【作者】 向延芳；

【导师】 史常旭；

【作者基本信息】 第三军医大学 ， 妇产科学， 2008， 博士

【摘要】 每个睾丸支持细胞(Sertoli细胞)与30~50个生殖细胞相联系,Sertoli细胞与生精细胞共同构成睾丸的精曲小管, Sertoli细胞不仅在结构上为生精细胞的分化成熟提供支架,供给生精过程所需的能量及营养物质;同时可分泌如转运蛋白类、调节蛋白类、生长因子类、类固醇类等数十种物质参与生精细胞的分化成熟,具有精细的协调作用,保证精子发生的正常有序进行。Sertoli细胞之间接近基底水平的紧密连接是血睾屏障主要结构成分,在精子发生过程中,细线前期和细线期精母细胞必须不断从曲细精管上皮的基底部穿越血睾屏障向腺腔部分迁移,并分化为单倍体精细胞,生殖细胞的迁移过程包含了细胞间紧密连接(TJs)和黏附连接(AJs)精细的动力学变化:时相性“关闭”和“开放”。如果能干扰Sertoli细胞间的连接,势必使正常的精子排放受到影响。这可能成为男性避孕措施的有效途径。闭锁因子是组成紧密连接的关键膜蛋白,该蛋白由4个跨膜结构域,2个胞外环,1个胞内环,1个小NH2-末端胞质结构域,1个大的COOH-末端胞质结构域组成。其第1个胞外环发挥细胞黏附作用,其第2个胞外环可干扰上皮细胞株中紧密连接屏障的装配。它的任何改变均可破坏TJs,从而破坏AJs,而TJs在精子的成熟和释放中起着关键性的作用。细胞间的TJs广泛存在于各类组织细胞中,如果非靶向地干扰闭锁因子功能,可能导致机体其它组织细胞间TJs屏障的非特异性功能损害,出现严重副作用。为此,将对应闭锁因子的多肽与靶向作用于Sertoli细胞的特异性分子耦合,通过特异性靶向干扰睾丸内Sertoli细胞间TJs蛋白的功能状态,才能特异性抑制或干扰精子发生和排放过程,达到可逆性避孕目的。卵泡刺激素(FSH)是女性卵泡成熟和男性精子发生必须的糖蛋白。在男性,FSH受体仅存在于Sertoli细胞胞膜上,FSH由α、β两个亚单位组成,其中α-亚单位与LH、HCG等激素具有共同序列,而β-亚单位具有种属和激素特异性。本研究选择仅有受体结合活性,但无激素生物活性的FSH-β亚单位中的部分序列,与编码闭锁因子中第二个胞外环发挥紧密连接功能的相应基因序列进行基因重组,通过毕赤酵母真核表达获得融合蛋白,以期获得可靶向干扰Sertoli细胞功能,从而干扰精子生成达到避孕的目的。本研究内容主要分三个部分:①pPIC9KFSH–Occludin酵母表达载体的构建。利用RT-PCR技术,从小鼠新鲜脑组织中提取总RNA扩增获得FSH-β亚单位,同法从小鼠新鲜睾丸组织中提取总RNA扩增occludin。通过部分重叠PCR扩增,获得含FSH-β亚单位特定基因序列和闭锁因子第2个胞外环序列的耦合片段,NotⅠ+EcoRⅠ双酶切pPIC9K质粒及目的片段后分别铺胶回收,连接过夜。所构建载体均转化大肠杆菌,快速抽提阳性质粒DNA,酶切及测序鉴定。②FSH–Occludin蛋白在毕赤酵母中表达、纯化及活性初步鉴定。构建载体以电穿孔法转染酵母菌GS115,依次进行G418筛选,PCR筛选鉴定、Mut筛选及小量摇瓶培养实验,寻找高拷贝菌株与表达蛋白的最佳收获时段,筛选结束后大量摇菌,于最佳时段收获上清液,经超滤浓缩、分子筛纯化后将所收集样品冷冻抽干备用。纯化后蛋白用SDS-PAGE、Western-Blot鉴定,并用免疫组化法检测其生理活性。③FSH-occludin融合蛋白避孕效应的研究。本研究将FSH-occludin融合蛋白皮下注射Balb/c小鼠,取各时期动物血清,应用酶联免疫法检测性激素睾酮和FSH的浓度并监测血清抗体滴度的变化,并用光镜和电镜观察了融合蛋白所导致睾丸组织的免疫性病理变化,动物免疫结束后合笼检测其免疫性避孕效应与可逆性。研究的主要实验方法及结果如下:1.通过生物信息学分析设计合成引物,采用RT-PCR技术分别从小鼠脑组织和睾丸组织成功扩增出FSH和Occludin的cDNA序列,PCR扩增出FSH-β亚单位33-100aa(68 aa)及Occludin 207-228aa(22 aa)和197aa-241aa(45 aa)区域的3个目的片段,通过部分重叠双重PCR法获得闭锁因子第二个胞外环序列和FSH-β亚单位基因序列的耦合片段,酶切连接后构建了2种载体(pPIC9K-6822与pPIC9K-6845),本研究将编码FSH-β亚单位上36及39位氨基酸上的TGT(半胱氨酸)突变成TCT(丝氨酸)使其与受体的结合不受影响,但减少了其生物激动剂的活性。经测序证实完全符合预想设计。2.将表达载体采用单交换的整合方式电转化毕赤酵母菌株GS115,筛选高拷贝重组转化子,经SDS-PAGE和Western blot分析证实获得了重组蛋白,完成了FSH-Occludin融合蛋白的酵母表达、纯化及免疫组化生物活性鉴定。3.将融合蛋白免疫Balb/c雄鼠,对照组注射PBS。免疫后实验组出现特异性IgG抗体,血清中促卵泡生成激素激素(FSH)及睾酮(T)水平各组无明显差异。实验组小鼠睾丸曲细精管管腔内的精子数以及附睾尾部精子数相对PBS组明显减少,电镜下观察到免疫组支持细胞与生精细胞的紧密连接被打开,失去依托关系,生精细胞之间有较大间隙。结果证实重组蛋白可靶向性干扰Sertoli细胞的TJs,实验组受孕率、产仔率明显低于对照组且其避孕效应具有可逆性。重组蛋白具有良好的靶向性,90d后免疫组的受孕率及产仔数均有不同程度的恢复符合可逆性避孕的研究设想。由此实验结果可以得出Occludin的第2个胞外环确是可以干扰细胞之间的TJs,而且第2个胞外环中的外部区域在紧密连接中有着非常重要的作用。融合蛋白能靶向干扰Sertoli cell的TJs从而干扰精子生成且其避孕效应具有可逆性。本研究的实施为开辟一种安全、高效、可逆的理想避孕途径奠定理论和实验基础。更多还原

【Abstract】 Background:Sertoli cell provides mechanical and nutritional support to about 30~50 developing germ cells at different stages of the seminiferous epithelial cycle during spermatogenesis. Furthermore,adjacent Sertoli cells in the seminiferous epithelium form an almost impermeable barrier near the basement membrane known as the blood–testis barrier (BTB).This BTB creates a unique microenvironment that sequestered virtually all the events associated with postmeiotic germ cell maturation from the systemic circulation. It also restricts the access of nutrients, hormones, electrolytes, and other biological substances from the interstitium to the developing germ cells behind the BTB.Tight junctions (TJs) between Sertoli cells at the basal compartment form the BTB.At the late stage VIII through early stage IX, preleptotene and leptotene spermatocytes must traverse the BTB, where junctions are rapidly broken and subsequently reassemble to allow the spematocytes to pass through.The translocation of germ cells across the seminiferous epithelium involves not only the dynamic modulation of TJs at the BTB, but also the extensive restructuring of actin-based adherens junctions (AJs) between Sertoli cells as well as between Sertoli and germ cells. thereafter, so that differentiating germ cells could move towards the adluminal compartment for further development.Several studies have demonstrated the unique function of occludin in TJs. Occludin, a 65 kDa protein,was the first TJ-integral membrane protein identified in multiple epithelia,Each occludin molecule consists of four transmembrane domains with a long carboxyl-terminus and a short amino-terminus in the cytoplasm,one intracellular loop,and two extracellular loops.The first extracellular domain is involved in cell-cell adhesion function. The second extracellular loop apparently is needed for the assembly and sealing of TJs, any change of occludin can destroy TJs then AJs,and lead to germ cell fall off from epithelial,TJs plays a key role in spermatogenesis by causing mature sperm to be released from the surrounding Sertoli cells. The integrity of the epithelial cell layer(s) is maintained by intercellular junctional complexes composed of tight junctions (TJs), adherens junctions, and desmosomes,if non-target interfere the function of Occludin would induce TJs functional lesion in other tissues. So specific interfering the function of TJs should be a new choice in developing male contraceptive.Follicle-stimulating hormone (FSH) is the central hormone of mammalian reproduction and FSH action is necessary for gonadal stimulation at puberty and gamete production during the fertile phase of life.FSH effects on gametogenesis by binding to a specific receptor that is located only on the surface of Sertoli cells in the testis and granulosa cells in the ovary. FSH is consists of two subunits, the a- and theβ-subunits, the a-subunit is non covalently joined to theβ-subunit to form a biologically active hormone, but it is theβ-subunit, that confers hormone specificity.Object:In this study,we choose receptor binding region of FSH-βand the secondary extracellular domain of Occludin as objective fragment and recombination. To obtain fusion protein through Pichia Pastoris expression technology and expect to target Sertoli cell then interfere its TJs . It should provide an attractive candidate for development of a contraceptive vaccine.Methods:There are three parts in our study:①The construction of pPIC9KFSH-Occludin yeast expression vector. Firstly, the full length cDNA encoding FSH and Occludin was amplified respectively from a mouse brain and testicle by RT-PCR method .Then mutated the aim amino acids by Site-Direct Mutation and partial overlapping double PCR get recombination gene .Finally, the recombination gene was subcloned into Not I and EcoR I sites of pPIC9K vector. The recombinant expression vectors were verified by DNA sequence test.②The Pichia yeast expression、purification of FSH-Occludin protein and the primary test of its biological activity. The verified recombinant vector was transformed into Pichia strain GS115 by electroporation. The positive clones grown on the HIS drop out medium were selected and confirmed by PCR analysis. The transformants with multiple inserts were screened in vivo by their resistance to G418, and Mut+ phenotype by Mut screening. A time course study of expression was performed to optimize the protein yield.Large baffled flasks were used to scale up the expression, and the culture supernatant were harvested on the time point obtained from previous study.The culture supernatant containing the recombinant protein were demineralized and dialysis, purified by Sephadex G-50 column, and froze in -70℃.The purified protein was confirmed by SDS-PAGE and Western blot analysis. Its biological activity was tested by immunohistochemical assay.③Study immune contraception effects of the FSH-occludin fusion protein . The adult male Balb/c mice were immunized with the fusion protein, detected the sepcific antibody、testosterone(T)and FSH concentrations in mice serum, to observe histopathologic damage、pregnant rates and number of offsprings.Results:1.The amplified full length cDNA encoding FSH and Occludin were exactly the same sequence as Genbank data(NM 008045 and NM 008756). Two amino acid of FSH sequence (36aa and 39aa)were mutated by Site-Direct Mutagenesis technology in order to decrease biology activity.The mutants were confirmed by sequence analysis that there are mutated as design.Two different pPIC9K FSH-Occludin [FSH-β(33aa-100aa) and the second extracellular loop of occludin (207-228aa)、(197-241aa) respectively] yeast expression vectors were obtained. These vectors were also confirmed by sequence analysis.2.Two different FSH-Occludin fusion proteins were obtained from the culture supernatant of yeast expression system. After purified,they were used to test their biological activity.3.The adult male Balb/c mice were immunized with FSH-occludin fusion protein, Two fusion protein can effectively inducing specific humoral antibody response and effected on experiment group mice fertilization without disturbing the hormone balance. The fusion proteins can targeting interfere with Sertoli cell, the sperm cell density in seminiferous tubules and the tail of epididymis of experiment group was obviously reduced than control group.There were big interspace between the Sertoli cells of the experimental group, TJs between Sertoli cells had been broken,Sertoli cells did not support germ cell and basal lamina of seminiferous tubules dissolved partly.The pregnant rate and the number of offspring in experimental group were obviously fewer than control group and contraception effect is reversible. Conclusin:1.In summary, occludin is a useful marker to monitor inter-Sertoli TJs assembly .The second external loop of occludin in particular the outermost region of the loop, apparently is important to confer to the TJs functionality and the inter-Sertoli TJs permeability barrier.2.the use of an occluding peptide or other peptide-based reagents homologous to selected TJs or AJs proteins to impair spermatogenesis may provide a potential approach to arrest spermatogenesis.The contraceptive effect of the two fusion proteins is reversible, though disrupt the BTB. Therefore, our research may pave the way for a safty、efficacious and reversible contraceptive method.更多还原