Development of Gene Transfer Systems into Placental Cells for Studies of Drug Transport Mechanism in Blood-Placenta Barrier

开发胎盘细胞基因转移系统以研究血胎盘屏障中的药物转运机制

• 批准号: 15590139
• 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: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590139/
• 关键词: placenta; trophoblast cell; adenovirus vector; coxsackievirus and adenovirus receptor; integrin; fiber-modified adenovirus vector; gene deliverv system; transgene oxpression; trohoblast cell; アデノウイルスベクター; coxsackievirus and adenovirus recepter /

The transfer of genes of interest is a useful method for studying placental biology. Recombinant adenovirus (Ad) vector is run efficient vector for Transgene ne expression. An interaction between the fiber of Ad and the coxackievirus and adenovirus receptor (CAR) on the cell membrane is the first step in infection. We developed fiber-modified Ad vectors and showed that they improved transgene activity un several cell Inns when compared to wild-type vector: Subsequently, we investigated the ability of three fiber-modified Ad vectors to transduce human choriocarcinorna cell lines; (JEG-3, JAR and BeWo) used as in vitro models of human placenta, and rat trophoblast cell lines (Rcho-1, TR-TBT 18d-1 and TR-TBT 18d-2) We compared the transgene efficiency of wild-type Ad-L2 vector, Ad-RGD(HI)-L2 vector containing an Arg-Gly- Asp motif, Ad-K7(C)L2 vector containing a 7 tandem lysine motif, and Ad-RGD(HI)K7(C)-L2 vector containing both motifs in the fiber. The luciferase gene as a reporter gene … More was used. In the human and rodent trophoblast cell lines, Ad-RGD(HI)-L2 had the greatest infectious potential, followed by Ad-RGD(HI)K7(C)-L2. Ad-K7(C)L2 fund Ad-L2. Compared to the amount of luciferase produced by wild-type vector Ad-RGD(HI)-L2 mediated 8-fold the amount of luciferase in JEG-3 cells. 14-fold in JAR cells, 77-fold im BeWo cells, 5-fold in Rcho-1,19-fold in Tr-TBTa8d-1 and 15-fold in TR-TBT 18d-2. These results indicate that Ad-RGD(HI) is a potential recombinant Ad vector for transgene expression in some trophoblast cell lines. Conventional Ad type 5 vectors have a narrow range of tropism and are limited by the size of the transgene that can be packaged. To overcome these limitations, we developed an Ad vector (Ad5/35 vector) containing a chimeric Ad type 5 amd 35 fiber protein. We evaluate the ability of the Ad5/35 vector to transfer genes into human trophoblast cell lines (JAR, JEG-3 and BeWo cells) above mentioned. It was found thatt expression of CD-46, which are recptors for Ad5/35 vector, are higher than that of CAR in all 3 trophoblast cell lines, as determined by flow cytometry. Sunsequently, we compared the trnasducing activity of Ad5 vector and Ad5/35 vector that each epressed luciferase as a reporter gene. Ad5/35 vector had superior gene transfer activity than the conventional Ad vector in all three trophoblast cell lines (1.8-fold in JAR cells, 5-fold in BeWo cells, 6-fold in JEG-3 cells). This, Advector that contains chinnerictype 5 and 35 fiber protein can be a powerful tool for gene trnasfer experiments in human trophoblast cell lines. Less

目的基因的转移是研究胎盘生物学的有用方法。重组腺病毒(Ad)载体是高效表达Ne基因的载体。腺病毒纤维与细胞膜上的柯萨奇病毒和腺病毒受体(CAR)相互作用是感染的第一步。我们开发了纤维修饰的Ad载体，与野生型载体相比，它们在几种细胞中的转基因活性都有所提高：随后，我们研究了三种纤维修饰的Ad载体转导人绒毛膜癌细胞的能力；将野生型Ad-L2载体、含有Arg-Gly-Asp基序的Ad-RGD(HI)-L2载体、含有7个串联赖氨酸基序的Ad-K7(C)L2载体和含有两种基序的Ad-RGD(HI)K7(C)-L2载体的转基因效率进行了比较。荧光素酶基因作为报告基因…使用了更多。在人和啮齿动物滋养层细胞系中，Ad-RGD(HI)-L2的侵染力最大，其次是Ad-RGD(HI)K7(C)-L2。AD-K7(C)L2基金Ad-L2。与野生型载体Ad-RGD(HI)-L2介导的荧光素酶产量相比，JEG-3细胞的荧光素酶产量提高了8倍。在JAR细胞中为14倍，在BeWo细胞中为77倍，在Rcho-1中为5倍，在Tr-TBTa8d-1中为19倍，在tr-TBT 18d-2中为15倍。这些结果表明，Ad-RGD(HI)是一种在某些滋养细胞系中进行转基因表达的潜在的重组Ad载体。传统的Ad5型载体的趋向性范围很小，并且受到可包装的转基因大小的限制。为了克服这些限制，我们开发了一种含有嵌合的Ad5和35纤维蛋白的Ad载体(Ad5/35载体)。我们评估了Ad5/35载体将基因转移到上述人滋养层细胞系(JAR、JEG-3和BeWo细胞)的能力。流式细胞仪检测发现，作为Ad5/35载体受体的CD-46在3种滋养层细胞系中的表达均高于CAR。因此，我们比较了Ad5载体和Ad5/35载体各自以荧光素酶为报告基因的转导活性。Ad5/35载体在三种滋养层细胞系中的基因转移活性均优于常规载体(在JAR细胞中为1.8倍，在BeWo细胞中为5倍，在JEG-3细胞中为6倍)。这种含有Chinneric5型和35型纤维蛋白的Ad载体可以作为人滋养层细胞系基因转移实验的有力工具。较少

项目成果

Comparison of transgene expression mediated by several fiber -modifleed Adenovirus vectors in trophoblast cells.

滋养层细胞中几种纤维修饰的腺病毒载体介导的转基因表达的比较。

• 发表时间: 2005
• 期刊: Placenta (in press)
• 作者: Naoya Koizumi;Masuo Kondoh;Hiroyuki Mizuguchl;Tsuyoshi Nakanishi;Akane Masuyama;Fumie Ida;Makiko Fujii;Takao Hayakawa;Emi Nakashima;Keiiehi Tanaka;Yoshiteru Watanabe
• 通讯作者: Yoshiteru Watanabe

Efficient gene transfer into human trophoblast cells with adenovirus vector containing chimeric type 5 and 35 fiber protein.

• DOI: 10.1248/bpb.27.2046
• 发表时间: 2004-12
• 期刊: Biological & pharmaceutical bulletin
• 影响因子: 2
• 作者: N. Koizumi;H. Mizuguchi;M. Kondoh;M. Fujii;T. Hayakawa;Yoshiteru Watanabe

小泉直也, 水口裕之, 宇都口直樹, 渡辺善照, 早川堯夫: "Generation of fiber-modified adenovirus vector containing heterologous peptides in both the HI loop and C terminal of the fiber knob."The Journal of Gene Medicine. 5. 267-276 (2003)

Naoya Koizumi、Hiroyuki Mizuguchi、Naoki Utsuguchi、Yoshiteru Watanabe、Takao Hayakawa：“纤维修饰腺病毒载体的生成，在纤维旋钮的 HI 环和 C 末端均含有异源肽。”基因医学杂志 5. 267-。 276（2003）

Expression profiles of zinc transpoters in rodent placenta models

啮齿动物胎盘模型中锌转运蛋白的表达谱

• 发表时间: 2004
• 期刊: Toxicology Letters 154
• 作者: Nagayoshi Asano;Masuo Kondoh;Chiaki Ebihara;Makiko Fujii;Tsuyoshi Nakknishi;Michael J. Soares;Erni Nakashima;Keiiehi Tanaka;Masao Sato;Yoshiteru Watanabe
• 通讯作者: Yoshiteru Watanabe

Comparison of transgene expression mediated by several fiber-modified adenovirus vectors in trophoblast cells.

滋养层细胞中几种纤维修饰腺病毒载体介导的转基因表达的比较。

• 发表时间: 2005
• 期刊: Placenta 26・10
• 作者: 岩松優;日出間純;熊谷忠;Koizumi N et al.
• 通讯作者: Koizumi N et al.