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中华产科急救电子杂志 ›› 2022, Vol. 11 ›› Issue (01) : 46 -52. doi: 10.3877/cma.j.issn.2095-3259.2022.01.010

实验研究

选择适用于研究滋养层细胞合体化过程的内参基因的实验研究
郑婉珊1, 殷茵2, 涂兆伟3, 曹彬4,()   
  1. 1. 广州医科大学附属第三医院妇产科 广东省产科重大疾病重点实验室 广州重症孕产妇救治中心 510150;厦门大学医学院生殖调控与生殖健康研究福建省高等学校重点实验室 361102
    2. 南京医科大学附属第一医院妇产科 210029
    3. 广州医科大学附属第三医院妇产科 广东省产科重大疾病重点实验室 广州重症孕产妇救治中心 510150
    4. 厦门大学医学院生殖调控与生殖健康研究福建省高等学校重点实验室 361102
  • 收稿日期:2021-08-26 出版日期:2022-02-18
  • 通信作者: 曹彬
  • 基金资助:
    国家重点研发计划项目(2018YFC1004400); 国家自然科学基金项目(81971414); 福建省自然科学基金项目(2020J06003)

Selection of internal references gene for studying trophoblast syncytialization

Wanshan Zheng1, Yin Yin2, Zhaowei Tu3, Bin Cao4,()   

  1. 1. Department Obstetrics and Gynecology, The Third Affiliated Hospital of Guangzhou Medical University, Guangdong Province Key Laboratory of Major Obstetric Diseases, Guangzhou Medical Center for Critical Pregnant Women, Guangzhou 510150, China; Fujian Provincial Key Laboratory of Reproductive Health Research, School of Medicine Xiamen University, Xiamen 361102, China
    2. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
    3. Department Obstetrics and Gynecology, The Third Affiliated Hospital of Guangzhou Medical University, Guangdong Province Key Laboratory of Major Obstetric Diseases, Guangzhou Medical Center for Critical Pregnant Women, Guangzhou 510150, China
    4. Fujian Provincial Key Laboratory of Reproductive Health Research, School of Medicine Xiamen University, Xiamen 361102, China
  • Received:2021-08-26 Published:2022-02-18
  • Corresponding author: Bin Cao
引用本文:

郑婉珊, 殷茵, 涂兆伟, 曹彬. 选择适用于研究滋养层细胞合体化过程的内参基因的实验研究[J]. 中华产科急救电子杂志, 2022, 11(01): 46-52.

Wanshan Zheng, Yin Yin, Zhaowei Tu, Bin Cao. Selection of internal references gene for studying trophoblast syncytialization[J]. Chinese Journal of Obstetric Emergency(Electronic Edition), 2022, 11(01): 46-52.

目的

筛选并确定适用于滋养层细胞合体化研究的内参基因。

方法

利用人绒毛膜癌细胞BeWo和人滋养细胞干细胞比较了11个候选内参基因的稳定性,并使用GeNorm、NormFinder和BestKeeper三种软件程序来确定最合适用于研究滋养层细胞合体化的内参基因。

结果

TBP和RPS13为稳定表达的内参基因,而常用的GAPDH(glyceraldehyde-3-phosphate dehydrogenase)和β-actin(actin, beta)在滋养层细胞合体化过程中表达水平稳定性不佳。

结论

在利用实时荧光定量聚合酶链反应进行滋养层细胞的合胞化研究时,TBP和RPS13是首选的内参基因。

Objective

To screen and identify reference genes suitable for studying on trophoblast cells.

Methods

The stability of 11 candidate reference genes were compared using BeWo and human trophoblast stem cells (hTSCs). Three software programs, GeNorm, NormFinder, and BestKeeper, were used to determine the most suitable internal reference genes for the study of trophoblast cell syncytialization.

Results

TBP and GRPS13 were stable reference genes, while GAPDH and β-actin expression levels were not stable during trophoblast cell syncytialization.

Conclusions

TBP and RPS13 were recommended as the preferred internal reference genes for trophoblast syncytialization studies using RT-PCR.

图1 BeWo和hTSCs模型中滋养层的合体化效率。诱导合体化后,BeWo细胞(A)和hTSCs(B-C)中E-cad(绿色)和hCG-β(红色)的代表性免疫荧光染色;细胞核经DAPI染色;比例尺50 μm。A: BeWo细胞(A)分别用DMSO/FSK(50 μmol/L)处理72 h;B~C: hTSCs分别利用增殖培养液和分化培养液培养72 h
图2 BeWo和hTSCs模型滋养层合胞过程中内参基因的qPCR分析结果。A~B:为BeWo和hTSCs的所有样本中候选的内参基因的Cq值;n=3;柱子上的线代表中位数,柱子代表了上四分位数和下四分位数
表1 NormFinder、GeNorm和BestKeeper三种程序对BeWo细胞中候选内参基因稳定性的排名比较
表2 NormFinder、GeNorm和BestKeeper三种程序对hTSCs中候选内参基因稳定性的排名比较
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