免疫磁珠分离羊水来源OCT-4阳性胎儿干细胞的实验研究  

Experimental research on OCT-4 positive fetal stem cells derived from amniotic fluid

作　　者：朱哲[1] 潘骏[2] 张亮[1] 耿红全[3] 谢华[3] 陈方[1] 

机构地区：[1]上海交通大学医学院附属新华医院泌尿外科 [2]上海市儿科医学研究所 [3]上海交通大学医学院附属新华医院小儿泌尿外科,200092

出　　处：《中华小儿外科杂志》2007年第11期598-601,共4页Chinese Journal of Pediatric Surgery

基　　金：上海市科学技术委员会基金(05jc14040)

摘　　要：目的探讨免疫磁珠分离羊水来源OCT-4阳性胎儿干细胞的可行性。方法采用免疫磁珠细胞分选法去除羊水细胞中CD44和SSEA4阳性细胞，使OCT-4阳性胎儿干细胞间接得到分离，体外培养扩增后，观察细胞生长特性，免疫荧光染色计算OCT-4阳性胎儿干细胞得率，Real—TimePCR比较分离前后OCT-4表达量的差异，免疫组化检测NANOG、AKP、MAP-2、NGFR和Myosin等细胞因子的表达。结果经免疫磁珠分离的细胞接种12h内贴壁生长，形态呈梭形，部分呈多角形．融合后形成辐射状排列。免疫荧光染色OCT-4阳性细胞得率为（90．15±8．25）％，Real—TimePCR检测结果显示分离后细胞OCT-4的表达量较未分离细胞和M义有显著性差异（P〈0．01）。原代培养可获得（1～2）×10^7个细胞，3代可获得（2～4）×10^8个细胞．传至10代以后细胞的增殖能力下降。免疫组化结果显示OCT-4阳性胎儿干细胞表达NANOG、AKP、MAP-2和NGFR等细胞因子。结论免疫磁珠细胞分选法可以从羊水中分离出OCT-4阳性的胎儿干细胞，分离后的细胞保持干细胞原有特性及生物学特征，可作为组织工程种子细胞。Objective To investigate the possibility of isolating OCT-4 positive stem cells derived from amniotic fluid during the second-trimester of gestation by immunomagnetic beads method. Methods CD44 and SSEA4 positive stem cells were removed by immunomagnetie beads method： OCT-4 positive fetal stem cells were isolated indirectly and expanded in culture medium in vitro. The characteristic of proliferation and growth was observed and the positive cells were assessed by OCT-4 immunofluorescence staining. The expression of OCT-4 was compared with MSC and primary amnio- cytes by Real-Time PCR. Immunochemistry stain was also used to detect the expression of cell factors such as NANOG,AKP,MAP-2,NGFR and Myosin etc. Results The isolated cells that adhered to the culture dishes within 12 h after inoculation showed a spindle or polygon shape with radiate array form in confluence. The OCT-4 positive cells were 90. 15 ± 8.25% with significant difference from MSC and primary amniocytes （P〈0. 01 ）. Approximately （1 ~2） × 10^7 cells were obtained after primary culture and the number was （2~4） × 10^8 after the third passage, but the proliferate ability of cells in the tenth generation decreased gradually. The OCT-4 positive cells demonstrated NANog, AKP, MAP-2 and NGFR. factor expressions. Conclusions OCT-4 positive fetal stem cells could be isolated from the amniotic fluid by immunomagnetic beads cell sorting, yet maintain the characteristic similar to those of stem cells. These method can be used as a novel source for tissue engineering.

关 键 词：羊水 OCT-4阳性胎儿干细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]