Functional analysis and application of cancer treatment of unknown gene express in spermatogonial stem cells and cancer cells

我们通过cDNARDA消减技术分离到精原细胞中特异表达的PTP-X。PTP-X mRNA在睾丸和睾丸肿瘤细胞系的各个发育阶段都有稳定的表达。因此，我们对PTP-X基因的表达调控和生物学功能进行了研究。我们通过Northern印迹分析比较了睾丸肿瘤、肿瘤细胞系和野生型睾丸的基因表达，但没有检测到剪接和表达的变化。我们将短干扰RNA(SiRNA)导入胚胎干细胞(ES细胞)，从类胚体(EBS)中分离出SSEA-1阳性细胞。这些细胞在维甲酸的存在下被分化为生殖细胞系。我们检测了与生殖细胞发育有关的基因的表达。在精原细胞和精母细胞中表达的基因没有差异。相反，在敲除细胞中精子细胞特异性基因的表达减少。接下来，我们检查了由siRNA ES细胞产生的siRNA嵌合体睾丸。用免疫组织化学方法分析14周时冰冻的siRNA嵌合体和野生睾丸组织中生殖细胞标志物的表达模式。GATA-1阳性细胞存在于曲细精管，而Dmc1和SCP1阳性细胞存在于曲细精管，体内外结果均表明PTP-X参与了生精分化。

We have isolated PTP-X that is specifically expressed in spermatogonial cells by cDNA RDA subtraction. PTP-X mRNA is expressed at a consistent level during all developmental stage of the testis and testicular tumor cell lines. So, we investigated the regulation of expression and the biological function of PTP-X gene. We compared gene expression between testicular tumors, tumor cell lines and wild type testis by northern blot analysis, but not detected alteration of splicing and expression.We introduced short interference RNA (siRNA) into embryonic stem (ES) cells, and isolated SSEA-1 positive cells from embryoid bodies (EBs). These cells were differentiated to germ cell line in the presence of retinoic acid.We examined the expression of genes implicated in germ cell development.There are no difference expression levels of genes that are expressed in the spermatogonia to spermatocyte. In contrast, expressions of spermatid specific genes were reduced in knock down cells.Next, we examined siRNA chimera testis produced from siRNA ES cells. Expression pattern of germ cell markers was analyzed by immunohistochemistry on sections from frozen siRNA chimera testis or wild testis at 14 weeks. GATA-1 positive cells were presence at seminiferous tubules, but Dmc1 and SCP1 positive cells were presence at lumen.Both in vitro and in vivo results indicate that PTP-X involve in differentiation of spermatogenesis.