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催产素受体基因沉默的大鼠子宫平滑肌原代细胞模型的构建及鉴定 被引量:2

Establishment and identification of an in vitro model with oxytocin receptor gene knockdown in primary smooth muscle cells from rats uterus
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摘要 目的建立催产素受体(oxytocin receptor,OXTR)基因沉默的细胞模型,为催产素受体相关的研究提供技术基础。方法设计3个阳性RNA干扰(RNA interference,RNAi)序列(Oxtr-RNAi-1、Oxtr-RNAi-2、Oxtr-RNAi-3)及一个阴性序列(con-RNAi),合成短发夹RNA(short hairpin RNA,shRNA)干扰片段用于实验。以慢病毒为载体,构建绿色荧光蛋白(green-fluorescent protein,GFP)标记的Oxtr干扰体系。将Oxtr-RNAi慢病毒感染原代大鼠子宫平滑肌细胞,通过观察GFP荧光表达比较各组的转染效率,并用Western blot和荧光定量PCR分别检测OXTR蛋白和mRNA表达水平,来比较各组的干扰效率。结果慢病毒感染5d后,感染Oxtr-RNAi-1病毒的细胞GFP荧光表达细胞比例最高,OXTR蛋白和mRNA水平最低。结论成功构建了沉默Oxtr的子宫平滑肌细胞,为进一步研究催产素受体相关的分子机制奠定实验基础。 Objective To establish a cell model of oxytocin receptor(OXTR)gene silencing and provide a technical basis for the study on OXTR.Methods Three positive RNA interference(RNAi)sequences(Oxtr-RNAi-1,Oxtr-RNAi-2,Oxtr-RNAi-3)and one negative sequence(Con-RNAi)were designed to synthesize short hairpin RNA(shRNA)for the experiment.The GFP-labeled Oxtr interference system was then established with lentivirus as the vector.The prepared Oxtr-RNAi lentivirus was used to infect primary smooth muscle cells from rats uterus.The efficiency of infection was accessed by measuring the GFP fluorescence and the efficiency of Oxtr gene silencing was accessed by detecting the levels of OXTR protein and mRNA with western blot and real-time PCR.Results After 5 days of virus infection,the primary smooth muscle cells infected with Oxtr-RNAi-1 viruses showed the highest rate of GFP fluorescence and lowest level of protein and mRNA of OXTR.Conclusion The Oxtr-silenced uterine smooth muscle cells were successfully constructed.The establishment of this cell line will support further studies on the molecular mechanisms associated with oxytocin receptor.
作者 荣翠平 刘晓茜 孙逸 侯雪芹 Rong Cuiping;Liu Xiaoqian;Sun Yi;Hou Xueqin(The Second Clinical Medical College,Guangzhou University of Chinese Medicine,Guangzhou,Guangdong 510006,China;Institute of Pharmacology,Shandong First Medical University&Shandong Academy of Medical Sciences,Tai’an,Shandong 271016,China)
出处 《中国组织化学与细胞化学杂志》 CAS CSCD 2020年第6期534-538,共5页 Chinese Journal of Histochemistry and Cytochemistry
基金 国家青年基金项目(81703901) 山东省重点研发项目(2019GSF108069) 山东省自然科学基金(ZR2016HB56)。
关键词 原代培养 子宫平滑肌细胞 催产素受体 基因沉默 转染 Primary culture uterine smooth muscle cells oxytocin receptor gene silencing transfection
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