Development of Gene Transfer Systems into Placental Cells for Studies of Drug Transport Mechanism in Blood-Placenta Barrier
| Project/Area Number |
15590139
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Medical pharmacy
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| Research Institution | Showa Pharmaceutical University |
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Principal Investigator |
WATANABE Yoshiteru Showa Pharmaceutical University, Professor, 薬学部, 教授 (70175131)
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| Co-Investigator(Kenkyū-buntansha) |
KONDOH Masuo Showa Pharmaceutical University Assistant, Professor, 薬学部, 講師 (50309697)
FUJII Makiko Showa Pharmaceutical University Associate, Professor, 薬学部, 助教授 (50199296)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | 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 / CAR / ヘパラン硫酸 |
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| Research Abstract |
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
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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
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Report
(3 results)
Research Products
(12 results)
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[Journal Article] Comparison of transgene expression mediated by several fiber-modified adenovirus vectors in trophoblast cells.2005
Author(s)
小泉直也, 近藤昌夫, 水口裕之, 中西剛, 増山茜, 井田史江, 藤井まき子, 早川尭夫, 中島恵美, 田中慶一, 渡辺善照
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Journal Title
Description
「研究成果報告書概要(和文)」より
Related Report
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[Journal Article] Comparison of transgene expression mediated by several fiber -modifleed Adenovirus vectors in trophoblast cells.2005
Author(s)
Naoya Koizumi, Masuo Kondoh, Hiroyuki Mizuguchl, Tsuyoshi Nakanishi, Akane Masuyama, Fumie Ida, Makiko Fujii, Takao Hayakawa, Emi Nakashima, Keiiehi Tanaka, Yoshiteru Watanabe
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Journal Title
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] Comparison of transgene expression mediated by several fiber-modified adenovirus vectors in trophoblast cells.2005
Author(s)
小泉直也, 近藤昌夫, 水口裕之, 中西剛, 増山茜, 井田史江, 藤井まき子, 早川尭夫, 中島恵美, 田中慶一, 渡辺善照
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Journal Title
Related Report
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