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Studies on Drug Permeation in Placental Barrier by RNA Interference

RNA干扰药物在胎盘屏障渗透的研究

基本信息

批准号：
17590136
负责人：
WATANABE Yoshiteru
金额：
$ 2.3万
依托单位：
Showa Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590136/
关键词：
placental barrier drug permeation RNA interference gene delivery system transgene expression trophoblast cell pharmacology 遺伝子薬物透過機構

项目摘要

To elucidate drug permeation mechanism by transporters in placental barrier, we positively studied the establishment of RNA interference (RNAi) technique using viral and nonviral vector systems. In a preliminary study, RNAi using fiber-modified Ad vectors, it was suggested that the combination of fiber-modified Ad vectors containing polylysine peptide and RNAi is an effective tool for study of biological and physiological mechanism such as adipogenesis in adipose. Ad vector-mediated RNAi for peroxisome proliferator-activated receptor (PPAR) γ should also be useful to clarify the biological role of the PPAR γ pathway in various tissues. This finding led us to investigate RNAi using Ad vectors in placental barrier functions. On the other hand, we studied transgene activity of Ad vector containing Arg-Gly-Asp (RGD) motif in the fiber protein (Ad-RGD vector) during differentiation of human cytotrophoblast BeWo cells into syncytiotrophoblast-like cells. Ad-RGD vector had no influence on dif … More ferentiation and had a 2.5 fold higher transduction activity than that of the conventional Ad vector. Thus, Ad-vRGD vector can be a powerful tool for gene transfer experiments in syncytiotrophoblast cells. Subsequently, we investigated characteristics of transcription-regulatory elements for gene expression from plasmid vectors in human trophoblast cell lines BeWo and JAR cells. It was found that insertion of intron elements enhanced transcriptional activities in the following order: intron A>hybrid β-globin-immunoglobin intron>no intron. We also found that gene expression level can be controlled by selecting the expression cassette. These results may be useful for further studies on construction of siRNA expression cassette by polymerase II promoter. For induction of transporters, we observed an induction of zinc transporters by forskolin in human trophoblast BeWo cells. A function of transporter in placenta during pregnancy, the mechanism of zinc release from the placenta into fetal circulation is not well understand. Treatment of BeWo cells with forskolin, a representative inducer of differentiation of BeWo cells cytotrophoblast into syncytiotrophoblast, resulted in morphological changes of BeWo cells and elevated Zn transporter 1, 2 and 4 mRNA levels. Several findings in this project should be useful for studies aimed at analysis of transporters in placenta using RNAi. We will continue further studies on RNAi of transporters by delivery of viral and nonviral vectors. Less

为了阐明转运蛋白在胎盘屏障中的药物渗透机制，我们积极研究了利用病毒和非病毒载体系统建立RNA干扰(RNAi)技术。在初步的研究中，利用纤维修饰的Ad载体进行RNAi的研究表明，含有聚赖氨酸肽的纤维修饰的Ad载体与RNAi相结合是研究脂肪生成等生物和生理机制的有效工具。腺病毒载体介导的过氧化物酶体增殖物激活受体γ的RNA干扰也有助于阐明过氧化物酶体增殖物激活受体γ通路在各种组织中的生物学作用。这一发现导致我们使用Ad载体研究RNAi在胎盘屏障功能中的作用。另一方面，我们研究了纤维蛋白中含Arg-Gly-Asp(RGD)基序的Ad载体(Ad-RGD载体)在人细胞滋养层细胞BeWo向合体滋养层样细胞分化过程中的转基因活性。Ad-RgD载体对DIF-…无影响转导活性是常规载体的2.5倍。因此，Ad-vRGD载体可以作为合体滋养层细胞基因转移实验的有力工具。随后，我们研究了人滋养层细胞系BeWo和JAR细胞中转录调控元件对基因表达的调控特性。研究发现，内含子元件的插入增强转录活性的顺序如下：内含子A和GT；杂交β-珠蛋白-免疫球蛋白内含子&GT；没有内含子。我们还发现，基因的表达水平可以通过选择表达盒来控制。这些结果为进一步研究聚合酶II启动子构建siRNA表达框奠定了基础。在转运蛋白的诱导方面，我们观察到Forskolin对人滋养层细胞BeWo细胞锌转运蛋白的诱导作用。作为妊娠期间胎盘转运蛋白的一种功能，锌从胎盘释放到胎儿循环的机制尚不清楚。BeWo细胞滋养层细胞分化为合体滋养层细胞的典型诱导剂Forsklin作用于BeWo细胞后，细胞形态发生改变，锌转运蛋白1、2和4mRNA水平升高。该项目中的几个发现对于利用RNAi分析胎盘转运蛋白的研究应该是有用的。我们将继续通过病毒和非病毒载体的传递来进一步研究转运蛋白的RNAi。较少