中文摘要：

目的构建高效表达的DAZL重组腺病毒载体系统，观察DAZL基因对人脐带间充质干细胞（HUMSCs）向男性生殖细胞分化的影响。方法1．用Gateway腺病毒系统制备含DAZL及EGFP基因的重组腺病毒pAd-DAZL-EGFP。2．用组织块贴壁法获得HUMSCs，用Ad-EGFP-DAZL感染第3代生长良好的HUMSCs，观察感染效率及细胞生长情况，确定最佳感染复数（MOI）和感染时间。3．通过形态学、反转录（RT）-PCR和免疫荧光鉴定DAZL基因异位表达诱导HUMSCs向男性生殖细胞方向分化的结果。结果成功构建表达DAZL的腺病毒，病毒滴度为1．0×10^9pfu／mL。最佳的感染条件为：MOI=100，感染时间：8h，该条件下的感染率为（43．00±1．52）％。HUMSCs在感染DAZL重组腺病毒后形态发生了明显的变化，第4—6天由成纤维细胞变成圆形，少数变圆的细胞在2～3周后变成蝌蚪状，阴性对照组及空白对照组细胞则未发生类似变化。RT—PCR和免疫荧光均证实实验组表达精原细胞的标志STELLA（DDPA3）、VASA（DDX4）、c—kit、DAZL和OCT4（POU5F1），阴性对照组及空白对照组则只表达内参基因。结论DAZL重组腺病毒感染HUMSCs后能诱导HUMSCs向男性生殖细胞方向分化，证实DAZL基因是精子形成过程中的关键基因。

英文摘要：

Objective To construct recombinant adenovirus vector encoding DAZL and evaluate the effect of DAZL transfection on proliferation and differentiation of human umbilical cord Wharton＇s jelly-derived mesenchymal stem ceils （HUMSCs） into germ cells. Methods 1. The recombinant adenovirus vector pAd-DAZL-EGFP contained DAZL and EGFP gene was established by the Gateway system. 2. Isolating, culturing and purifying HUMSCs with adherent method. HUMSCs of the third generation were infected and cultured by pAd-DAZL-EGFP to gain the best multiplicity of infection（MOI） and incubated time. 3. Differentiation of HUMSCs into germ cells was valued after HUMSCs were transfected by pAd-DAZL-EGFP by morphology, reverse transcription（RT）-PCR and immunofluo rescence. Results The recombinant adenovirus vector was successfully established with tilter of 1.0 × 109 pfu/mL. The best incubated time was 8 h while the best MOI was 100. The efficiency of infection was （43.0 ± 1.52） % on this infected condition. The shapes of HUMSCs changed greatly from fibroblast to the round on day 4 to day 6 after infection, some even familiar to the tadpole after 2 to 3 weeks. Moreover, these ceils expressed the known molecular markers of spermatogonial cells, such as STELLA （DDPA3）, VASA （DDX4）, c-kit, DAZL and OCT4 both in mRNA and protein levels, but the negative control group and the blank control group didn＇t. The controls only express the housekeeping gene. Conclusion HUMSCs infected by pAd-DAZL-EGFP can be differentiated into germ cells successfully,which proves that DAZL play a critical role in germ cell development and differentiation.