胚龄及分离方法对小鼠胚胎生殖细胞扩增效率的影响

Influences of embryo age and isolation method on amplification efficiency of mouse embryonic germ cells

目的：应用原始生殖细胞进行全能干细胞的研究具有与胚胎干细胞同样广阔的前景,尤其适合于难以从囊胚内细胞团中分离培养胚胎干细胞的动物。但胚胎生殖细胞体外培养条件一直是制约这方面发展的瓶颈。实验设计了体外有效扩增小鼠原始生殖细胞的方法,拟搭建稳定高效的原始生殖细胞培养平台。方法：实验于2006-10/2007-08在重庆医科大学组织胚胎学教研室重庆市生物化学分子药理学重点实验室完成。①实验材料：清洁级昆明孕鼠由重庆医科大学动物实验中心提供,见阴栓计为0.5d胚龄,实验过程中对动物处置符合动物伦理学标准。②实验方法：取胚龄8．5d胎鼠尾端尿囊及周围组织，取胚龄9．5～10．5d胎鼠后肠及周围组织，取胚龄11．5d，12．5d胎鼠生殖嵴及周围组织，分别经0．25％胰酶一0．02％EDTA消化并接种于0．1％明胶包被的培养皿中。比较不同妊娠时间点取材对原代、F1代胚胎生殖细胞克隆形成的影响。采用SSEA-1免疫组织化学法计数细胞阳性率，计数胚胎生殖细胞克隆联合MTT法比较胚龄11．5d，12．5d两个时间点原始生殖细胞的增殖能力。比较上述两种取材培养方法对胚龄11．5d，12．5d原始生殖细胞扩增的影响。采用碱性磷酸酶染色、免疫组化检测SSEA一1、免疫荧光检测Nanog，体外分化实验鉴定分化能力。结果：①胚龄11．5d，12．5d胎鼠原代和F1代集落形成率较胚龄8．5～10．5d高，扩增效率显著提高（P〈0．01）。②免疫组织化学法检测分离的胚龄11．5d，12．5d原始生殖细胞SSEA-1阳性率差异不显著（P〉0．05）。原代第3天克隆计数显示，胚龄11．5d胎鼠高于12．5胚龄胎鼠（P〈0．01），MTT数据显示11．5胚龄组稍高于12．5胚龄组。③两种方法对胚龄11．5d，12．5d胎鼠胚胎生殖细胞集落形成无显著性差异（P〉O．05），但生长方式有所不同。④克隆胚胎生殖细胞呈典型的胚胎干细胞样集落生长，能稳定传代；碱性磷酸酶、SSEA-1、Nanog表达强阳性：体外能分化为囊状类胚体。结论：胚龄11．5d和12．5d胎鼠，尤其是胚龄11．5d胎鼠扩增原始生殖细胞效率较高，在这两个时间点运用与周围组织共培养法和生殖嵴分离培养法均适宜。

AIM：Studies on totipotential stem cells using primordial germ cells （PGCs） have the same application prospect as embryonic stem cells, especially for the animals difficult to isolate embryonic stem cells from blastula. In vitro culture condition of embryo germ cells is the key to control this. This study designs a method to effectively amplify PGCs of mice in vitro and establishes an effective culture method of PGCs. METHODS： Experiments were carried out in the Department of Histology and Embryology of Chongqing Medical University and Key Laboratory of Biochemistry and Molecular Pharmacology from October 2006 to August 2007. ①Clean Kunming pregnant mice （embryos 0.5 dpc） were provided by Animal Experimental Center of Chongqing Medical University. Experimental procedures met the animal ethical standards. ②Allantois and surrounding tissues of 8.5 day post coitum （dpc） embryos, the hindhut and surrounding tissues of 9.5 dpc and 10.5 dpc embryos, the gonadal ridges and surrounding tissues of 11.5 dpc and 12.5 dpc embryos were collected and digested with 0.25%pancreatin-0.02%EDTA, then the cells were cultured in the plastic petridishes which are pretreated with 0.1% gelatine. The formation ratio of primary and the first passaged Embryonic germ （EG） clones were compared among those different time points. The SSEA-I positive ratio of isolated cells was compared between the two days by immunohistochemical method. The assays of clone numbers counting and MTT were used to compare the different proliferation ability of PGCs from the 11.5 dpc and 12.5 dpc embryos. The effects of expanding PGCs from 11.5 dpc and 12.5 dpc embryos by those two ways above were compared. The EG clones were analyzed by the expression of alkaline phosphatase and the immunologic marker SSEA- 1, the undifferentiated marker Nanog and the differentiation ability in vitro were also tested. RESULTS.- ①The formation ratio of primary and the first passaged EG clone from I 1.5 and 12.5 dpc embryos was higher than that from 8.5 to 10.5 dpc embryos and efficiency of expansion was increased （P 〈 0.01）.②The SSEA-1 positive ratio of isolated PGCs from 11.5 and 12.5 dpc embryonic gonadal ridges had no significant difference by immunohistochemical method （P 〉 0.05）. On the third day, the number of the primary EG clones from I 1.5 dpc embryos was higher than that from 12.5 dpc embryos （P 〈 0.01）. MTT showed the same results. ③The EG clones formation ratio of I 1.5 dpc and 12.5 dpc embryos by two different ways was similar （P 〉 0.05）, but the two groups had different characteristics. ④The EG clones have representative morphology with high level of alkaline phosphatase activity and SSEA-1, Nanog expression, which can differentiate into the cystic embryoid body. CONCLUSION： 1 1.5 dpc and 12.5 dpc mouse embryos, especially 11.5 dpc embryos are the optional time points to expand PGCs efficiently. Co-culturing with the tissue and isolating the gonadal ridges both of these two ways are practicable.