HIF-3αノックアウトとHIF-2αノックダウンを組み合わせたマウスに基づく肺胞発生解析

Analysis of the alveolar development based on

the mice with the combined HIF-3α knockout

and HIF-2α knockdown

著者（英）

ZULKIFLI AMIN FIRMAN

year

2019

その他のタイトル

HIF-3αノックアウトとHIF-2αノックダウンを組み

合わせたマウスに基づく肺胞発生解析

学位授与大学

筑波大学 (University of Tsukuba)

学位授与年度

2018

報告番号

12102甲第8882号

URL

http://hdl.handle.net/2241/00156659

氏 名 FIRMAN ZULKIFLI AMIN

学 位 の 種 類

博士（医学）

学 位 記 番 号

博甲第 ８８８２ 号

学 位 授 与 年 月

平成 ３１年 １月 ３１日

学位授与の要件

学位規則第４条第１項該当

審 査 研 究 科

人間総合科学研究科

学 位 論 文 題 目

Analysis of the alveolar development based on

the mice with the combined HIF-3α knockout and HIF-2α

knockdown（HIF-3α ノックアウトと HIF-2α ノックダウンを組

み合わせたマウスに基づく肺胞発生解析）

主

査

筑波大学教授 博士（獣医学） 杉山文博

副

査

筑波大学講師 博士（理学） 小林麻己人

副

査

筑波大学講師 博士（医学） 丸島愛樹

副

査

筑波大学助教 博士（薬学） 船越祐司

論文の内容の要旨

Abstract of thesis

The doctoral dissertation of Mr. Firman Zulkifli Amin describes his study on the role of HIF-3 and

HIF-2αin the development of murine alveoli. The following is the abstract of his dissertation.

（目的 Purpose）

Hypoxia-inducible factors (HIFs; HIF-1α, HIF-2α, and HIF-3α) act as regulators of the molecular hypoxic response; in a study examining normal alveolarization in fostered newborn rats, HIFs promoted alveolar development and regeneration by preventing and repairing oxygen-induced alveolar damage. A previous study that used HIF-3α knockout mice showed impaired lung remodeling exhibited by the walls of the secondary septa in subdivided alveoli, and immunostaining of alveolar endothelial cells presented an increase in defective space in the inter-alveolar septa and hyperplasia of endothelial cells during the maturation of alveolar formation in these knockout mice. Additionally, another study revealed that these HIF-3α−/− mice showed impairments in lung endothelial cells presented by slow growth and a decreased number of tubes formed by endothelial cells. Furthermore, a different but related study demonstrated that impaired expression of HIF-2α in HIF-2α knockdown mice (HIF-2αkd/kd_{) induced compensatory}

expression of HIF-1α. To further investigate a role of HIFs in alveolar development, HIF-3α−/− mice were interbred with HIF-2αkd/+ _{mice, resulting in the double mutant mice. In the present study, the author investigated the findings}

from HIF-3α−/−:: HIF-2αkd/kd _{double mutant mice to uncovers more insights in alveolar development.}

（対象と方法 Materials and Methods）

Firstly, the wild-type (WT) and mutant mouse lines were maintained on the C57BL/6J genetic background. The

HIF-2αkd/kd _{mice and HIF-}3α-/- _{(single mutant) mice were each generated. Concerning this, the mating of single mutant}

and HIF-2αkd/+ _{mice resulted in the combined HIF-}3α-/- _{and HIF-}2αkd/kd _{mice, which were called the double-mutant}

mice. Then the author dissected the lungs from each of the WT and double-mutant mice at postnatal day 0 (P0) for hematoxylin and eosin staining and immunological staining with CD31, HIFs, vascular endothelial cadherin (VE-cadherin), and vascular cell adhesion molecule (VCAM-1) antibodies. The lungs from each of the WT, HIF-2αkd/kd_,

single-mutant, and double-mutant mice were dissected at postnatal 6-week-old for cell culture. Also, the gene expression of HIFs and their target genes were analyzed by quantitative reverse transcription polymerase chain reaction (qPCR).

（結果 Results）

No mice with both HIF-3α-/- _{and HIF-}2αkd/kd _{died immediately after birth. The mice with both HIF-}3α-/- _and

HIF-2αkd/kd _{exhibited impaired lung alveolar structure and HIFs expression. Transcriptional analysis of lung cells revealed}

the depressed expressions of HIF-1α, HIF-2α, VE-cadherin, VCAM-1, Tie-2, and Ang-2 in the double mutant mice compared to that in the WT mice. The endothelial cell numbers were also decreased in the double mutant mice as opposed to the WT mice.

（考察 Discussion）

In this study, the author proposed the insightful ideas of the role of both HIF-3α and HIF-2α in lung development, which involved in proliferation of endothelial cells and expression of angiogenic factors. Further studies are necessary to elucidate the underlying mechanism of how HIF-3α and HIF-2α regulate the lung endothelial cells as well as lung development. Nevertheless, it is apprehensible that studies of HIF-dependent responses on pulmonary vascular and airway epithelium are equally important. Thus, the future perspective based on the present study will need to aim at elucidating the functional role of double mutations consisted of both HIF-3α-/- _{and HIF-}2αkd/kd _{in lung endothelial}

cells and alveolar epithelial cells of the double mutant mice.

審査の結果の要旨

Abstract of assessment result

（批評 General Comments）

In this study, the author generated HIF-3α-/- _{and HIF-}2αkd/kd _{double mutant mice to disclose HIFs’ function in alveolar}

development. The findings by the author provide valuable insight for functional contribution of both HIF-3α and HIF-2α in alveolar development, and implicate their involvement in endothelial cell proliferation and angiogenic factor expression, which raises interesting question to be addressed in future research in this field.

The final examination committee conducted a meeting as a final examination on December 04, 2018. The applicant provided an overview of dissertation, addressed questions and comments raised during Q&A session. All of the committee members reached a final decision that the applicant has passed the final examination.

（結論 Conclusion）

The final examination committee approved that the applicant is qualified to be awarded Doctor of Philosophy in Medical Sciences.