【作者】 李玉艳；

【导师】 史常旭；

【作者基本信息】 第三军医大学 ， 妇产科， 2002， 硕士

【摘要】 受精过程包括精子成熟、附着、结合、顶体反应、穿卵、融合、皮质反应、双方配子的带反应和核聚变等过程，而抗精子抗体可通过降低精子速度、减少顶体反应率、抑制精卵结合等途径来影响精子受精功能，导致免疫性不孕。利用精子抗原来制作避孕疫苗，已成为免疫避孕的研究热点。精子蛋白SP17位于精子顶体部位，与精子顶体跨膜蛋白Cyritestin一样，均在精-卵透明带结合、精-卵膜结合和融合过程中发挥重要作用。本研究利用已经获得的含SP17和Cyritestin表位的人工合成35肽（P3肽）和抗P3肽兔血清，探讨抗P3抗体与人精子的结合和对受精过程的影响及其程度，评价人工合成的P3肽对人精子功能的影响，旨在对其生物学特性进行深入研究。实验分两部分进行。第一部分运用试剂盒，将P3多肽通过EDC与KLH载体蛋白结合，免疫BALB/C小鼠，收获血清经过柱纯化，获得抗P3抗体，用FITC直接标记，通过流式细胞仪检测抗体与精子结合的阳性率，即抗体与人精子的结合率，用荧光显微镜观察抗体与人精子的结合部位，并且采用免疫组化方法，以抗Cyritestin单抗为阳性对照，观察含抗P3抗体的兔血清与人精子的结合部位。第二部分将人精子与含抗体血清共同孵育，利用CASA系统检测人精子活动力，用FITC-PSA法及考马斯亮蓝法检测顶体反应率，通过SPA法检测穿卵率，评价抗体对人精子功能的影响及其程度。抗体经纯化、标记后与精子表面的结合率可达97.7%，抗体血清与人精子孵育后，用羊抗人IgG检测阳性率从49.2%降至36.4%，荧光显微镜见精子头颈部着色，部分见赤道板或顶端荧光增强，免疫组化染色见精子头部着深褐色，与阳性对照一致。抗体血清与人精子孵育后运动速度、各项运动精子百分率及其他各项指标均显著下降，并且与抗体浓度变化有一定关系，<WP=7>精子顶体反应率及穿卵率也显著下降。结论：P3肽在精子头部表面有表达，特异性抗P3抗体可与其高度结合；抗P3抗体可显著影响精子的活动力、顶体反应和穿卵率，可干扰精子受精功能，因此P3肽对人精子受精功能有影响，具有一定的开发前景。更多还原

【Abstract】 There are many events in fertilization such as maturation of egg and sperm, attachment, binding, acrosomal reaction, penetration, fusion, cortical reaction, zona reaction and nuclear fusion of both gamete. It is well known that anti-sperm antibody could impair fertility in couples by resulting in immunological infertility through damaging sperm motility, reducing acrosome reaction, inhibiting sperm penetration zona pellucida, and so on. Many special sperm antigens have been utilized to prepare contraception vaccine for controlling fertilization, which is focused in immunological contraception research.Sperm protein Sp17 is one of sperm antigens on acrosomal cap, and can bind specially with ovum zona pellucida. Cyritestin, a cross-membrane protein of acrosome, as Sp17 can also effect on sperm penetration. Both Sp17 and Cyritestin play important role in process of sperm-zona pellucida or sperm-member of egg binding and fusion. This study utilized P3 multiple peptides which was synthetic polypeptide containing both epitopes of Sp17 and Cyritestin and anti-P3 multiple peptides serum of rat. Our study is aimed to investigate the biological property of anti-P3 antibody, and its efficiency against sperm-egg conjugation and anti-fertilization. This research is divided into two parts. In part one, we conjugated the P3 peptide and carrier protein KLH with EDC, and immunized BALB/C mouse with the purified compound P3-KLH. Serum were collected, anti-P3 antibody was purified using immobilized diaminodipropylamine column. The purified anti-P3 antibody was then labeled with FITC, and used to detect the sperm’s positive percent by flow cytometry. Then we confirmed the binding site of antibody<WP=5>against P3 peptide by immunofluorescence and immunocytochemistry techniques separately.In part two, after swim-up collection, the motile sperm were incubated with antiserum against P3 peptide and control serum. Parameters of sperm motility, including sperm head amplitude, linearity, straight and curvilinear velocity, motility percentage, were evaluated and quantified using Computer-aided semen analysis system. Acrosomal reaction rate was determined by FITC-PSA fluorescence stain and Coomassie’s blue stain. The penetrate capacity was assessed by hamaster zona-free egg penetration assay. Brown reaction appeared on the sperm head surface by pre-incubating with antibody serum and also with the positive control. The positive rate of sperm conjugating with FITC-anti-P3 antibody is 97.7%. The conjugating positive rate of FITC-goat-anti-human IgG with sperm was reduced from 49.2% to 36.4% by acquired antiserum. After incubation with acquired antibody serum, all the parameters of sperm quality (motility) were decreased significantly, including sperm velocity, percent motility, ALH, LIN, the rate of AR, the rate of SPA. Our results suggested that: on the surface of sperm head, there is an epitope as P3 peptide, and anti-P3 antibody could conjugate with sperm specially and excellently; the anti-P3 antibody serum could reduce the motility, acrosome reaction and penetrate capacity of sperm and disturb sperm’s fertilization ability. So, the potential practicability of synthetic P3 peptide needs to be further determined.更多还原