干细胞体外自发分化和相关基因表达状况的研究  

RESEARCH ON SPONTANEOUS DIFFERENTIATION OF STEM CELLS AND RELATED GENE EXPRESSION PROFILE

作　　者：钱坤[1] 陈红[2] 章汉旺[1] 靳镭[1] 李豫峰[1] 刘群[1] 张苏明[2] 朱桂金[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院生殖医学中心,武汉430030 [2]华中科技大学同济医学院附属同济医院神经科,武汉430030

出　　处：《解剖学报》2007年第1期81-84,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30370505);同济医院基金资助

摘　　要：目的在人胚胎干细胞(hESC)体外自发分化过程中,检测子宫内膜发育相关基因的表达以及子宫内膜样组织结构,子宫内膜样细胞的存在。方法悬浮法培养类胚体14d,10%中性甲醛固定,常规石蜡包埋切片,取具有子宫内膜样组织结构的切片行雌激素受体(ER)免疫组织化学染色。hESC贴壁分化10d,免疫荧光双标检测雌激素受体/波形蛋白(角蛋白)的表达。hESC贴壁分化5d,RT-PCR方法检测子宫内膜发育相关基因:Wnt4、Wnt7a、Wnt5a、Hoxa10、Hoxa11、ER的表达。结果hESC贴壁分化5d后可检测到Wnt4、Wnt7a、Wnt5a、Hoxa10、Hoxa11、ER的表达。hESC贴壁分化10d后,部分细胞同时表达ER/波形蛋白(角蛋白)。培养14d类胚体内存在子宫内膜样的腺体结构,部分腺体结构ER表达阳性。结论hESC自发分化过程中,可能部分细胞具有自发分化为子宫内膜干细胞,子宫内膜细胞的倾向,但需对整个发育过程进行进一步鉴定。Objective To investigate the expression of endometrial developing-related genes, the endometrium-like structure and cells during spontaneous differentiation of hESCs. Methods （ 1 ） Embryoid bodies were cultured in suspension for 14days, fixed in 10% neutral-buffered formalin and embedded in paraffin. Estrogen receptor （ER） was detected by immunocytochemical staining. （2） hESCs were cultured in a 35 mm plastic dish and differentiated spontaneously for 10 days. The expression of ER and Vimentin/Keratin was detected by double immunolluorescence histochemistry. （3） hESCs were cultured in a 35 mm plastic dish and differentiated for 5 days. The expressions of endometrial developing-related genes including Wnt4, Wnt7a, Wnt5a, Hoxa10, Hoxa11 and ER were detected by RT-PCR. Results （1） The endometrium-like structures were detected in 14-day-old EBs and some were positive for ER staining. （2） Some spontaneously differentiated cells from hESCs for 10 days were positive for ER together with Vimentin/Keratin. （3）Wnt7a, Wnt4, Hoxa10, Hoxa11 and ER were detected by RT-PCR in hESCs which were differentiated spontaneously for 5 days. Conclusion During the spontaneous differentiation of hESCs, some cells are liable to differentiate into endometrial stem cells and endometrium-like cells. However, further identifications of the whole development process are needed to be done in the future.

关 键 词：子宫内膜 子宫内膜干细胞 逆转录聚合酶链反应 免疫荧光双标组织化学 分化 人胚胎干细胞 

分 类 号：R329.1[医药卫生—人体解剖和组织胚胎学]