JPH0272866A - Human vascular endothelical cell-derived transforming cell - Google Patents
Human vascular endothelical cell-derived transforming cellInfo
- Publication number
- JPH0272866A JPH0272866A JP63224215A JP22421588A JPH0272866A JP H0272866 A JPH0272866 A JP H0272866A JP 63224215 A JP63224215 A JP 63224215A JP 22421588 A JP22421588 A JP 22421588A JP H0272866 A JPH0272866 A JP H0272866A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- vascular endothelial
- endothelial cells
- derived
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、永久増殖可能なヒト血管内皮細胞由来形質転
換細胞、その作成方法及びその用途に関するものである
。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to human vascular endothelial cell-derived transformed cells that can proliferate permanently, a method for producing the same, and uses thereof.
更に詳細には、本発明は、ヒト正常血管内皮細胞の特徴
を有し、血栓溶解剤組織由来プラスミノ−ゲン活性化因
子（以下しＰＡと略す）の工業的製造、血管収縮因子・
血管弛緩因子の試験、あるいは血管内皮細胞産生生理活
性物質の大量生産業上の利用分野にとってきわめて有効
なヒト血管内皮細胞由来形質転換細胞、その作成方法及
びその用途に関するものである。More specifically, the present invention relates to the industrial production of thrombolytic tissue-derived plasminogen activator (hereinafter abbreviated as PA), which has the characteristics of human normal vascular endothelial cells, and the vasoconstrictor.
The present invention relates to human vascular endothelial cell-derived transformed cells that are extremely effective for testing vascular relaxing factors or for mass production of physiologically active substances produced by vascular endothelial cells, methods for producing the same, and uses thereof.
（従来の技術）
虚血性心疾患や脳血管障害をはじめとする血管系の病気
が、とくに先進諸国で増加の一途をたどっている。これ
らの虚血性疾患は動脈硬化性病変に基づいており、長寿
社会をむがえて、血管病変の克服はますます重要な課題
となってきている。(Prior Art) Vascular diseases such as ischemic heart disease and cerebrovascular disorders are on the rise, especially in developed countries. These ischemic diseases are based on arteriosclerotic lesions, and as we live longer in our society, overcoming vascular lesions has become an increasingly important issue.
血管には、内皮細胞、中膜平滑筋細胞及び線維芽綱胞な
どが存在するが、血液と直に接触しているのは血管内皮
細胞だけである。この血管内皮細胞は様々な特異機能を
有しており、それによって様々な血液成分及び血圧等の
変化に対応していると考えられている。血管内皮細胞は
、血液凝固・線溶系、血圧、物質透過、血管新生、脂質
代謝及び癌転移に深く関与しており、例えばｔ、ＰＡを
はじめとして、プロスタサイクリン、アンチトロンビン
■、α、−マクログロブリン、ヘパラン硫酸、プロティ
ンＳ、スロンボモジュリン、ブラスミノーゲンアクチベ
ーターインヒビター、フォンビルブランドファクター、
ティシュ−ファクター、血小板活性因子（ＰＡＦ）及び
血管弛緩物質（ＥＤＲＦ）などの生理活性物質を産生ず
ることが知られている。Blood vessels contain endothelial cells, medial smooth muscle cells, fibroblasts, etc., but only vascular endothelial cells are in direct contact with blood. These vascular endothelial cells have various specific functions, and are thought to be able to respond to changes in various blood components, blood pressure, and the like. Vascular endothelial cells are deeply involved in the blood coagulation/fibrinolytic system, blood pressure, substance permeation, angiogenesis, lipid metabolism, and cancer metastasis. Globulin, heparan sulfate, protein S, thrombomodulin, blasminogen activator inhibitor, von Willebrand factor,
It is known to produce physiologically active substances such as tissue factor, platelet activating factor (PAF) and vasorelaxant (EDRF).
近年、細胞培養技術の進歩により、ヒト血管内皮細胞の
培養方法が確立され、上記分野において広く基礎研究に
用いられている。しかしながら、ヒト正常血管内皮細胞
は増殖に生長因子を必要とし、２０回程度***するとフ
ライシスをむがえ死んでしまうため、培養の比較的困難
な細胞である。In recent years, with advances in cell culture technology, methods for culturing human vascular endothelial cells have been established and are widely used in basic research in the above fields. However, human normal vascular endothelial cells require growth factors for proliferation, and after dividing approximately 20 times, they undergo phytosis and die, so they are relatively difficult cells to culture.
従来より、該内皮細胞の増殖機能を獲得するために、Ｓ
Ｖ４０により、形質転換を起こす試みがなされているが
、形質転換された細胞は、増殖機能を獲得するものの、
元来その細胞が保有する機能が、失われるのが常である
［実験医学、第４巻、７号、５７９頁−５８５頁（１９
８６）参照］。従って該内皮細胞の工業的な応用や生理
活性物質の大量生産への応用が難しかった。Conventionally, in order to acquire the proliferative function of endothelial cells, S
Attempts have been made to induce transformation using V40, but although the transformed cells acquire a proliferation function,
The functions originally possessed by the cell are usually lost [Experimental Medicine, Vol. 4, No. 7, pp. 579-585 (19
86)]. Therefore, it has been difficult to apply the endothelial cells industrially or to the mass production of physiologically active substances.
（発明が解決しようとする課題）
前記のヒト血管内皮細胞を工業的に応用、あるいは生理
活性物質の大量生産へ応用する為には、ヒト正常血管内
皮細胞の性質を残したまま永久増殖性を付与し、即ちセ
ルライン化し、がっ、培養を安価で容易にする為に血管
内皮細胞生長因子の要求性を無くする技術を開発しなけ
ればならない。(Problems to be Solved by the Invention) In order to apply the above-mentioned human vascular endothelial cells industrially or to the mass production of physiologically active substances, it is necessary to maintain permanent proliferation while retaining the properties of normal human vascular endothelial cells. In order to provide the vascular endothelial cell growth factor, that is, to make it into a cell line and to make it inexpensive and easy to culture, it is necessary to develop a technology that eliminates the requirement for vascular endothelial cell growth factors.
（課題を解決する為の手段）
本発明者らは、このような問題点を解決すべく鋭意研究
を重ねた結果、ヒト血管より分離した正常な内皮細胞を
ＳＶ４０ ＤＮＡでトランスホームさせた形質転換細
胞が、ヒト正常血管内皮細胞の特徴を残したまま内皮細
胞生長因子の要求性が無くなり、さらに、永久増殖性を
獲得したことを見い出し、本発明を完成した。すなわち
、本発明は、ヒト正常血管内皮細胞をＳＶ４０ ＤＮ
Ａでトランスホームした形質転換細胞、その作成方法及
びその用途を要旨とするものである。(Means for Solving the Problems) As a result of extensive research to solve these problems, the present inventors have developed a transformation method in which normal endothelial cells isolated from human blood vessels were transformed with SV40 DNA. The present invention was completed based on the discovery that the cells retain the characteristics of normal human vascular endothelial cells, lose the requirement for endothelial cell growth factors, and furthermore acquire permanent proliferation ability. That is, the present invention uses human normal vascular endothelial cells as SV40 DN.
This article summarizes the transformed cells transformed with A, the method for producing them, and their uses.
次に、本発明の細胞株の細胞学的性質を示す。Next, the cytological properties of the cell line of the present invention will be shown.
■）細胞の形態：敷石状（ヒト正常血管内皮細胞とほぼ
同様の形状をしている。）
２）細胞の由来、ヒトより分離した血管内皮細胞
３）継代培養：永久増殖可能
４）生長因子要求性：血管内皮細胞生長因子やヘパリン
を含まない培地で増殖可能である。■) Cell morphology: cobblestone-like (almost the same shape as normal human vascular endothelial cells) 2) Origin of cells, vascular endothelial cells isolated from humans 3) Subculture: Permanent proliferation possible 4) Growth Factor requirement: Can grow in a medium that does not contain vascular endothelial cell growth factor or heparin.
５）機能：ヒト正常血管内皮細胞とほぼ同様の組織由来
プラスミノーゲン活性化因子産生能を示す。5) Function: Shows ability to produce tissue-derived plasminogen activator almost similar to normal human vascular endothelial cells.
６）血液凝固箱■因子産土能：血管内皮細胞の特徴であ
る血液凝固第ＶＩＩＩ因子を産生する。6) Blood coagulation box ■ Factor-producing ability: Produces blood coagulation factor VIII, which is a characteristic of vascular endothelial cells.
７）ＳＶ４０ ＤＮＡの染色体における存在：ＳＶ４
０ ＤＮＡが細胞の染色体に挿入されている。7) Presence of SV40 DNA in chromosome: SV4
0 DNA has been inserted into the chromosome of the cell.
８）ＳＶ４０ ＤＮＡウィルスの遊離・本細胞株から
のＳＶ４０ ＤＮＡウィルスの遊離は認められていな
い。8) Release of SV40 DNA virus - Release of SV40 DNA virus from this cell line was not observed.
９）コロニーの形式：ペトリ皿上ではコロニを形成する
が、軟寒天中では形成しない。9) Colony format: Colonies are formed on Petri dishes, but not in soft agar.
１０）凍結保存・−７０゛Ｃないし一１９６℃できわめ
て長期間保存可能である。10) Cryopreservation - Can be stored for an extremely long period at -70°C to -196°C.
本細胞株は全く未知のものであり、世界に類例を見ない
ものである。This cell line is completely unknown and unprecedented in the world.
本発明の細胞株を得るには、例えば、次のごとき方法を
採用すればよい。To obtain the cell line of the present invention, for example, the following method may be employed.
すなわち、まず、ヒト血管内皮細胞を取得するには、例
えば、ジャーナル・オブ・クリニカル・インヴエスティ
ゲーション（Ｊ、Ｃ１１ｎ、Ｉｎｖｅｓｔ、）第５２巻
、２７４５頁−２７５６頁（１９７３年）に記載の方法
に従って行なえばよい。その概要は、血管（ヒト血管で
あればいずれの組織でもよいが、例えばサイ帯静脈は入
手が容易で好適である。）を出来るだけ無菌的に採取し
、洗浄後、トリプシン処理により細胞を結合組織より分
離して集めることによって得られる。得られたヒト正常
血管内皮細胞は、２０％牛脂児血清、ヘパリン及び内皮
細胞生長因子を含む通常の細胞培養用培地で培養するこ
とができる。That is, first, to obtain human vascular endothelial cells, for example, the method described in Journal of Clinical Investigation (J, C11n, Invest,) Vol. 52, pp. 2745-2756 (1973) is used. Just follow the instructions. The outline is to collect a blood vessel (any human blood vessel tissue may be used, but for example rhinoceros vein is suitable as it is easy to obtain) as sterilely as possible, wash it, and bind cells by trypsin treatment. Obtained by separating it from tissue and collecting it. The obtained normal human vascular endothelial cells can be cultured in a normal cell culture medium containing 20% tallow serum, heparin, and endothelial cell growth factor.
該細胞培養用培地としては、例えば、ＢＭＥ培地、ＭＥ
Ｍ培地（アール、アルファ、ダルベツコ、１１ｉｇｈ−
ＧＥＭ）、ハム培地（Ｆ−１０，Ｆ−１２）、イスコツ
培地、１１９培地、Ｌ−１５培地、マツコイ５Ａ培地、
ＮＣＴＣ１３５培地、ウィリアムスＥ培地、ウェイマウ
ス培地等の該細胞の培養可能な培地であれば、いずれの
培地でもよいが、特に１１９培地が好ましい。Examples of the cell culture medium include BME medium, ME
M medium (R, Alpha, Dulbecco, 11igh-
GEM), Ham's medium (F-10, F-12), Iskot medium, 119 medium, L-15 medium, Matsukoi 5A medium,
Any medium may be used as long as the cells can be cultured, such as NCTC135 medium, Williams E medium, Weymouth medium, etc., but 119 medium is particularly preferred.
該細胞を培養するに当り、上記培地に数％、好ましくは
５％程度の炭酸ガスを含有せしめることを必要とする。In culturing the cells, the medium needs to contain several percent, preferably about 5%, of carbon dioxide gas.
かかる培地のｐＨは、６ないし８であり。The pH of such a medium is between 6 and 8.
特に中性付近が好都合である。培養は、３０〜４０℃で
可能であり、特に、３７℃付近が好ましい。培養時間は
使用する培地、ｐＨ５温度条件等により一概にはいえな
いが、通常、４〜５日の培養により、該細胞は継代する
ことができる。In particular, near neutrality is convenient. Cultivation is possible at 30 to 40°C, particularly preferably around 37°C. Although the culture time cannot be determined unconditionally depending on the medium used, pH5 temperature conditions, etc., the cells can usually be subcultured after 4 to 5 days of culture.
一方、形質転換能を有するＳＶ４０ ＤＮＡを用意す
る。該ＳＶ４０ ＤＮＡとして、さらに好ましくは、
ＳＶ４０の複製原点が欠失してウィルスが増殖できない
変異株ＤＮＡが使用できる。この変異株ＤＮＡは、アメ
リカ・コールドスプリングハーバ−研究所のボルツマン
博士の報告した方法〔コールド・スプリング・ハーバ−
・シンボジア・オン・クオンティタティブ・バイオロジ
イー（ＣｏｌｄＳｐｒｉｎｇｌｌａｒｂｏｒ Ｓｙｍｐ
ｏｓｉａ ｏｎ ＱｕａｎｔｉｔａｔｉｖｅＢｉｏｌｏ
ｇｙ） 、第４４巻、２９３頁−３００頁（１９８０年
）参照〕で調製される。このＳＶ４０ ＤＮＡは通常
の遺伝子工学的方法で、大量に生産、抽出及び精製して
調製できる。On the other hand, SV40 DNA having transformation ability is prepared. More preferably, the SV40 DNA is
A mutant strain DNA in which the replication origin of SV40 is deleted and the virus cannot proliferate can be used. This mutant strain DNA was obtained using the method reported by Dr. Boltzmann of the Cold Spring Harbor Laboratory in the United States [Cold Spring Harbor Laboratory].
・Symbodia on Quantitative Biology (ColdSpringllarbor Symp
osia on Quantitative Biolo
gy), Vol. 44, pp. 293-300 (1980)]. This SV40 DNA can be prepared by producing, extracting, and purifying it in large quantities using conventional genetic engineering methods.
次に、培養しておいたヒト正常血管内皮細胞に対して、
上記トランスホーミング能を有するＤＮＡを導入する。Next, on cultured human normal vascular endothelial cells,
The DNA having the above-mentioned transforming ability is introduced.
ＤＮＡの該細胞への導入は、通常用いられている方法、
例えばリン酸カルシウム法、レーザー光線法、電気穿孔
法、マイクロインジェクション法、トランスフェクショ
ン法、リポソーム法、プロトプラスト融合法、赤血球ゴ
ースト法又はウィルスを利用した方法等で行なわれるが
、好ましくは、ＤＥＡＥ−デキストラン−ＤＭＳＯショ
ック法〔コールド・スプリング・ハーバ−・シンボジア
・オン・クオンティタティブ・バイオロジイー（Ｃｏｌ
ｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｓｙｍｐｏｓｉａｏ
ｎ Ｑｕａｎｔｉｔａｔｉｖｅ Ｂｉｏｌｏｇｙ） 、
第４４巻、２９３頁−３００頁（１９８０年）参照〕が
用いられる。このようにして、外来ＤＮＡが導入された
細胞は、数回継代された後に、クローニングされる。継
代する培地は、通常用いられる培地でよいが、好ましく
は、１回目は血管内皮細胞生長因子とヘパリンを含む培
地で、２回目以降はそれらを含まない培地が望ましい。The DNA can be introduced into the cells by a commonly used method,
For example, it is carried out by a calcium phosphate method, a laser beam method, an electroporation method, a microinjection method, a transfection method, a liposome method, a protoplast fusion method, a red blood cell ghost method, a method using a virus, etc., but preferably a DEAE-dextran-DMSO shock. [Cold Spring Harbor Symbosia on Quantitative Biology (Col.
d Spring Harbor Symposiao
n Quantitative Biology),
44, pp. 293-300 (1980)] is used. In this way, cells into which foreign DNA has been introduced are passaged several times and then cloned. The medium for passage may be a commonly used medium, but preferably a medium containing vascular endothelial cell growth factor and heparin for the first passage, and a medium that does not contain them for the second and subsequent passages.
クローニングは、通常の希釈法その他で行なわれ、その
後に細胞の性質を調べることによって目的とする細胞株
を得ることができる。Cloning is carried out using conventional dilution methods and the like, and the desired cell line can then be obtained by examining the properties of the cells.
形質転換されたヒト血管内皮細胞は、前記のヒト血管内
皮細胞の培養条件に準じて培養することができる。培養
により得られる培養液からは、ｔＰＡを初めとする前記
の生理活性物質を分離、精製することができる。その代
表例としてｔＰＡの精製方法を示すと、その概要は、例
えば、ジャーナル・オブ・バイオケミストリー（Ｊ、Ｂ
ｉｏｌ、Ｃｈｅｍ、 、 ）第２５６巻、７０３５頁〜
７０４１頁（１９８１年）に記載の方法に準じて、培養
液をＺｎ−キレートクロマトグラフィー、ｃｏｎＡクロ
マトグラフィー、ゲル濾過等を用いて行うことができる
。Transformed human vascular endothelial cells can be cultured according to the culture conditions for human vascular endothelial cells described above. The above-mentioned physiologically active substances including tPA can be separated and purified from the culture solution obtained by culturing. As a typical example, the method for purifying tPA is summarized in, for example, the Journal of Biochemistry (J, B.
iol, Chem, ) Volume 256, Page 7035~
The culture solution can be subjected to Zn-chelate chromatography, conA chromatography, gel filtration, etc. according to the method described on page 7041 (1981).
本細胞株は、少なくとも１００代以上継代できる永久増
殖性を獲得しており、内皮細胞生長因子の要求性も無く
なっている。一方、本細胞株は、後記の実施例２で見ら
れるように、ヒト正常血管内皮細胞に見られる特徴を有
している。This cell line has acquired the ability to proliferate permanently and can be passed for at least 100 generations, and has no requirement for endothelial cell growth factors. On the other hand, this cell line has characteristics found in normal human vascular endothelial cells, as seen in Example 2 below.
従って、該形質転換細胞を用いることにより、例えば、
ガン細胞を用いることなく、血栓溶解剤↑、ＰＡを工業
的に生産したり、あるいは血管内皮細胞がつくる各種生
理活性物質を大量産生させるなどの応用が可能となる。Therefore, by using the transformed cells, for example,
Applications such as the industrial production of thrombolytic agents ↑ and PA without the use of cancer cells, and the mass production of various physiologically active substances produced by vascular endothelial cells will become possible.
（実施例） 次に、本発明を実施例により具体的に説明する。(Example) Next, the present invention will be specifically explained using examples.
実施例１
ヒトサイ帯静脈をＭＥＭ培地で洗浄後、０．１％トリプ
シンで静脈を満たし、３７℃で１５分間保温する。その
後、細胞懸濁液をとり出して、１１０００ｒｐ、３分間
の遠心により洗浄した後、２０％牛脂児血清、血管内皮
細胞生長因子及びヘパリンを含有する１９９培地で３７
℃で培養する。二の内皮細胞にＤＥＡＥ−デキストラン
−ＤＭＳＯショック法で、アメリカ・コールドスプリン
グハーバ−研究所のボルツマン博士の確立したＳＶ４０
の複製原点を欠損し、ウィルスが増殖できない変異株Ｓ
Ｖ４０ ＤＮＡのリニアＤＮＡを、細胞に導入した。Example 1 After washing a human rhinoceros vein with MEM medium, the vein is filled with 0.1% trypsin and kept at 37° C. for 15 minutes. Thereafter, the cell suspension was taken out, washed by centrifugation at 11,000 rp for 3 minutes, and then incubated with 199 medium containing 20% beef tallow serum, vascular endothelial cell growth factor, and heparin for 3 hours.
Incubate at °C. SV40, which was established by Dr. Boltzmann of the Cold Spring Harbor Laboratory in the United States, was applied to two endothelial cells using the DEAE-dextran-DMSO shock method.
Mutant strain S that lacks the replication origin and is unable to reproduce the virus.
Linear DNA of V40 DNA was introduced into the cells.
以下に、その詳細な方法を示す。無血清培地１９９培地
で培養したヒト血管内皮細胞に上記のＳＶ４０ ＤＮ
Ａｌ０μｇを、ＤＥＡＥ−デキストランとともに添加す
る。The detailed method is shown below. The above SV40 DN was added to human vascular endothelial cells cultured in serum-free medium 199 medium.
Add 0 μg of Al along with DEAE-dextran.
６時間後に、培養上清を除去し、１０％ＤＭＳＯで１分
間インキュベートして、細胞を１９９培地で洗浄する。After 6 hours, culture supernatants are removed, cells are washed with 199 medium by incubation with 10% DMSO for 1 minute.
２０％牛脂児血清、内皮細胞生長因子及びヘパリンを含
有する１９９培地で３７℃で培養後、培地を生長因子を
含まないものに変えてさらに６回継代する。その後、希
釈法によってクローニングを行ない、目的のヒト血管内
皮細胞由来形質転換細胞Ｓ Ｖ −１−＃２を得た。な
お、本細胞は、永久増殖性を獲得し、内皮細胞生長因子
要求性が無くなっていた１本細胞株の顕微鏡写真を第１
図に示す。After culturing at 37° C. in 199 medium containing 20% tallow serum, endothelial cell growth factor, and heparin, the medium is changed to one that does not contain growth factors, and the cells are passaged six more times. Thereafter, cloning was performed by a dilution method to obtain the target human vascular endothelial cell-derived transformed cell SV-1-#2. This cell line has acquired permanent proliferation and no longer requires endothelial growth factors.
As shown in the figure.
この方法に従って得られた、ヒト血管内皮細胞由来形質
転換細胞の１株を、Ｓ Ｖ−１−＃２と命名し。One strain of human vascular endothelial cell-derived transformed cells obtained according to this method was named SV-1-#2.
昭和６３年８月３日に、工業技術院微生物工業技術研究
所に寄託の手続を行い、ｒ微工研菌寄第１０１７４号（
ＦＥＲＭ Ｐ−１０１７４）Ｊとして受は入れられた。On August 3, 1988, the deposit procedure was carried out at the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the
The acceptance was accepted as FERM P-10174)J.
この５Ｖ−１−＃２は、７０℃ないし一１９６℃でほぼ
永久的に保存可能であって、たえず頒布可能な状態にお
かれている。This 5V-1-#2 can be stored almost permanently at 70°C to -196°C and remains ready for distribution.
この形質転換細胞株５Ｖ−１−３２は、通常用いられる
細胞培養用培地で増殖可能である。例えば、牛胎児血清
を１０％含有する１９９培地を培地として用い、３７℃
、炭酸ガス濃度５％含有空気下でよく増殖する。This transformed cell line 5V-1-32 can be grown in a commonly used cell culture medium. For example, using 199 medium containing 10% fetal bovine serum as a medium,
, grows well in air containing 5% carbon dioxide gas concentration.
実施例２
実施例１のヒト血管内皮細胞由来の形質転換細胞につい
て、血液凝固第■因子の産生を検討した。Example 2 The production of blood coagulation factor (I) was investigated using the transformed cells derived from the human vascular endothelial cells of Example 1.
血液凝固第■因子は、血管内皮細胞が特異的に産生ずる
もので、血管内皮細胞の同定に使われる指標である。形
質転換細胞５Ｖ−１−＃２を固定後、蛍光色素を結合し
た抗第■因子モノクローナル抗体で、蛍光染色したとこ
ろ、細胞表面に黄色の蛍光が観察され（第２図参照）、
本細胞株が第■因子を産生していることが確認された。Blood coagulation factor (III) is specifically produced by vascular endothelial cells and is an indicator used to identify vascular endothelial cells. After fixing the transformed cells 5V-1-#2, they were fluorescently stained with an anti-factor II monoclonal antibody conjugated with a fluorescent dye, and yellow fluorescence was observed on the cell surface (see Figure 2).
It was confirmed that this cell line produced factor Ⅰ.
実施例３ 形質転換細胞５Ｖ−１−＃２のｔＰＡ産生能を調べた。Example 3 The tPA production ability of the transformed cell 5V-1-#2 was examined.
培地中のし°ＰＡ量の濶定は、１２０μＱのサンプル−
６０μＱのプラスミノーゲン（２ｖ／ｍｕ）−４２０μ
Ｑのフィブリンクロット液を酵素反応液として、濁度の
低下を指標とするフィブリン懸濁法で行なった。第３図
に示すように、５Ｖ−１−６２株は、ヒト正常血管内皮
細胞と同じ程度のｔＰＡを産生じていることが確認され
た。To determine the amount of PA in the medium, 120 μQ of sample
60μQ plasminogen (2v/mu) - 420μ
The fibrin suspension method was carried out using the fibrin clot solution of Q as the enzyme reaction solution and using the decrease in turbidity as an indicator. As shown in FIG. 3, it was confirmed that the 5V-1-62 strain produced tPA to the same extent as human normal vascular endothelial cells.
実施例４
形質転換細胞Ｓ Ｖ−１−＃２における５Ｖ４０ＤＮＡ
の存在様式を検討した。Ｓ Ｖ−１−＃２株より、常法
によって染色体ＤＮＡを抽出し、制限酵素ＰｖｕＩＩで
切断後、ＳＶ４０全ゲノムＤＮＡをプローブとして、サ
ザンハイプリダイゼーションを行った。第４図に示すよ
うに、２００７ｂｐと１４４６ｂｐの位置にＳＶ４０
ＤＮＡのバンドが観察され、確かにＳＶ４０ ＤＮ
ＡＪ（ＳＶ−１−＃２株の染色体ＤＮＡに挿入されてい
ることが確認され、本枕が自然変異株で無いことが示さ
れた。Example 4 5V40 DNA in transformed cell S V-1-#2
We examined the mode of existence of Chromosomal DNA was extracted from the SV-1-#2 strain by a conventional method, digested with restriction enzyme PvuII, and then subjected to Southern hybridization using SV40 whole genome DNA as a probe. As shown in Figure 4, SV40 is located at 2007bp and 1446bp.
A DNA band was observed, and it was definitely SV40 DN.
It was confirmed that AJ (SV-1-#2) was inserted into the chromosomal DNA of the strain, indicating that Honmakura is not a natural mutant strain.
実施例５
形質転換細胞Ｓ Ｖ−１−６２を、継代培養を繰り返し
て永久増殖性を検討した。継代は以下のように行なった
。まず培養上清を棄て、ＰＢＳ（Ｎａｉ１８ｇ／（１，
ＫＣＱ ０．２ｇ／Ｑ、 Ｎａ、ＨＰＯ，１，１５ｇ
／１２、Ｋｌｌ、ＰＯ４０，２ｇ ／ Ｑ ）で２度細
胞を洗浄後、トリプシン液（ＰＢＳ液中に、０．１％ト
リプシン及び０．０２％ＥＤＴＡを含む）で、３７℃、
３分間処理をする。ｌＯ％牛脂児血清を含有する１９９
培地で、反応をとめて、ピペッティングによって細胞を
はがす。１１００Ｏｒｐ、３分間遠心して細胞を集め、
上清をすてる。ＰＢＳで、細胞を洗浄後、ｌＯ％牛脂児
血清を含有する１９９培地で細胞を懸濁して、培養フラ
スコ中３７℃、炭酸ガス濃度５％含有空気下で培養する
。１９９培地（ギブコ社製）の組成は、以下のようであ
る。〔モーガン・ジエイ・エフ（Ｍｏｒｇａｎ Ｊ、Ｆ
、）ら。プロシーディング・オブ・ザ・ソサエティ・フ
ォア・エクスベリメンタル・バイオロジイー・アンド・
メデイシン（Ｐｒｏｃ、ｓｏｃ、Ｅｘｐ。Example 5 Transformed cell SV-1-62 was repeatedly subcultured to examine its permanent proliferation ability. Passage was carried out as follows. First, discard the culture supernatant and add PBS (Nai18g/(1,
KCQ 0.2g/Q, Na, HPO, 1.15g
After washing the cells twice with PBS solution containing 0.1% trypsin and 0.02% EDTA at 37°C,
Process for 3 minutes. 199 containing lO% tallow serum
Stop the reaction with medium and detach the cells by pipetting. Collect cells by centrifuging at 1100 Orp for 3 minutes.
Discard the supernatant. After washing the cells with PBS, the cells are suspended in 199 medium containing 10% tallow serum and cultured in a culture flask at 37° C. in air containing 5% carbon dioxide. The composition of 199 medium (manufactured by Gibco) is as follows. [Morgan J.F.
,)and others. Proceedings of the Society for Experimental Biology and
Medicine (Proc, soc, Exp.
Ｂｉｏｌ、Ｍｅｄ、） 、第７３巻、１頁（１９５０年
）参照〕培地１９９
成 分
無水塩化カルシウム
硝酸鉄（ｍ）丸木和物
塩化カリウム
無水硫酸マグネシウム
塩化ナトリウム
ｉ！、ｄｉ水素７トリウ１
硫酸アデニン
てず〈ｄぢ云苗′酸
アデニル酸
コレステロール
デオキシリボース
Ｄ−グルコース
グルタチオン（還元体）
グアニン塩酸塩
ヒボキサンチンナトリウム塩
フェノール レッド
リボース
酢酸ナトリウム
チミン
トウィーン８０Ｑ
ウラシル
キサンチンナトリウム塩
ＤＬ−アラニン
Ｌ−アルギニン塩酸塩
ＤＬ−アスパラギン酸
頚ル
２００．００
０．７２
４００．００
９７．６７
６８００．００
１４０．００
１０．００
１．００
０．２０
０．２０
０．５０
１０００．００
０．０５
０．３０
０．３５４
２０．００
０．５０
５０．００
０．３０
２０．００
０．３０
０．３４４
５０．００
？０．００
６０．００
（続き）
成分
Ｌ−システィン塩酸塩−水和物
Ｌ−システィン塩酸塩
ＤＬ−グルタミン酸−水和物
Ｌ−グルタミン
グリシン
Ｌ−ヒスチジン塩酸塩−水和物
し−ヒドロキシプロリン
ＤＬ−イソロイシン
ＤＬ−ロイシン
Ｌ−リジン塩酸塩
ＤＬ−メチオニン
ＤＬ−フェニルアラニン
Ｌ−プロリン
ＤＬ−セリン
ＤＬ−スレオニン
ＤＬ−トリプトファン
し−チロシンニナトリウム塩
ＤＬ−バリン
アスコルビン酸
α−トコフェロールリン酸
二ナトリウム塩
ｄ−ビオチン
カルシフェロール
Ｄ−パントテン酸カルシウム
塩化コリン
葉酸
ｉ−イノシトール
鴫
０．１１
２６．００
１５０．００
ｔｏｏ、ｏ。Biol, Med, ), Vol. 73, p. 1 (1950)] Culture medium 199 Ingredients Anhydrous calcium chloride Iron nitrate (m) Marukiwazate Potassium chloride Anhydrous magnesium sulfate Sodium chloride i! , di hydrogen 7 triu 1 adenine sulfate acid adenylate cholesterol deoxyribose D-glucose glutathione (reduced form) guanine hydrochloride hypoxanthin sodium salt phenol red ribose sodium acetate thymine 80Q uracil xanthine sodium Salt DL-alanine L-arginine hydrochloride DL-aspartic acid neck 200.00 0.72 400.00 97.67 6800.00 140.00 10.00 1.00 0.20 0.20 0.50 1000. 00 0.05 0.30 0.354 20.00 0.50 50.00 0.30 20.00 0.30 0.344 50.00 ? 0.00 60.00 (Continued) Ingredients L-Cystine hydrochloride-hydrate L-Cystine hydrochloride DL-Glutamic acid-hydrate L-Glutaminyl glycine L-Histidine hydrochloride-hydrate-Hydroxyproline DL- Isoleucine DL-Leucine L-Lysine Hydrochloride DL-Methionine DL-Phenylalanine L-Proline DL-Serine DL-Threonine DL-Tryptophan-Tyrosine Disodium Salt DL-Valine Ascorbic Acid α-Tocopherol Phosphate Disodium Salt d-Biotin Calcium Ferol D-calcium pantothenate choline chloride folic acid i-inositol 0.11 26.00 150.00 too, o.
５０．００
２１．８８
１０．００
４０．００
１２０ 、００
７０．００
３０．００
５０．００
４０．００
５０．００
６０．００
２０．００
５７．８８
５０．００
０．０５
０．０１
０．０１
Ｏｌｌｏ
０．０１
０．５０
０．０１
０．０５
（続き）
戊」穿
メナジオン
ナイアシン
ナイアシンアミド
ｐ−アミノ安息香酸
ビリドキサル塩酸塩
ピリドキシン塩酸塩
リボフラビン
チアミン塩酸塩
ビタミンＡ酢酸塩
５匹
０．０Ｉ
Ｏ，０２５
０，０２５
０，０５
０，０２５
０，０２５
０，０１
０，０１
Ｏ１１４
本細胞株は、細胞の分離から逆算して、１００回程度分
裂を繰り返してもまだ増殖をしているところから、永久
増殖性を獲得していると考えられる。50.00 21.88 10.00 40.00 120 ,00 70.00 30.00 50.00 40.00 50.00 60.00 20.00 57.88 50.00 0.05 0.01 0. 01 Ollo 0.01 0.50 0.01 0.05 (continued) Menadione niacin niacinamide p-aminobenzoic acid pyridoxal hydrochloride pyridoxine hydrochloride riboflavin thiamine hydrochloride vitamin A acetate 5 animals 0.0I O, 025 0,025 0,05 0,025 0,025 0,01 0,01 O114 This cell line is still proliferating even after dividing approximately 100 times, counting backwards from cell separation. It is thought that it has acquired the ability to reproduce permanently.
（発明の効果）
本発明のヒト血管内皮細胞来由形質転換細胞番よ、ヒト
正常血管内皮細胞の特徴を有しながら、かつ内皮細胞生
長因子要求性が無く、更に永久増殖性を獲得しているの
で、ｔＰＡをはじめとする生理活性物質の生産あるいは
それらの試験及び研究番こ応用することができる。従っ
て、これまでヒト血管内皮細胞の長期継代培養ができな
かった為に、少量しか得られなかった生理活性物質が大
量生産できるようになり、それら物質の医薬品への応用
あるいは試験及び研究ｌ＼の応用が可能となった。(Effects of the Invention) The human vascular endothelial cell-derived transformed cell number of the present invention has the characteristics of normal human vascular endothelial cells, does not require endothelial cell growth factors, and has acquired permanent proliferation ability. Therefore, it can be applied to the production of physiologically active substances such as tPA, as well as their testing and research. Therefore, it has become possible to mass-produce physiologically active substances that could only be obtained in small quantities because long-term subculturing of human vascular endothelial cells was not possible, and the application of these substances to pharmaceuticals or testing and research. became possible to apply.
第１図は、本発明の方法のよって得られたヒト内皮細胞
由来形質転換細胞株５Ｖ−１−１ｔ２の顕微鏡写真を示
す。
第２図は、本発明の方法によって得られた形質転換細胞
５Ｖ−１−６２を、蛍光色素を結合した抗第■因子モノ
クローナル抗体で、蛍光染色した顕微鏡写真を示す。
第３図は１本発明の方法によって得られた形質転換細胞
５Ｖ−１−＃２の培養上清中のｔ、ＰＡ活性（・）及び
ヒト正常血管内皮細胞の培養上清中のｔＰＡ活性（○）
を測定した結果を示す。
第４図は、本発明の方法によって得られた形質転換細胞
５Ｖ−１−＃２の染色体ＤＮＡを制限酵素Ｐｖｕｎで切
断後、ＳＶ４０全ゲノムＤＮＡをプローブとするサザン
ハイプリダイゼーションの結果を示す。FIG. 1 shows a micrograph of the human endothelial cell-derived transformed cell line 5V-1-1t2 obtained by the method of the present invention. FIG. 2 shows a photomicrograph of the transformed cells 5V-1-62 obtained by the method of the present invention, fluorescently stained with an anti-factor Ⅰ monoclonal antibody conjugated with a fluorescent dye. Figure 3 shows t, PA activity (·) in the culture supernatant of transformed cells 5V-1-#2 obtained by the method of the present invention, and tPA activity (·) in the culture supernatant of human normal vascular endothelial cells. ○)
The results are shown below. FIG. 4 shows the results of Southern hybridization using SV40 whole genome DNA as a probe after cutting the chromosomal DNA of the transformed cell 5V-1-#2 obtained by the method of the present invention with the restriction enzyme Pvun.
Claims (3)
形質転換細胞 １）細胞の形態：敷石状（ヒト正常血管内皮細胞とほぼ
同様の形状をしている。） ２）細胞の由来：ヒトより分離した血管内皮細胞 ３）継代培養：永久増殖可能 ４）生長因子要求性：血管内皮細胞生長因子やヘパリン
を含まない培地で増殖可能である。 ５）機能：ヒト正常血管内皮細胞とほぼ同様の組織由来
プラスミノーゲン活性化因子産生能を示す。 ６）血液凝固第VIII因子産生能：血管内皮細胞の特徴で
ある血液凝固第VIII因子を産生する。 ７）ＳＶ４０ＤＮＡの染色体における存在：ＳＶ４０Ｄ
ＮＡが細胞の染色体に挿入され ている。 ８）ＳＶ４０ＤＮＡウィルスの遊離：本細胞株からのＳ
Ｖ４０ＤＮＡウィルスの遊離は認められていない。 ９）コロニーの形式：ペトリ皿上ではコロニーを形成す
るが、軟寒天中では形成しない。１０）凍結保存：−７
０℃ないし−１９６℃できわめて長期間保存可能である
。(1) Transformed cells derived from human vascular endothelial cells with the following cytological properties 1) Cell morphology: cobblestone-like (approximately the same shape as normal human vascular endothelial cells) 2) Origin of cells: Vascular endothelial cells isolated from humans 3) Subculture: Permanent proliferation possible 4) Growth factor requirement: Vascular endothelial cells can proliferate in a medium that does not contain growth factors or heparin. 5) Function: Shows ability to produce tissue-derived plasminogen activator almost similar to normal human vascular endothelial cells. 6) Ability to produce blood coagulation factor VIII: Produce blood coagulation factor VIII, which is a characteristic of vascular endothelial cells. 7) Presence of SV40 DNA in chromosome: SV40D
NA is inserted into the chromosome of the cell. 8) Release of SV40 DNA virus: S from this cell line
No release of V40 DNA virus was observed. 9) Colony format: Colonies are formed on Petri dishes, but not in soft agar. 10) Cryopreservation: -7
It can be stored for a very long time at 0°C to -196°C.
ランスホームすることを特徴とするヒト血管内皮細胞由
来形質転換細胞の作成方法。(2) A method for producing transformed cells derived from human vascular endothelial cells, which comprises transforming human vascular endothelial cells with SV40-derived DNA.
培養、採取することを特徴とする血栓溶解剤組織由来プ
ラスミノーゲン活性化因子の生産方法。(3) A method for producing a thrombolytic tissue-derived plasminogen activator, which comprises culturing and collecting the transformed cells according to claim 1 in a cell culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63224215A JPH0272866A (en) | 1988-09-07 | 1988-09-07 | Human vascular endothelical cell-derived transforming cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63224215A JPH0272866A (en) | 1988-09-07 | 1988-09-07 | Human vascular endothelical cell-derived transforming cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0272866A true JPH0272866A (en) | 1990-03-13 |
Family
ID=16810318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63224215A Pending JPH0272866A (en) | 1988-09-07 | 1988-09-07 | Human vascular endothelical cell-derived transforming cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0272866A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992017569A1 (en) * | 1991-04-04 | 1992-10-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Immortalization of endothelial cells |
-
1988
- 1988-09-07 JP JP63224215A patent/JPH0272866A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| MICROBIOLOGY AND IMMUNOLOGY * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992017569A1 (en) * | 1991-04-04 | 1992-10-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Immortalization of endothelial cells |
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