple antigen peptide from the reptive sequenceof the plasmodiue malarise circumsporzoite protein induces a specific antibody response in mice of various H-2 haplotype[J]. Eur J Immunol, 1990, 20 (7):1619- 1622.
30. 曹胜利,菜孟深. 含两种不同肽段的血吸虫多抗原肽疫苗的合成与生物活性[J].药学学报, 1999, 34(10)751-754.
31. 汪海丹,丁淑香. 病毒 T 细胞表位和 T 细胞疫苗的研究进展[J]. 国外医学.预防诊断、治疗用生物制品分册, 1999, 22(4):152-156.
32. 潘勇, 顾正, 左嘉客. 局部避免和避孕疫苗[J]. 生殖与避孕, 1999, 19(6)328-334.
33. 何畏, 史常旭, 梁志清, 等. 多价精子抗原表位多肽-免疫刺激复合物的抗生育作用研究[J]. 重庆医学, 2002, 31(7)555-556.
34. Russell MW, Moldoveanu Z, White PL, et al. Salivary,nasal,genital,and systemic antibody responsesin monkeys immunized intranasally with a bacterial protein antigen and the cholera toxin B subunit[J]. Infect Immun, 1996, 64:1272 .
35. Borges M, Bose P, Frank HG, et al. A two-color fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines[J]. Placenta, 2003, 24(10): 959- 964.
36. Das.M, Xu.B, Rote.NS, et al. Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast[J]. Placenta, 2004, 25(5):396-407.
37. 成令忠. 组织学与胚胎学[M]. 第四版. 北京, 人民出版社, 1996, 265-267.
38. James C, Keith JD. Placental syncytin expression in normal and preeclampsia pregnancies[J]. AmJ Obstet Gynecol, 2002, 187(4):1122-1124.
39. Knerr I, Huppertz B, Weigel C, et al. Endogenous retroviral syncytin:compilation of experimental research on syncytin and its possible role in normal and disturbed human placentogenesis[J]. Mol Hum Reprod, 2004, 10(8):581-588.
40. Huppertz B, Tews DS, Kaufmann P. Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle[J]. Int Rev Cytol, 2001, 205:215-253.
41. Vignery A.Osteoclasts and giant cells:macrophage-macrophage fusion mechan- ism[J]. Int J Exp Pathol, 2000, 81(5):291-304.
42. Lee A, Morrow JS, Fowler VM. Caspase remodeling of the spectrin membrane skeleton during lens development and aging[J]. J Biol Chem, 2001, 276(23):20735 -20742.
43. Potgens AJ, Schmitz U, Bose P, et al.Mechanisms of syncytial fusion:a review[J]. Placenta Trophoblast Research, 2002, 23(6):S107-S113.
44. Gilpin BJ, Loechel F, Mattei MG, et al. A novel, secreted form of human ADAM
12(meltrin alpha)provokes myogenesis in vivo[J]. J-Biol-Chem, 1998, 273(1):157-166.
45. Frendo JL, Olivier D, Cheynet V, et al. Direct involvement of HERV-W Env glycol- protein in human trophoblast cell fusion and differiation[J]. Mol Cell Biol, 2003, 23(10):3566-3574.
46. Frendo JL, Cronier L, Bertin G, et al. Involvement of connexin 43 in human trophoblast cell fusion and differentiation[J]. J Cell Sci, 2003, 116(16):3413-3421.
47. Stoute JA, Ballou WR, Kolodny N, et al. Induction of humoral immune response against Plasmodiumfalciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library[J]. Infect Immun, 1995, 63:934-941.
48. Cortese R, Monaci P, Nicosia A, et al. Identification of biologically active peptides using random libraries displayed on phage[J]. Curr Opin Biotechnol, 1997, 6:73-80.
49. 王雁玲,庄临之.人胎盘滋养层细胞的功能分化及其调节[J].生殖与避孕, 1999, 19(6):335-341.
50. Sha M, Lee XH, Li XP, et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[J]. Nature, 2000, 403:785- 789.
1. Huppertz B, Tews DS, Kaufmann P. Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle[J]. Int Rev Cytol, 2001, 205: 215- 253.
2. Vignery A.Osteoclasts and giant cells:macrophage-macrophage fusion mecha- nism[J]. Int J Exp Pathol, 2000, 81(5):291-304.
3. Lee A, Morrow JS, Fowler VM. Caspase remodeling of the spectrin membrane skeleton during lens development and aging[J]. J Biol Chem, 2001, 276(23): 20735 -20742.
4. Potgens AJ, Schmitz U, Bose P, et al. Mechanisms of syncytial fusion:a review [J]. Placenta, 2002, 23(Suppl A):S107-S113.
5. Benirschke K, Kaufmann P. Pathology of the human placenta[M]. 4th ed.New York: Springer Verlag, 2000.
6. Daleke DL, Lyles JV. Identification and purification of aminophospholipid flippases[J]. Biochimica et Biophysica Acta, 2000, 1486(1):108-127.
7. Sims PJ, Wiedmer T. Unraveling the mysteries of phospholipid scrambling. Thrombosis and Haemostasis[J], 2001, 86(1):266-275.
8. Das.M,Xu.B, Rote. NS, et al. Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast[J]. Placenta, 2004, 25(5):396-407.
9. Adler RR, Ng AK, Rote NS. Monoclonal antiphosphatidylserine antibody inhib- its intercellular fusion of the choriocarcinoma line, JAR[J]. Biology of Reproduction, 1995, 53(4):905-910.
10. Black S, Kadyrov M, Huppertz B, et al. Syncytial fusion of human trophoblast depends on caspase8[J]. Cell-Death-Differ, 2004,11(1):90-98.
11. Blond JL, Beseme F, Duret L,et al. Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family[J]. J Virol, 1999, 73(2): 1175-1185.
12. Borges M, Bose P, Frank HG, et al. A two-color fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines[J]. Placenta, 2003, 24(10): 959-964.
13. Sha M, Lee XH, Li XP, et al. Syncytin is a captive retroviral envelope protein involvedin human placental morphogenesis[J]. Nature, 2000, 403(6765-6771):785-789.
14. Frendo JL, Olivier D, Cheynet V, et al. Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differiation[J]. Mol Cell Biol, 2003, 23(10):3566-3574.
15. Kudo Y, Boyd CA. Changs in expression and function of syncytin and receptors , amino acid transport system B(0)(ASCT2),in human placental choriocarcinoma BeWo cells during syncytialization[J]. Placenta, 2002, 23(7):536-541.
16. Gong R,Peng X,Kang S, et al. Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W[J]. Biochem- Biophys- Res-Commun. 2005, 331(4):1193-200.
17. Gilpin BJ, Loechel F, Mattei MG, et al. A novel, secreted form of human ADAM
12(meltrin alpha)provokes myogenesis in vivo[J]. J-Biol-Chem, 1998, 273(1): 157-166.
18. Cronier L, Defamie N, Malassine A, et al. Connexin expression and gap junctio- nal intercellular communication in human first trimester trophoblast[J]. Mol-Hum-Reprod, 2002, 8(11):1005-1013.
19. Cronier L, Frendo JL, Malassine A, et al. Requirement of gap junctional intercellular communication for human villous trophoblast differentiation [J]. Biol-Reprod, 2003, 69(5):1472-1480.
20. Frendo JL, Cronier L, Bertin G, et al. Involvement of connexin 43 in human trophoblast cell fusion and differentiation[J]. J Cell Sci, 2003, 116(16): 3413-3421.
21. Frank HG, Bose P, Kaufmann P, et al. Evaluation of fusogenic trophoblast surface epitopes as targets for immunecontraception [J]. Contraception, 2005, 71(4):282-293.
22. James C, Keith JD. Placental syncytin expression in normal and preeclampsia pregnancies[J]. AmJ Obstet Gynecol, 2002, 187(4):1122-1124.
23. Knerr I, Huppertz B, Weigel C, et al. Endogenous retroviral syncytin: compilation of experimental research on syncytin and its possible role in normal and disturbed human placentogenesis[J]. Mol Hum Rep rod, 2004, 10(8): 581-588.
24. Bose P, Kadyrov M, Goldin R, et al. Aberrations of early trophoblast differen- tiation predispose to pregnancy failure:lessons from the anti-phospholipid syndrome[J]. Placenta, 2006, 27(8):869-875.
1. Sette A, Fikes J. Epitope-based vaccines:an update on epitope identification, vaccine design and delivery[J]. Curr-Opin-Immunol. 2003, 15(4):461-470.
2. Hou Y, Gu XX. Development of peptide minotopes of lipooligosaccharide from nontypeable haemophilus influenzae as vaccine candidates[J]. J Immunol, 2003, 170(8):4373-4379.
3. Frank HG, Bose P, Albieri-Borges A, Kaufmann P, et al. Evaluation of fusogenic trophoblast surface epitopes as targets for immune contraception[J]. Contraception, 2005, 71(4):282-293.
4. Le Doussal JM, Piqueras B, DoganI, et al. Phage display of peptide/major histocompatibility complex[J]. J-Immunol-Methods, 2000, 241(2):147-158.
5. Scheibenbogen C, Sun Y, Keilholz U, et al. Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL2-based treatment[J]. Int J Cancer, 2002, 98 (3):409-414.
6. Panigada M, Sturniolo T,Besozzi G,et al. Identification of a promiscuous T-cell epitope in Mycobacteriumtuberculosis Mce proteins[J]. Infect Immun, 2002, 70(1):79-85.
7. Del Guercio MF, Alexander J, Kubo RT, et al. Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper Epitopes(PADRE) for antibody responses in vivo[J]. vaccine, 1997, 15(4): 441-448.
8. Nardin EH, Calvo-Calle JM, Oliveira GA, et al. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types [J]. J Immunol, 2001, 166(1): 481-489.
9. Livingston BD,Newman M,Crimi C, et al. Optimization of epitope processing enhances immunogenicity of DNA vaccines[J]. Vaccine, 2001, 19(32):4652-4660.
10. Livingston B, Crimi C, Newman M, et al. A rational strategy to design multiepitope immunogens-based multiple HTL epitopes[J]. J Immunol, 2002, 168 (11):5499-5506.
11. Velders MP,Weijzen S, Eiben GL, et al. Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNAvaccine[J]. J-Immunol, 2001, 166(9):5366-5373.
12. Partidos C, stanley C, steward M.The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides[J]. Eur J Immunol, 1992, 22(10):2675-2680.
13. Deliyannis G, Jackson DC, Ede NJ,et al. Induction of long-term memory CD8R T cells for recall of viral clearing responses against influenza virus[J]. J Virol, 2002, 76(9): 4212-4221.
14. BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines yesterday,today, and tomorrow[J]. Lancet Infec Dis, 2002, 2(7):425-431.
15. Kawamura KS, Su R-C, Nguyen LT, et al. In vivo generation of cytotoxic T cells from epitopes displayed on peptide-based delivery vehicles[J]. J Immunol, 2002, 168(11): 5709-5715.
16. 何畏, 史常旭, 梁志清, 等. 多价精子抗原表位多肽-免疫刺激复合物的抗生育作用研究[J].重庆医学, 2002, 31(7):555-556.
17. Alexander J, Oseroff C, Dahlberg C, et al. A decaepitope polypeptide primes for multiple CD8R IFN- and Th lymphocyte responses: evaluation of multi- epitope polypeptides as a mode for vaccine eelivery[J]. J Immunol, 2002, 168(12):6189-6198.
18. Zwaveling S, Ferreira Mota SC, Melief CJ, et al. Established human papill- omavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides[J]. J Immunol, 2002, 169(1):350-358.
19. Barton GM, Medzhitov R. Toll-like receptor signaling pathways[J]. Science, 2003, 300 (5625):1524-1525.
20. La Rosa C, Wang Z, Diamond DJ, et al. Preclinical development of an adjuvant free peptide vaccine with activity against CMV pp65 in HLA transgenic mice[J]. Blood, 2002, 100(10):3681-3689.
21. Chikh GG, Kong S, Bally MB, et al. Efficient delivery of antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8R T cells[J]. J Immunol, 2001, 167(11): 6462-6470.
22. Leifert JA, Harkins S, Whitton JL. Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity[J]. Gene Ther, 2002, 9(21):1422-1428.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |