Stra 8基因的激活与精原干细胞的特异性分化研究

Activation of Stra 8 Gene During the Differentiation of Spermatogonial Stem Cells

摘要:

视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现，在雄性生殖腺发育过程中有一组基因，它们可以被视黄酸特异性的诱导活化，称为Stra（Stimulated by Retinoic Acid）基因。从鼠源分离得到的Stra8 基因编码一种细胞质蛋白，该基因只特异性的在成熟雄性生殖细胞中表达，其功能被认为与精子形成有关。为研究Stra8 基因的表达特性，我们从小鼠的基因组中克隆了Stra8 基因的启动子序列（1.4kb）。将Stra8 基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前，构建成由Stra8 基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中，如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等，通过荧光显微镜观察发现，绿色荧光蛋白只在小鼠精原干细胞中表达，表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞，经G418筛选2周后，用视黄酸诱导，12h培养后，有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8 的转录，还有生殖细胞特异表达基因CyclinA8 和Oct4的转录，这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。

Abstract:

Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse，Stra8 is restrictively expressed in male germ line cells，and its function is related to the development of sperm. In order to investigate the feature of Stra8gene expression，the 1.4kb (-1407～+7) promoter region of Stra8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4kb promoter region，and the reporter gene of EGFP that was regulated by 1.4kb Stra8 promoter. To investigate the specificity of Stra8promoter，the vector pStra8-EGFP was transfected into undifferentiated mouse stem cells such as ES-129，bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells，which indicated that Stra8 gene was specially regulated in testis tissue. As the gene marker，vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks，the mMSC cells were induced by retinoic acid. After 12 hours induction，some induced cells started to express GFP protein，which was observed under the fluorescence microscope. At the same time，several stem cell specificity biomarkers such as Oct4，and spermatogonial stem cell biomarkers such as CyclinA2and Stra8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.