CAJ | 학술논문

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。
목적：구건 pSilencer4.1/HtrA1질립,은정전염인자양세포주 HPT-8。방법면역조화 SABC 방법검측HtrA1재 HPT-8세포중적표체；구건 pSilencer4.1/HtrA1질립；장 pSilencer4.1/HtrA1질립、pSilencer4.1공백질립전염 HPT-8,관찰전염후세포 HtrA1적표체정황。결과HPT-8약90%좌우적세포포질피염성종황색,포핵미견명현염색。전염 pSilencer4.1/HtrA1질립적 HPT-8세포적 G418사선질량농도위200μg/mL。RT-PCR、Western blot법감정전염세포 HtrA1적표체,결과현시전염 Psilence4.1/HtrA1중조질립적세포중 HtrA1표체수평교공백질립、정상세포명현하강(P 균<0.05)。공백질립조、HPT-8조간흡광도치무통계학차이(P >0.05)。결론성공구건료은정적、HtrA1침묵표체적자양세포주 HPT-8/pSilencer4.1-HtrA1,위하일보적연구전정료실험기출。
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 200 μg/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.