HtrA1表达被沉默的滋养细胞株HPT-8/pSilencer4.1-HtrA1的构建  

Construction of trophoblastic cell line HPT-8/pSilencer4.1-HtrA1 with silenced expression of HtrA1

作　　者：宗璐[1] 苟文丽[1] 郭玉琳[1,2] 

机构地区：[1]西安交通大学医学部第一附属医院妇产科,陕西西安710001 [2]榆林市第二医院妇产科,陕西榆林719000

出　　处：《西安交通大学学报（医学版）》2015年第6期749-752,757,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省自然科学基础研究项目(No.2014JQ2-8047);中央高校基本科研业务费专项资金资助(No.XJJ2014072)~~

摘　　要：目的构建pSilencer4.1/HtrA1质粒,稳定转染人滋养细胞株HPT-8。方法免疫组化SABC方法检测HtrA1在HPT-8细胞中的表达;构建pSilencer4.1/HtrA1质粒;将pSilencer4.1/HtrA1质粒、pSilencer4.1空白质粒转染HPT-8,观察转染后细胞HtrA1的表达情况。结果 HPT-8约90%左右的细胞胞质被染成棕黄色,胞核未见明显染色。转染pSilencer4.1/HtrA1质粒的HPT-8细胞的G418筛选质量浓度为200μg/mL。RT-PCR、Western blot法鉴定转染细胞HtrA1的表达,结果显示转染Psilence4.1/HtrA1重组质粒的细胞中HtrA1表达水平较空白质粒、正常细胞明显下降(P均<0.05)。空白质粒组、HPT-8组间吸光度值无统计学差异(P>0.05)。结论成功构建了稳定的、HtrA1沉默表达的滋养细胞株HPT-8/pSilencer4.1-HtrA1,为下一步的研究奠定了实验基础。Objective To construct the plasmid pSilencer4.1/HtrA1 and stably transfect human trophoblastic cell line HPT-8.Methods We detected the expression of HtrA1 in cell line HPT-8 with immunohistochemical SABC,constructed the plasmid pSilencer4.1/HtrA1,transfected human trophoblastic cell line HPT-8 with plasmid pSilencer4.1/HtrA1 and negative plasmid pSilencer4.1,and observed the cell expression after transfection.Results About 90% of cellular cytoplasm of HPT-8 was dyed brown while the nucleus was negatively stained.Selective concentration of G418 was 200μg/mL for HPT-8 to be transfected pSilencer4.1/HtrA1.With RT-PCR and Western blot methods,the expression of HPT-8 transfected plasmid Psilence4.1/HtrA1 was significantly downregulated compared with that of cells with negative plasmid and normal cells（P 0.05）.There was no significant difference in absorbance value between blank plasmid group and HPT-8 group（P〉0.05）.ConclusionWe successfully constructed stable trophoblastic cell line HPT-8/pSilencer4.1-HtrA1 with silenced expression of HtrA1,which lays foundation for further research.

关 键 词：滋养细胞 HPT-8 HTRA1 质粒 

分 类 号：R714[医药卫生—妇产科学]