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- 2015
HtrA1表达被沉默的滋养细胞株HPT-8/pSilencer4.1-HtrA1的构建
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Abstract:
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。
ABSTRACT: 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). Conclusion We successfully constructed stable trophoblastic cell line HPT-8/ pSilencer4.1-HtrA1 with silenced expression of HtrA1, which lays foundation for further research
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