【作者】 张平；

【导师】 邢万金；

【作者基本信息】 内蒙古大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 近来有研究报道说明出生后的幼年和成年小鼠卵巢表面上皮（ovariansurface epithelium,OSE）有生殖干细胞（germline stem cells,GSCs）和卵泡更新过程。这些报道引发了许多质疑和争论。我们对大鼠卵巢进行了类似研究以检查大鼠卵巢内是否也具有GSCs和卵泡更新过程。我们选择大鼠的vasa类似物RVLG（rat vasa-like gene）N端的部分片段作为抗原制备抗体识别大鼠卵巢中的生殖细胞。首先克隆了RVLG基因的cDNA5’端约627 bp片段,序列测定结果表明,我们所克隆的RVLG cDNA片段比GenBank中报道的大鼠RVLG cDNA（NM₀01077647）相应部位多60 bp。与大鼠RVLG基因序列（NW₀01084795.1）比对后确定所多出来的60 bp系第7内含子的5’端和第8内含子的3’端两处选择性剪接的结果,导致第7外显子3’端多出45 bp,第9外显子5’端多出15bp。进一步克隆RVLG cDNA全长序列后发现第一次克隆中出现的60 bp只保留了第7外显子3’端的45 bp,而没有第9外显子5’端的15 bp。将该片段插入原核表达载体pGEX-4T1,转入大肠杆菌BL21（DE3）中成功地诱导表达了GST-pRVLG融合蛋白,目的蛋白占菌体总蛋白的10%以上。用纯化后的GST-pRVLG融合蛋白免疫昆明（KM）小鼠,最后给小鼠腹腔注射S180细胞制备了抗RVLG的腹水多克隆抗体。Westernblot鉴定RVLG腹水多克隆抗体的特异性表明制备的抗体可特异性地识别RVLG蛋白。我们研究了幼年和成年期大鼠卵巢表面是否存在可以维持新生卵子发生的GSCs,以及新生卵子发生所需的一些减数分裂标记基因的表达情况。卵巢组织切片观察表面上皮没有找到大的卵圆形细胞,免疫组织化学检查卵巢皮层也没有找到RVLG阳性细胞及RVLG、BrdU双阳性细胞。RT-PCR检测出生后大鼠卵巢,也没有发现减数分裂特异性基因Scp1、Scp3和Spo11的转录。Westernblot和免疫组织化学法均未能检测到减数分裂进入标记DMC1、STRA8及SCP3。我们的实验未能观察到成年大鼠卵巢上皮有处于分裂期的生殖细胞和进入减数分裂的细胞,不能支持出生后的大鼠卵巢内存在GSCs和新的卵子发生的猜想。更多还原

【Abstract】 Recently several reports provided evidences that indicate ovarian surface epithelium（OSE） of juvenile and adult mice contains germline stem cells（GSCs） that sustain postnatal follicular renewal.But their reports have met with skepticism and doubts.Here we carried out some relevant researches in rat to examine whether GSCs and neo-oogenesis exist in adult rat ovaries.In order to identify germ cells in ovary,we prepared a mouse anti-RVLG polyclonal antibody.A 627-bp cDNA fragment of RVLG（rat vasa-like gene） was cloned and sequence analysis showed that it was 60-bp longer than that released in the GenBank（NM₀01077647）.Aligment with the RVLG-containing genomic DNA sequence（NW₀01084795.1） indicated that 45-bp fragment is located at the 3’ of exon 7,other 15-bp at the 5’ of exon 9,both resulting from an alternative splicing of the RVLG pre-mRNA in its intron 7 and intron 8.To further study the alternative splicing pattern in the whole ORF,we cloned the full-length of RVLG cDNA.Sequence analysis revealed the existence of the 45-bp,but the other 15-bp is absent.The R VLG cDNA fragment was inserted into the prokaryotic expression vector pGEX-4T-1,and transformed into E.coli BL21（DE3） to express the GST-pRVLG fusion protein.The GST-pRVLG fusion protein was expressed in E.coli BL21 （DE3） at a high level which accounts for more than 10%of the total bacterial cellular protein.The mouse anti-rat RVLG polyclonal antibody was generated by immunizing KM mouse with purified GST-pRVLG fusion protein followed by celiacly injecting the mouse with S180 cells.The antibody recognizes RVLG protein specifically and its titer is about 1:20000.We analyzed the presence of GSCs in the ovarian surface epithelium（OSE） and the expression of meiotic marker genes,which are required for neo-oogenesis, in juvenile and young adult rat ovaries.No large ovoid cells were observed in the OSE,and the RVLG expressing,BrdU incorporating germ cells were not identified in the postnatal ovarian cortex by immunohistochemical analysis.Transcription of SCP1,SCP3 and SPO11,three early meiotic-specific and oogenesis-associated genes,was undetectable in postnatal rat ovaries using RT-PCR.The early meiocytes expressing meiotic entry markers such as DMC1,STRA8 and SCP3, were not observed using Western blotting and immunohistochemical methods in juvenile and young rat ovarian cortex.Taken together,we didn’t observe proliferating germline cells and meiosis in the postnatal rat ovary.Thus our data didn’t support the postulation that GSCs and neo-oogenesis may exist in the postnatal rat ovary.更多还原