【作者】 李迎春；

【导师】 王心如；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2010， 博士

【摘要】 随着世界人口的快速增长和社会文明的进步,越来越多的男性愿意承担避孕的义务,但是与此相矛盾的是可供男性选择的避孕方法很少,而且现有男性避孕方法均具有一定的局限性。所以迫切需要研制出可供男性选用、高效、安全和可逆的避孕方法。人们从精子发生的调控过程中筛选出合适的靶抗原用作免疫避孕疫苗,其中卵泡刺激素受体(Follicle Stimulating Hormone Receptor,FSHR)特异性表达于睾丸支持细胞,是卵泡刺激素(Follicle Stimulating Hormone,FSH)调节精子发生所必需的受体,是个合适的免疫避孕靶标。本研究旨在制备FSHR多肽疫苗,并对其进行免疫效应研究,为中试生产和进一步的灵长类动物实验提供技术支持和实验基础;此外通过流行病学研究,探讨FSHR基因多态与原发性男性不育及精子凋亡的相关性,阐明FSHR影响男性生育的可能生物学机制,寻找男性不育的易感性生物标志物,将有助于男性不育患者的病因学分析,为疫苗的深入研究奠定理论基础。第一部分FSHR(1-57aa)多肽的制备及其免疫效应研究本研究旨在体外制备高纯度的FSHR多肽,并初步明确其免疫效应。我们运用PCR法,以人睾丸组织cDNA为模板获得人FSHR基因片段,将其连入pET28a(+)载体His6-Tag的下游,并在FSHR基因片段和His6-Tag之间引入EK酶切位点,1mM IPTG诱导原核蛋白表达;对表达的蛋白进行纯化、复性和EK酶切处理。本研究最终获得了以包涵体形式存在、纯度大于95%、不带His6-tag的FSHR多肽。为了证实重组FSHR多肽的免疫原性及免疫效应,本研究将重组FSHR多肽配伍氢氧化铝佐剂免疫雄性Balb/c小鼠,观察其免疫效应。ELISA法在血清中可检测到特异性抗FSHR抗体,最高效价可达1:10000;同时附睾精子数量下降、睾丸体积减小;TUNEL法检测发现各级生精细胞和精子凋亡增加;血清睾酮水平无明显改变。上述结果显示基因工程表达的高纯度重组人FSHR多肽与氢氧化铝佐剂配伍后免疫雄性小鼠能产生较好的免疫原性,为研制适宜男性避孕的FSHR多肽疫苗提供了理论和实践基础。第二部分FSHR基因多态与男性不育相关性研究本研究首先以病例-对照为基础,探讨了中国汉族人群中FSHR基因单核苷酸多态性(SNPs)与原发性男性不育症发病风险的关联。在五组原发性男性不育的病人(171例精液参数正常,101例原发性无精症,15例原发性少精症,152例原发性弱精症,95例原发性少弱精症)和285例生育正常的男性对照中,通过Taqman探针和聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法对FSHR基因-29(G>A)、Thr307Ala(A>G)和Asn680Ser (A>G)三个功能性多态性位点进行基因分型。以单因素和多因素Logistc回归模型分析FSHR基因多态与原发性男性不育症的相关性。然后用TUNEL法检测337例病人的精子凋亡率,通过单纯病例研究探讨FSHR基因多态与精子凋亡之间的关联性。结果显示:(1)Thr307Ala(A>G)和Asn680Ser(A>G)两个多态位点与原发性男性不育有关,其AG基因型对男性不育具有保护作用。与携带AA基因型相比,携带AG基因型的个体罹患原发性弱精症的风险显著降低(OR=0.56,95% CI=0.37-0.85;OR=0.55,95% CI=0.36-0.83)。当合并Thr307Ala(A>G)和Asn680Ser(A>G)的AG和GG基因型时,携带G等位基因(AG+GG)降低了弱精症男性不育的风险(OR=0.58,95% CI=0.39-0.86, P = 0.024;OR=0.57,95% CI=0.38-0.85, P = 0.018)。这两个多态位点联合分析发现,与Thr307-Asn680单倍型相比,Ala307-Ser680单倍型显著降低了男性不育的发病风险(OR=0.70;95% CI=0.51-0.96)。(2)与GG基因型相比,G-29A AG基因型携带者精子凋亡率显著降低(P=0.021)。我们得出初步结论:Thr307Ala(A>G)和Asn680Ser(A>G)的遗传多态与中国汉族人群的弱精症男性不育具有一定的关联性;-29(A>G)多态与精子凋亡具有一定的关联性,但需进一步进行功能研究并在不同人群中进行验证。更多还原

【Abstract】 As the rapid increase in population around the world and the development of social civilization, a majority of men would be willing to use a male contraceptive. In contrast, advances in male-directed methods have been confined to refinements in the type of condom and technique of vasectomy. Thus, it has become more and more urgent to develop a safe, effective, reversible and acceptable contraceptive for males. Researchers chose suitable target antigens based on the regulation of spermatogenesis as the vaccines. FSHR is expressed exclusively in testicular Sertoli cells and mediates the action of FSH during the spermatogenesis, so it is shown to be a suitable immunocontraception target. The present study aimed to produce the FSHR peptides vaccine and research its immune effect, which could provide the technical and theoretical bases for pilot scale productin and primate trial. Furthermore, we investigated whether polymorphisms in FSHR were associated with the risk of developing idiopathic male infertility and sperm apoptosis, which would help us analysize the etiology of male infertility and provide the theoretical bases for contraception vaccine.Part I The production of FSHR peptides and its immune effectThe present study aimed to produce the FSHR peptides vaccine and research its immuno-antifertility effects. In this research, an FSHR (1-57aa) cDNA was generated by PCR using the human testis cDNA as template, and then the cDNA was cloned into vector pET-28a(+) downstream of His6-Tag. To cut the His6-Tag, we added the enterokinase (EK) recognition site between His6-Tag and our target protein. Subsequently, the prokaryotic proteins were expressed under induction of 1 mM IPTG. After Ni-NTA purification, renaturation and EK digestion, we obtained FSHR peptides, which were existed in form of inclusion bodies. The purity of FSHR peptides is above 95%.To study the immune effects, we used the recombinant FSHR peptides, which were mixed with the aluminum adjuvant, to immunize male Balb/c mice. The specific anti-FSHR IgG could be detected in serum with the highest titre around 1:10000. Meanwhile, reduced sperm count, smaller testis and germ cells apoptosis were observed.In conclusion, the high purified FSHR peptides prepared by genetic engineering have powerfully immunogenicity after mixed with the aluminum adjuvant in mice, which could favor further research in pilot scale production and non-human primate trial. Part II Study on the association of polymorphisms in FSHR geneThis study aimed to investigate the effects of the FSHR polymorphisms on idiopathic male infertility in Han-Chinese. Three FSHR polymorphisms, -29(G>A), Thr307Ala(A>G) and Asn680Ser(A>G) were analyzed using Taqman allelic discrimination assay and PCR-RFLP in 285 fertile controls and 534 infertile patients. Association between the polymorphisms and risk of male infertility were estimated by univariate and multivariate logistic regression analysis. Furthermore, to analyze the association between FSHR polymorphisms and sperm apoptosis, we measured sperm apoptosis by TUNEL assay using flow cytometry in 337 infertile patients. The results showed that the ThrAla307 (TA307) and AsnSer680 (NS680) genotypes were associated with a significantly decreased risk of idiopathic asthenospermia (OR= 0.56 and 0.55, 95% CI = 0.37-0.85 and 0.36-0.83, P = 0.024 and P = 0.018 respectively) compared with TT307 and NN680 respectively. Haplotypic analysis showed a significantly decreased risk of asthenospermia associated with the A307-S680 haplotype compared with the T307-N680 haplotype (OR= 0.70, 95% CI = 0.51-0.96). However, no significant association was found between the -29(G>A) polymorphism and idiopathic male infertility. Analysis showed that patients with -29(G>A) AG genotype were associated with lower sperm apoptosis rate when compared to those with GG genotype (P=0.021). Our results suggest that the FSHR polymorphisms may contribute to the pathogenesis of idiopathic male infertility in the Han-Chinese population, but future mechanistic and epidemiologic studies are needed to confirm these findings更多还原