人羊膜上皮细胞的分离培养及其免疫原性初步研究.pdfVIP

· · 国际生殖健康/ 计划生育杂志2011 年7 月第30 卷第4 期 J Int Reprod Health蛐Fam Plan ，July 2011, Vol. 30, No. 4 272 ·论著· 人羊膜上皮细胞的分离培养及其免疫原性初步研究* ▲ ▲ △ 彭 琳 王 建 卢光琇 【摘 要】 目的：分离培养人羊膜上皮细胞（HAEC），通过与同种异体淋巴细胞混合培养探讨HAEC 的免疫原性，为进一步的临床研究提供指导。方法：体外分离培养HAEC，透射电镜观察细胞超微结构，反 转录聚合酶链反应（RT- PCR）和细胞免疫荧光方法检测上皮细胞相关基因及细胞表面标记细胞角蛋白 CK18表达情况。与同种异体外周血来源淋巴细胞建立混合淋巴细胞（the mixed lymphocyte reaction ，MLR） 反应体系，通过MTT法检测细胞增殖率。结果：分离得到的HAEC具有上皮细胞特异性的微绒毛结构，表达 上皮细胞相关基因CDH1，claudin 3和claudin 4 ，并表现为细胞角蛋白CK18阳性。MTT分析检测OD值结果显 示，在HAEC细胞与淋巴细胞浓度分别为1∶2 000 ，1∶200和1∶20时各组的OD值分别为0.074±0.022 ，0.049± 0.012和0.019±0.011。结论：从羊膜中分离得到的HAEC表达CK18 ，且免疫原性较低。 【关键词】 细胞培养技术；上皮细胞；羊膜；细胞，培养的；逆转录聚合酶链反应；荧光免疫测定 The Isolation and Immunogenicity of Human Amniotic Epithelial Cells PENG Lin ▲, WANG Jian ▲, LU Guang-xiu. Institute of the Reproductive and Stem Cell Engineering, Central South University, Changsha 410078, China （PENG Lin ，WANG Jian，LU Guang-xiu）；National Center of Human Stem Cell Research and ▲ Engineering ，Changsha 410078, China (WANG Jian, LU Guang-xiu), Equal contribution Corresponding author : LU Guang-xiu, E-mail: lgxdirector@ 【Abstract 】 Objective ：To isolate the human amniotic epithelial cells and to investigate the inhibition of the isolated-cells to Lymphocyte. Methods ：Human amniotic epithelial cells were isolated from amnion by using trypsin/EDTA. Ultrastructures of the isolated cells were observed by using TEM. Immunofluorescence was used for detecting CK18+ cells, and RT-PCR was performed to detect the gene expression of CDH1, claudin 3 and claudin 4. MTT was used to identify the proliferation of cells after co-culture for 96 h in vitro. Results ： The TEM showed that these isolated cells were pure epithelial cells with microvillus.