JP2007037434A - ARTIFICIAL PROTEIN EXPRESSED PANCREATIC beta-CELL-SPECIFICALLY, REPORTER GENE ENCODING THE PROTEIN AND METHOD FOR MEASURING AMOUNT OF PANCREATIC betaCELL - Google Patents
ARTIFICIAL PROTEIN EXPRESSED PANCREATIC beta-CELL-SPECIFICALLY, REPORTER GENE ENCODING THE PROTEIN AND METHOD FOR MEASURING AMOUNT OF PANCREATIC betaCELL Download PDFInfo
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- JP2007037434A JP2007037434A JP2005223696A JP2005223696A JP2007037434A JP 2007037434 A JP2007037434 A JP 2007037434A JP 2005223696 A JP2005223696 A JP 2005223696A JP 2005223696 A JP2005223696 A JP 2005223696A JP 2007037434 A JP2007037434 A JP 2007037434A
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Abstract
Description
本発明は、膵臓に存在するβ細胞量の定量に関するものであり、詳しくは、膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌されるレポータータンパク質を用い、その血中濃度等を測定することにより、生体内においてβ細胞量を定量的に推定するモデルを提供するものである。 The present invention relates to the quantification of the amount of β-cells present in the pancreas, and more specifically, using a reporter protein that is specifically and constitutively expressed in pancreatic β-cells and secreted without regulation, and the blood The present invention provides a model for quantitatively estimating the amount of β cells in a living body by measuring an intermediate concentration or the like.
本出願において「遺伝子」とは、ｍＲＮＡとして転写される配列に加え、その上流及び下流に位置し前記ｍＲＮＡの転写調節に関与している配列を含んだ塩基配列全体、と定義する。また本出願において、膵臓β細胞は、膵β細胞またはβ細胞と略す。 In the present application, the “gene” is defined as the entire base sequence including a sequence that is located upstream and downstream in addition to a sequence that is transcribed as mRNA and that is involved in transcriptional regulation of the mRNA. In the present application, pancreatic β cells are abbreviated as pancreatic β cells or β cells.
膵臓β細胞の量的変化を計測することは、糖尿病の発症予知や発症後の経過観察に対して有用であるが、生体内における測定は非常に困難であり、その方法も限定されている（特許文献１，２、非特許文献１）。
Measuring the quantitative change of pancreatic β cells is useful for predicting the onset of diabetes and for the follow-up after onset, but it is very difficult to measure in vivo and its method is limited (
インスリン分泌障害は、１型のみならず２型糖尿病の発症要因の一つとしても必須のものである。２型糖尿病では、インスリン抵抗性が同時に存在しうるが、インスリン分泌の亢進による代償が破綻してはじめて糖尿病を発症すると考えられる。
Insulin secretion disorders are indispensable not only as
インスリン分泌障害には２つの要素が考えられる。すなわち、β細胞機能の質的障害と、β細胞量の不足である。１型糖尿病はβ細胞量の減少によって発症する。２型糖尿病においてはこれら２つの要素がどの程度の比率で関与するかは明らかでないが、β細胞の質的障害（ブドウ糖応答性の障害）に加え、代償的に肥大または過形成する能力の障害も含めたβ細胞量も発症に影響を持つことは疑いない。また、β細胞量は糖尿病発症後も１次的、２次的に減少することが知られている。
Two factors can be considered for impaired insulin secretion. That is, the qualitative disorder of β cell function and the lack of β cell amount.
この様に、β細胞量の変化は糖尿病発症の予知や発症後の経過観察に有用であるが、実際には、これまでのところ動物モデルにおいてさえその測定は極めて困難である。直接的には膵摘出標本での組織学的検査が可能ではあるが、生体に行うことは難しい。従って、生体では定常状態または各種刺激下（例えば、グルカゴン刺激）でのインスリン分泌量等からβ細胞量を推測することが、現時点で取り得る数少ない方法である（特許文献３）。
上記の現状に鑑み、本発明者らは、糖尿病の発症機構に着目して研究を進め、ＭＯＤＹ（ｍａｔｕｒｉｔｙ ｏｎｓｅｔ ｄｉａｂｅｔｅｓ ｏｆ ｔｈｅ ｙｏｕｎｇ）の幾つかのタイプが膵β細胞に発現する転写因子の異常により発症することから、それらの一部ではインスリン分泌障害が関係することを示した（非特許文献２）。また本発明者らは、膵臓ランゲルハンス島からβ細胞が選択的に消失し、インスリン依存状態の糖尿病を発症する「Ｗｏｌｆｒａｍ症候群」の原因遺伝子「ＷＦＳ１」を世界に先駆けて同定し、その病態モデルとなるＷＦＳ１ノックアウトマウスを作成し、糖尿病の発症機序について研究を進めてきている（非特許文献３）。 In view of the above situation, the present inventors proceeded with research focusing on the pathogenesis of diabetes, and some types of MODY (maturity onset diabetics of the young) are caused by abnormal transcription factors expressed in pancreatic β cells. From the onset, it was shown that some of them are associated with impaired insulin secretion (Non-patent Document 2). In addition, the present inventors have identified, for the first time in the world, a causal model “WFS1” of “Wolfram syndrome” in which β-cells selectively disappear from pancreatic islets of Langerhans and develop diabetes in an insulin dependent state. The WFS1 knockout mouse is developed, and research on the pathogenesis of diabetes has been advanced (Non-patent Document 3).
これらの研究過程で、本発明者らは、膵β細胞量の変化を簡便に測定できる系の必要性と有用性に着目した。ここで重要なのは、β細胞量を増加させることは糖尿病の治療や予防の一手段となりうるが、この様な作用を持つ薬物や成長因子などの開発、スクリーニングには、簡便なβ細胞量の評価法が必須であるということである。すなわち本発明は、膵β細胞で構成的に発現され血液中に分泌される人工レポーター遺伝子を作成し、トランスジーンとして種々のレシピエント細胞に導入して、膵β細胞を簡便に計測する方法を提供することを課題とする。 In the course of these studies, the present inventors have focused on the necessity and usefulness of a system that can easily measure changes in pancreatic β cell mass. What is important here is that increasing the amount of β-cells can be a means of treating or preventing diabetes, but it is easy to evaluate the amount of β-cells for the development and screening of such drugs and growth factors. The law is essential. That is, the present invention provides a method for easily measuring pancreatic β cells by creating an artificial reporter gene that is constitutively expressed in pancreatic β cells and secreted into the blood, and introduced into various recipient cells as a transgene. The issue is to provide.
本発明は、膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌されるレポータータンパク質（及びそれをコードする遺伝子）、および前記タンパク質を用い、その血中濃度等を測定することにより、生体内においてβ細胞量を定量的に推定するモデルを提供することを目的とする。 The present invention specifically and constitutively expresses in pancreatic β cells and is secreted without regulation, and a reporter protein (and a gene encoding the same), and its blood concentration is measured using the protein. It is an object of the present invention to provide a model for quantitatively estimating the amount of β cells in a living body.
すなわち本発明の第１の態様は、膵β細胞で特異的に且つ構成的に発現され、調節を受けずに分泌することを特徴とする人工タンパク質を提供することにある。 That is, the first aspect of the present invention is to provide an artificial protein characterized in that it is specifically and constitutively expressed in pancreatic β cells and secreted without being regulated.
本発明の第２の態様は、第１の発明に緒稀有人工タンパク質としてヒトアルブミン遺伝子のシグナル配列にヒトインスリン遺伝子のＣ−ペプチド又はルシフェラーゼを融合させた物質を提供するにある。 A second aspect of the present invention is to provide a substance in which a human insulin gene C-peptide or luciferase is fused to a human albumin gene signal sequence as a rare artificial protein according to the first invention.
本発明の第３の態様は、上記第１の態様、第２の態様における膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌される人工タンパク質をコードしたレポーター遺伝子を提供する。膵β細胞特異的に発現するためのプロモーター配列を有することを特徴とする、レポーター遺伝子を提供する。 According to a third aspect of the present invention, there is provided a reporter gene encoding an artificial protein that is specifically and constitutively expressed in the pancreatic β cells in the first and second aspects and is secreted without being regulated. provide. A reporter gene characterized by having a promoter sequence for specific expression of pancreatic β cells is provided.
本発明の第４の態様は、第３の態様において膵β細胞特異的に発言するためのプロモーター配列を有するレポーター遺伝子を提供する。 The fourth aspect of the present invention provides a reporter gene having a promoter sequence for speaking specifically in pancreatic β cells in the third aspect.
本発明の第５の態様は、前記プロモーター配列がインスリンＩ遺伝子のプロモーター配列であるレポーター遺伝子を提供する。 According to a fifth aspect of the present invention, there is provided a reporter gene wherein the promoter sequence is an insulin I gene promoter sequence.
本発明の第６の態様は、前記レポーター遺伝子により発現される人工タンパク質が、種々の生理条件下でも、発現量が変化しないことを特徴とするレポーター遺伝子を提供する。 The sixth aspect of the present invention provides a reporter gene, wherein the artificial protein expressed by the reporter gene does not change in expression level even under various physiological conditions.
本発明の第７の態様は、前記第３乃至第６の態様として示したレポーター遺伝子を組み込んだベクターを提供する。 The seventh aspect of the present invention provides a vector incorporating the reporter gene shown as the third to sixth aspects.
本発明の第８の態様は、前記第７の態様において、ベクターとしてプラスミドまたはウイルスを用いられることを特徴とするベクターを提供する。 According to an eighth aspect of the present invention, there is provided the vector according to the seventh aspect, wherein a plasmid or a virus is used as the vector.
更に、本発明の第９の態様は、第８の態様におけるウイルスとしてレトロウイルスをベクターとすることを特徴とする発明である。 Furthermore, the ninth aspect of the present invention is an invention characterized in that a retrovirus is used as the vector in the eighth aspect.
本発明の第１１の態様は、前記第７の態様乃至第９の態様のいずれかによるレポーター遺伝子をゲノム内に組み込んだモデル動物であって、前記レポーター遺伝子がコードする人工タンパク質を、膵β細胞で特異的にかつ構成的に発現することを可能としたモデル動物を提供する。 An eleventh aspect of the present invention is a model animal in which a reporter gene according to any one of the seventh to ninth aspects is incorporated into a genome, wherein an artificial protein encoded by the reporter gene is expressed in pancreatic β cells A model animal that can be expressed specifically and constitutively is provided.
本発明の第１０の態様は、前記第１の態様又は第２の態様、すなわち膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌されることを特徴とする、人工タンパク質を測定することを特徴とする膵β緒細胞量の計測である方法。 The tenth aspect of the present invention is the artificial aspect characterized in that it is specifically and constitutively expressed in the pancreatic β-cell and secreted without being regulated, according to the first aspect or the second aspect. A method for measuring the amount of pancreatic β cord cells, characterized by measuring a protein.
本発明の第１２の態様は、膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌されることを特徴とする、人工タンパク質の発現量または血中濃度、及び前記タンパク質をコードしたレポーター遺伝子の発現量を計測することを特徴とする、膵β細胞量の定量法を提供する。 In a twelfth aspect of the present invention, the expression level or blood concentration of an artificial protein, which is specifically and constitutively expressed in pancreatic β cells and secreted without being regulated, and the protein A method for quantifying the amount of pancreatic β-cells, comprising measuring the expression level of a reporter gene that encodes pancreatic β.
本発明の第１３の態様は、前記第１０の態様、又は第１２の態様における膵β細胞量の定量法を用いた、膵β細胞を増加させる効果を持つ物質のスクリーニング方法を提供する。 A thirteenth aspect of the present invention provides a screening method for a substance having an effect of increasing pancreatic β cells, using the method for quantifying the amount of pancreatic β cells in the tenth aspect or the twelfth aspect.
本発明を利用した非侵襲的膵β細胞量定量法は、糖尿病発症、進展の病態の解明や発症予知のみならず、膵β細胞の減少を阻止する様な薬剤療法や再生医療の開発に寄与するものと期待される。 Noninvasive pancreatic β-cell quantification method using the present invention contributes to the development of drug therapy and regenerative medicine that prevent the decrease of pancreatic β-cells as well as elucidation and prediction of the pathogenesis of diabetes onset and progression Expected to do.
以下に本発明の実施の形態を詳述する。本発明の第1の態様は、膵β細胞で特異的に且つ構成的に発現され、調節を受けずに分泌されることを特徴とする人工タンパク質（レポーター分子）を提供する。本発明者らは、膵β細胞量の非侵襲的な計測を目的とし、膵β細胞で合成されるレポーター分子を用い、この濃度を測定することで、生体内において膵β細胞量が推定できるシステムを開発した。本発明のレポーター分子に必要とされる条件としては、（１）レポーター分子の発現が膵β細胞特異的である。（２）発現が構成的（ｃｏｎｓｔｉｔｕｔｉｖｅ）で、種々の条件下でも発現調節を受けない。（３）分子が構成的に血中に分泌される。（４）分泌されたレポーター分子は比較的安定で、且つ生理活性を持たない。（５）レポーター分子の血中濃度測定が容易である。の５点が挙げられる。 Hereinafter, embodiments of the present invention will be described in detail. The first aspect of the present invention provides an artificial protein (reporter molecule) that is specifically and constitutively expressed in pancreatic β cells and secreted without being regulated. The present inventors can estimate the amount of pancreatic β cells in vivo by measuring this concentration using a reporter molecule synthesized in pancreatic β cells for the purpose of noninvasive measurement of the amount of pancreatic β cells. A system was developed. The conditions required for the reporter molecule of the present invention are as follows: (1) Expression of the reporter molecule is specific to pancreatic β cells. (2) Expression is constitutive and is not subject to expression regulation under various conditions. (3) The molecule is constitutively secreted into the blood. (4) The secreted reporter molecule is relatively stable and has no physiological activity. (5) The blood concentration of the reporter molecule can be easily measured. Of 5 points.
本発明の第２から第６の態様においては、膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌されることを特徴とする、人工タンパク質をコードしたレポーター遺伝子が提供される。ここでいうレポーター遺伝子は、膵β細胞特異的に発現するためのプロモーター配列を有し、より詳しくは発現が構成的で、種々の生理条件下でも発現量が変化しない遺伝子であって、更に詳しくは膵β細胞を計測しようとする対象生物に由来しない遺伝子で、そのコードする遺伝子産物が生理活性を持たないレポーター遺伝子である。前記レポーター遺伝子を更に具体的に例示すれば、インスリンＩ遺伝子のプロモーター配列を有し、そのコードするタンパク質がアルブミン遺伝子のシグナル配列にヒトインスリン遺伝子のＣ−ペプチドを融合させ、更にＭｙｃタグを付加した人工タンパク質、またはアルブミン遺伝子のシグナル配列にルシフェラーゼを融合させ、更にＭｙｃタグを付加した人工タンパク質であるレポーター遺伝子である。本発明におけるプロモーターと人工タンパク質の組み合わせは、上述のレポーター遺伝子に必要とされる条件を満たしていればその種類に特に限定は無く、またその組み合わせが本発明を限定するものでは無い。 In the second to sixth aspects of the present invention, there is provided a reporter gene encoding an artificial protein, characterized in that it is specifically and constitutively expressed in pancreatic β cells and secreted without being regulated. Is done. The reporter gene referred to here has a promoter sequence for specific expression of pancreatic β cells, more specifically a gene whose expression is constitutive and whose expression level does not change even under various physiological conditions. Is a gene not derived from the target organism for which pancreatic β cells are to be measured, and its encoded gene product is a reporter gene having no physiological activity. More specifically, the reporter gene has a promoter sequence of insulin I gene, a protein encoded by the C-peptide of human insulin gene is fused to the signal sequence of albumin gene, and a Myc tag is added. It is a reporter gene that is an artificial protein or an artificial protein in which a luciferase is fused to the signal sequence of an albumin gene and a Myc tag is further added. The combination of the promoter and artificial protein in the present invention is not particularly limited as long as it satisfies the conditions required for the above-mentioned reporter gene, and the combination does not limit the present invention.
本発明の第７から第９の態様においては、膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌されることを特徴とする、人工タンパク質をコードしたレポーター遺伝子を組み込んだベクターが提供される。本発明におけるベクターは、詳しくは請求項２から請求項６のうちいずれか１項に記載のレポーター遺伝子を組み込んだベクターであり、より詳しくはプラスミドベクターまたはウイルスベクターのいずれかである。 In the seventh to ninth aspects of the present invention, a reporter gene encoding an artificial protein, which is specifically and constitutively expressed in pancreatic β cells and secreted without being regulated, is incorporated A vector is provided. Specifically, the vector in the present invention is a vector into which the reporter gene according to any one of claims 2 to 6 is incorporated, and more specifically, either a plasmid vector or a viral vector.
本発明の第１０の態様においては、請求項７に記載のベクターを用い、請求項２から請求項６のいずれか１項に記載のレポーター遺伝子をゲノム内に組み込んだモデル動物であって、前記レポーター遺伝子がコードする人工タンパク質を、膵β細胞で特異的にかつ構成的に発現することを可能とした、モデル動物が提供される。このモデル動物の例としてはマウスが挙げられ、このマウスはゲノムＤＮＡ内に前記レポーター遺伝子を組み込んだものである。このマウスを他の糖尿病モデルマウスと交配させるなどにより、膵β細胞量の変化を追跡し糖尿病発症の病態解明に寄与することが期待される。 In a tenth aspect of the present invention, there is provided a model animal using the vector according to claim 7 and incorporating the reporter gene according to any one of claims 2 to 6 in the genome, Provided is a model animal that can specifically and constitutively express an artificial protein encoded by a reporter gene in pancreatic β cells. An example of this model animal is a mouse, which is a mouse in which the reporter gene is incorporated into genomic DNA. This mouse is expected to contribute to elucidation of the pathogenesis of diabetes by tracking changes in the amount of β-cells by mating with other diabetes model mice.
本発明の第１１、第１２の態様においては、膵β細胞で特異的にかつ構成的に発現され、調節を受けずに分泌される人工タンパク質（レポーター分子）の、発現量または血中濃度、及びレポーター遺伝子の発現量を計測することを特徴とする、膵β細胞量の定量法が提供される。実施例で述べるとおり、計測方法としては、例えば分泌されたタンパク質をＥＬＩＳＡ法等によって定量するなどの方法が考えられる。 In the eleventh and twelfth aspects of the present invention, the expression level or blood concentration of an artificial protein (reporter molecule) that is specifically and constitutively expressed in pancreatic β cells and secreted without regulation, And a method for quantifying the amount of pancreatic β cells characterized by measuring the expression level of a reporter gene. As described in the examples, as a measuring method, for example, a method of quantifying secreted protein by ELISA or the like can be considered.
本発明の第１３の態様においては、請求項１１または１２に記載の膵β細胞量の定量法を用いた、膵β細胞を増加させる効果を持つ物質のスクリーニング方法が提供される。前述の通り、１型糖尿病では膵β細胞が経時的に減少、消失するが、２型糖尿病においてもまたその発症及び進展には膵β細胞量の減少が関わることが明らかになってきており、これを抑えるかまたは膵β細胞を増加させる効果を持つ物質が利用可能となれば、１型糖尿病のみならず２型糖尿病の治療に大きく寄与できると期待される。その様な物質のスクリーニングのために、例えば候補物質の投与前後におけるレポーター遺伝子または遺伝子産物を定量する、本発明の膵β細胞量の定量法は利用可能である。
In a thirteenth aspect of the present invention, there is provided a screening method for a substance having an effect of increasing pancreatic β cells, using the pancreatic β cell amount quantification method according to claim 11 or 12. As described above, pancreatic β-cells decrease and disappear over time in
以下に本発明の実施例を述べるが、本実施例は本発明を限定するものでは無い。 Examples of the present invention will be described below, but the present examples are not intended to limit the present invention.
（レポーター遺伝子の作成と発現） ヒトアルブミン遺伝子のｃＤＮＡ全長を、ｈｕｍａｎ ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ細胞株ＨｅｐＧ２よりｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ（ＲＴ−ＰＣＲ）法にて得た。また、ヒトＣ−ペプチドについては、合成オリゴヌクレオチドによりｃＤＮＡ作成を行った。これらのｃＤＮＡを用い、表1のプライマーを用いたＰＣＲ法により、合成遺伝子Ａｌｂ−ｈｃを作成した（図1）。また、発現された人工タンパク質の検出をより容易にするために、Ｍｙｃタグを挿入し、それぞれを発現するためのベクターを、プラスミドｐｃＤＮＡ３を基に構築した。作成した全てのＤＮＡについては、シークエンシングを行いその塩基配列を確認した。
（細胞培養） ＮＨＩ３Ｔ３線維芽細胞、Ｃｏｓ７細胞、ＨＥＫ２９３細胞及びＰｌａｔＥ細胞（東京大学医科学研究所・北村俊雄博士より供与）を、１０％（ｖ／ｖ）ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍを含むＤｕｌｂｅｃｃｏ‘ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ ｍｅｄｉｕｍ（２５ｍＭ ｇｌｕｃｏｓｅ，５０ｍｇ／ｍｌ ｓｔｒｅｐｔｏｍｙｃｉｎ，５０ｕｎｉｔｓ／ｍｌ ｐｅｎｉｃｉｌｌｉｎ）を用いて３７℃，５％ＣＯ２下で培養した。マウスインスリノーマ細胞株であるＭＩＮ６は、１５％（ｖ／ｖ）ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍを含む前記Ｄｕｌｂｅｃｃｏ‘ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ ｍｅｄｉｕｍ（含５０μＭ β−メルカプトエタノール）を用い、３７℃，５％ＣＯ２下で培養した。前述のレポーター遺伝子をリポフェクション法（ＦｕＧｅｎｅ，Ｒｏｃｈｅ Ｄｉａｇｎｏｔｓｔｉｃｓ，ＣＨ）によりＣｏｓ７及びＨＥＫ２９３細胞、ＰｌａｔＥ細胞に導入し、人工タンパク質Ａｌｂ−ｈｃの発現を、抗Ｍｙｃ抗体を用いてＷｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ法により確認した。トランスフェクションのコントロールとして、ｐｃＤＮＡ３−ＧＦＰを使用した。実験は、トランスフェクションから４８時間後の細胞を用いて行った。 (Cell culture) NHI3T3 fibroblasts, Cos7 cells, HEK293 cells and PlatE cells (provided by Dr. Toshio Kitamura, Institute of Medical Science, The University of Tokyo) 10% (v / v) Dulbecco's modified Eagle containing fat bovine serum It was cultured at 37 ° C. and 5% CO 2 using 's medium (25 mM glucose, 50 mg / ml streptomycin, 50 units / ml penicillin). MIN6, a mouse insulinoma cell line, uses the above-mentioned Dulbecco's modified Eagle's medium (containing 50 μM β-mercaptoethanol) containing 15% (v / v) fetal bovine serum at 37 ° C. and 5% CO 2 . Cultured. The aforementioned reporter gene was introduced into Cos7, HEK293 cells and PlatE cells by lipofection method (FuGene, Roche Diagnostics, CH), and the expression of artificial protein Alb-hc was confirmed by Western blotting method using anti-Myc antibody. PcDNA3-GFP was used as a transfection control. The experiment was performed using cells 48 hours after transfection.
（Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ ａｓｓａｙ） 細胞培養液をＫｒｅｂｓ−Ｒｉｎｇｅｒ ｂｉｃａｒｂｏｎａｔｅ（ＫＲＢ）−ＨＥＰＥＳ ｂｕｆｅｒ（１２０ｍＭ ＮａＣｌ，４．７ｍＭ ＫＣｌ，１．２ｍＭ ＭｇＳＯ４，１．２ｍＭ ＫＨ２ＰＯ４，２０ｍＭ ＮａＨＣＯ３，２０ｍＭ ＨＥＰＥＳ，２ｍＭ ＣａＣｌ２，ｐＨ７．４）に置き換え４時間培養を行った後、培養上清を一部回収した。Ｐｈｏｓｐｈａｔｅ−ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ（ＰＢＳ）で細胞を１回洗浄し、Ｌｙｓｉｓ ｂｕｆｆｅｒ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ，０．５％ ＮＰ４０，２５０ｍＭ ＮａＣｌ，３ｍＭ ＥＧＴＡ，３ｍＭ ＥＤＴＡ，１．０％ ａｐｒｏｔｉｎｉｎ）を加えて細胞溶解液を作成した。細胞溶解液のタンパク質量をＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｒｅａｇｅｎｔ（ＢｉｏＲａｄ，ＵＳＡ）を用いて測定した。上清及び細胞溶解液にＳａｍｐｌｅ ｂｕｆｆｅｒを加え加熱処理した後、１２％アクリルアミドゲルでＳＤＳ−ＰＡＧＥを行い、泳動後のゲルからＨｙｂｏｎｄ ｍｅｍｂｒａｎｅ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ，ＵＳＡ）にタンパク質を転写した。メンブレンを５％ Ｓｋｉｍ ｍｉｌｋ ｉｎ ０．１％ ＴＢＳ−Ｔ（０．１％ Ｔｗｅｅｎ ２０，１０ｍＭ Ｔｒｉｓ−ＨＣｌ，１５０ｍＭ ＮａＣｌ）でブロッキングした。１次抗体として抗Ｍｙｃ抗体を、２次抗体として抗マウスＩｇ抗体を用い、ＥＣＬ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ，ＵＳＡ）により検出した。
(Western blotting assay) Cell culture The Krebs-Ringer bicarbonate (KRB) -HEPES bufer (120mM NaCl, 4.7mM KCl, 1.2mM MgSO 4, 1.2mM KH 2 PO 4,
（レトロウィルスによる遺伝子発現） 融合遺伝子Ａｌｂ−ｈｃを、レトロウィルスベクターであるｐＭＸｓ（東京大学医科学研究所・北村俊雄博士より供与）へ組み込んだ。これをパッケージング細胞ＰｌａｔＥにリポフェクション法（ＦｕＧｅｎｅ）を用いて導入し、４８時間後、ウィルスを含む細胞上清を回収した。７０％ｃｏｎｆｌｕｅｎｔとなったＮＩＨ ３Ｔ３ ｆｉｂｒｏｂｌａｓｔをレトロウィルス液で１２時間培養した。感染４８時間後よりｐｕｒｏｍｙｃｉｎ ｓｅｌｅｃｔｉｏｎ（２μｇ／ｍｌ）を行い、安定発現細胞株を作製した。ＭＩＮ６細胞に対しても、同様の方法でレトロウィルスを用いた。但し、遺伝子の導入効率を上げるために初回感染から４８時間後に２回目の感染を行った。２回目の感染から４８時間後にｐｕｒｏｍｙｃｉｎ ｓｅｌｅｃｔｉｏｎ（１μｇ／ｍｌ）を行い、安定発現細胞株を作製した。 (Gene expression by retrovirus) The fusion gene Alb-hc was incorporated into a retrovirus vector pMXs (provided by Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo). This was introduced into the packaging cell Plat using the lipofection method (FuGene), and after 48 hours, the cell supernatant containing the virus was recovered. NIH 3T3 fibroblast which became 70% confluent was cultured in a retrovirus solution for 12 hours. Puromycin selection (2 μg / ml) was performed from 48 hours after infection to prepare a stable expression cell line. Retroviruses were used in the same manner for MIN6 cells. However, in order to increase the efficiency of gene transfer, the second infection was performed 48 hours after the first infection. Puromycin selection (1 μg / ml) was performed 48 hours after the second infection to produce a stable expression cell line.
（ヒトＣ−ペプチドＥＬＩＳＡキットによるレポーター分子の測定） Ｃｏｓ７、２９３細胞及びＮＩＨ ３Ｔ３ ｆｉｂｒｏｂｌａｓｔに融合遺伝子Ａｌｂ−ｈｃをトランスフェクションし、４８時間後に実験を行った。Ｍｅｄｉｕｍを吸引、Ｋｒｅｂｓ−Ｒｉｎｇｅｒ ｂｉｃａｒｂｏｎａｔｅ （ＫＲＢ−ＨＥＰＥＳ） ｂｕｆｆｅｒ（１２０ｍＭ ＮａＣｌ，４．７ｍＭ ＫＣｌ，１．２ｍＭ ＭｇＳＯ４，１．２ｍＭ ＫＨ２ＰＯ４，２０ｍＭ ＮａＨＣＯ３，２０ｍＭ ＨＥＰＥＳ，２ｍＭ ＣａＣｌ２，ｐＨ７．４）で細胞を２回洗浄、ｌｙｓｉｓ ｂｕｆｆｅｒ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ，０．１％ ＮＰ４０，２５０ｍＭ ＮａＣｌ，３ｍＭ ＥＧＴＡ，３ｍＭ ＥＤＴＡ，１．０％ ａｐｒｏｔｉｎｉｎ）を加え細胞溶解液を作製した。培養上清及び細胞溶解液を用いてＥＬＩＳＡ法（ヒトＣ−ペプチドＥＬＩＳＡキット，ＬＩＮＣＯ，ＵＳＡ）にてＣ−ペプチド濃度を測定した。融合タンパク質を発現した安定細胞株３Ｔ３ ｆｉｂｒｏｂｌａｓｔにおいては、６ｃｍ ｄｉｓｈに蒔き、８０％ ｃｏｎｆｌｕｅｎｔとなった状態で上記と同様に行った。 (Measurement of Reporter Molecules by Human C-Peptide ELISA Kit) Cos7, 293 cells and NIH 3T3 fibroblast were transfected with the fusion gene Alb-hc, and an experiment was performed 48 hours later. Medium is aspirated, Krebs-Ringer bicarbonate (KRB-HEPES) buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 20 mM NaHCO 3 , 20 mM HEPES, 2 mM CaCl 2 , pH 7 The cells were washed twice in 4), and lysis buffer (20 mM Tris-HCl, 0.1% NP40, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA, 1.0% aprotinin) was added to prepare a cell lysate. The C-peptide concentration was measured by ELISA (human C-peptide ELISA kit, LINCO, USA) using the culture supernatant and cell lysate. In the stable cell line 3T3 fibroblast that expressed the fusion protein, the cells were plated in a 6 cm dish and 80% confluent.
（Ｃｏｓ７，ＨＥＫ２９３細胞での融合タンパク質の発現） まず、３つの異なる位置にＭｙｃ−ｔａｇを付加した融合タンパク質Ａｌｂ−ｈｃを発現させるためのベクターを、ｐｃＤＮＡ３を用いて作製した（図２）。これら発現ベクターをＣｏｓ７細胞に導入し、Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ法にて融合タンパク質の発現を確認した。３０−３３ｋＤａにバンドが確認できたが、Ｃ−ペプチド部分の両端にｍｙｃ−ｔａｇを付加したＡｌｂ−ｈｃ（Ｃ：ｍｙｃ−ｃｐｅｐ−ｍｙｃ，Ｃ：ｍｃｍ）の検出が良好であり、以後これを実験に用いた（図３）。 (Expression of fusion protein in Cos7, HEK293 cells) First, a vector for expressing the fusion protein Alb-hc with Myc-tag added at three different positions was prepared using pcDNA3 (Fig. 2). These expression vectors were introduced into Cos7 cells, and the expression of the fusion protein was confirmed by Western blotting. Although a band was confirmed at 30-33 kDa, detection of Alb-hc (C: myc-cep-myc, C: mcm) with myc-tag added to both ends of the C-peptide portion was good, Used for the experiment (FIG. 3).
（Ｃｏｓ７細胞での融合タンパク質の分泌） 融合タンパク質Ａｌｂ−ｈｃを発現した細胞からＡｌｂ−ｈｃが分泌されているかどうか検討するため、ＫＲＢ−ＨＥＰＥＳ ｂｕｆｆｅｒで４時間培養し、上清の一部を用いてＷｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ法を行った（図４）。上清中にも、細胞溶解液中において検出されたバンドと同じサイズのバンドが検出された。Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ法の結果は、融合タンパク質Ａｌｂ−ｈｃが細胞から分泌されていることを示している。 (Secretion of fusion protein in Cos7 cells) In order to examine whether Alb-hc is secreted from cells expressing the fusion protein Alb-hc, the cells were cultured for 4 hours in KRB-HEPES buffer, and a part of the supernatant was used. The Western blotting method was performed (FIG. 4). A band having the same size as that detected in the cell lysate was also detected in the supernatant. The result of Western blotting shows that the fusion protein Alb-hc is secreted from the cells.
Ａｌｂ−ｈｃが既存の方法で測定可能かどうかを検討するために、ヒトＣ−ペプチド特異的抗体を用いたＥＬＩＳＡ法による検出を行った。細胞上清を用いて融合タンパク質の測定をヒトＣ−ペプチドＥＬＩＳＡアッセイキットにより試みた結果、培養液中のＣ−ペプチドは検出・測定可能であった。Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ法での結果と同様、Ａｌｂ−ｈｃ（Ｃ：ｍｃｍ）でより高い値を示した（図５）。更にＣｏｓ７細胞以外の細胞でも同様に発現分泌されるかどうかを確認するため、ＨＥＫ２９３細胞にＡｌｂ−ｈｃ：ｍｃｍを発現させ同様の実験を行った。ＨＥＫ２９３細胞においても培養上清中にＡｌｂ−ｈｃが分泌され、Ｃｏｓ７細胞と同等の濃度のヒトＣ−ペプチドが確認された（図６）。 In order to examine whether Alb-hc can be measured by an existing method, detection by ELISA using a human C-peptide specific antibody was performed. As a result of an attempt to measure the fusion protein using the cell supernatant using a human C-peptide ELISA assay kit, it was possible to detect and measure the C-peptide in the culture solution. Similar to the result of Western blotting, Alb-hc (C: mcm) showed a higher value (FIG. 5). Furthermore, in order to confirm whether or not the expression and secretion were similarly performed in cells other than Cos7 cells, Alb-hc: mcm was expressed in HEK293 cells, and the same experiment was performed. Also in HEK293 cells, Alb-hc was secreted into the culture supernatant, and human C-peptide having a concentration equivalent to that of Cos7 cells was confirmed (FIG. 6).
本発明のレポーター遺伝子、人工タンパク質及びベクターを利用し、トランスジェニックマウスを作製することにより、ヒトＣ−ペプチドをマウスで発現させ、これによってマウス膵β細胞量を推定する事が可能になると考えられる。またこうして得られたマウスを他の糖尿病モデルマウスと交配させる事により、膵β細胞量変化を追うことで糖尿病発症の病態解明に寄与すると考えられる。また本発明は、膵β細胞の減少を防止し、その再生や増殖を促進する糖尿病治療薬のスクリーニングにも応用可能である。 By producing a transgenic mouse using the reporter gene, artificial protein and vector of the present invention, it is considered that human C-peptide can be expressed in the mouse and thereby the amount of mouse pancreatic β cells can be estimated. . In addition, by mating the mouse thus obtained with other diabetic model mice, it is considered to contribute to the elucidation of the pathogenesis of diabetes by tracking changes in the amount of pancreatic β cells. The present invention can also be applied to screening for antidiabetic drugs that prevent the decrease of pancreatic β cells and promote their regeneration and proliferation.
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