JPWO2006129717A1 - Tumor marker for detecting pancreatic cancer and kit for detecting pancreatic cancer using the same - Google Patents

Tumor marker for detecting pancreatic cancer and kit for detecting pancreatic cancer using the same Download PDF

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JPWO2006129717A1
JPWO2006129717A1 JP2007519041A JP2007519041A JPWO2006129717A1 JP WO2006129717 A1 JPWO2006129717 A1 JP WO2006129717A1 JP 2007519041 A JP2007519041 A JP 2007519041A JP 2007519041 A JP2007519041 A JP 2007519041A JP WO2006129717 A1 JPWO2006129717 A1 JP WO2006129717A1
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明 外川
明 外川
重紹 高野
重紹 高野
宮崎　勝
勝 宮崎
文夫 野村
文夫 野村
毅 朝長
毅 朝長
一幸 曽川
一幸 曽川
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Abstract

早期膵癌の陽性率が高く、膵癌をより確実に検出する腫瘍マーカー及びそれを用いた膵癌検出キットを提供することを目的とする。ＡｐｏＣ−１蛋白からなる膵癌検出用腫瘍マーカー、およびＡｐｏＣ−１蛋白を検出するためのプロテインチップを含む膵癌検出用キットであることを特徴とする。An object of the present invention is to provide a tumor marker that has a high positive rate of early pancreatic cancer and more reliably detects pancreatic cancer, and a pancreatic cancer detection kit using the same. A pancreatic cancer detection kit comprising a tumor marker for detection of pancreatic cancer comprising ApoC-1 protein and a protein chip for detecting ApoC-1 protein.

Description

本発明は、膵癌検出用マーカー、及び、該マーカーを用いた膵癌検出用キットに関する。   The present invention relates to a marker for detecting pancreatic cancer and a kit for detecting pancreatic cancer using the marker.

膵癌の発生は、年々増加の傾向を辿っており、日本での発生率は１９８５年には１０万人中５．２人まで増加した。更に、１９９５年には膵癌による死亡数は１０万人中１４．７人となり、男性の癌による死因の第５位、女性の第７位を占めている（厚生統計協会：国民衛生の動向１９９７年）。   The incidence of pancreatic cancer has been increasing year by year, and the incidence in Japan increased to 5.2 out of 100,000 in 1985. Furthermore, the number of deaths from pancreatic cancer in 1995 was 14.7 out of 100,000, accounting for the fifth leading cause of cancer death among men and seventh among women (Health Statistics Association: National Health Trends 1997). Year).

膵癌は消化器癌のなかでも予後不良の癌として知られている。その理由としては、（１）早期発見が困難で、診断時既に進行癌となり切除率が低く、（２）生物学的悪性度が高く、早期に周囲神経叢、腹膜播種や肝転移をきたし、（３）有効な補助療法が確立されていないことなどが挙げられる。   Pancreatic cancer is known as a cancer with poor prognosis among digestive organ cancers. The reasons are as follows: (1) Early detection is difficult, advanced cancer is already diagnosed at the time of diagnosis, and the resection rate is low. (2) Biological malignancy is high. (3) An effective adjuvant therapy has not been established.

現状、局所進行膵癌に対しては拡大リンパ節郭清や門脈合併切除が試みられ、切除率の向上に寄与し、膵癌症例の１０〜１５％が外科的切除の対象となっている。しかしながら、生存率の改善には至っておらず、切除例でも５年生存率は５〜１０％といわれている。従って、膵癌を早期に診断し、外科的切除を行い治療成績の向上を図ることが、急務である。   Currently, extended lymph node dissection and portal vein combined resection are attempted for locally advanced pancreatic cancer, which contributes to the improvement of resection rate, and 10 to 15% of pancreatic cancer cases are targeted for surgical resection. However, the survival rate has not been improved, and the 5-year survival rate is said to be 5 to 10% even in resected cases. Therefore, it is urgent to diagnose pancreatic cancer at an early stage and perform surgical resection to improve treatment results.

また、膵癌は非常に悪性であり、膵臓と離れた部位に転移（遠隔転移）をきたしやすく、この遠隔転移（特に、肝臓）のため、多くの患者は外科的切除の対象とならない。手術不能の患者の予後は更に悪く、５年生存率はほぼ０％である。このため、できる限り早期に診断し、切除を行うことが最も効果的な治療である。従って、早期に膵癌患者の発見方法を開発することが何より重要である。   In addition, pancreatic cancer is very malignant and tends to metastasize (distant metastasis) to a site distant from the pancreas, and many patients are not subject to surgical resection because of this distant metastasis (particularly the liver). The prognosis for inoperable patients is even worse and the 5-year survival rate is almost 0%. For this reason, the most effective treatment is to diagnose and remove as early as possible. Therefore, it is most important to develop a method for finding pancreatic cancer patients at an early stage.

従来、膵癌の診断には画像診断としてＣＴスキャンや超音波検査を中心に行われている。しかしながら、この診断方法では膵癌は進行例として発見されることが大部分である。その理由は、例えば腹痛・背部痛・黄疸・食欲不振など膵癌自覚症状が出現した時には既に周囲臓器や遠隔転移をきたしている場合が多いためである。さらに、ＣＴスキャンや超音波検査では腫瘍径が２ｃｍ未満の小膵癌の発見は困難である。このため、外科的切除の対象となるのは全患者の１０〜１５％程度であり、仮に切除の対象となる患者でも高度に進行してしまっている患者が殆どである。   Conventionally, diagnosis of pancreatic cancer has been performed mainly by CT scan and ultrasonic examination as image diagnosis. However, in this diagnostic method, pancreatic cancer is mostly found as an advanced case. The reason for this is that, for example, when subjective symptoms of pancreatic cancer such as abdominal pain, back pain, jaundice, and anorexia appear, peripheral organs and distant metastases are often already present. Furthermore, it is difficult to find small pancreatic cancer having a tumor diameter of less than 2 cm by CT scan or ultrasonography. For this reason, about 10 to 15% of all patients are targeted for surgical resection, and even patients who are the target of resection are highly advanced.

更に一方で、膵癌の腫瘍の検出としてＣＡ１９−９、ＤＵＰＡＮ−２、ＳＰＡＮ−１、ＳＬＸ等各種腫瘍マーカーが用いられており、膵癌の診断に寄与している（例えば下記非特許文献１乃至４参照）。
張正和ら、“新しい腫瘍マーカーＣＡ１９−９の臨床的意義 膵癌症例を中心に”胆と膵、１９８５年、６巻、１１２９−１１３５頁 ＴＡＫＡＳＡＫＩら“Ｃｏｒｒｅｌａｔｉｖｅ Ｓｔｕｄｙ ｏｎ Ｅｘｐｒｅｓｓｉｏｎ ｏｆ ＣＡ１９−９ ａｎｄ ＤＵ−ＰＡＮ−２ ｉｎ Ｔｕｍｏｒ Ｔｉｓｓｕｅ ａｎｄ ｉｎ Ｓｅｒｕｍ ｏｆ Ｐａｎｃｒｅａｔｉｃ Ｃａｎｃｅｒ Ｐａｔｉｅｎｔｓ”Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ、１９８８年、４８巻、１４３５−１４３８頁 ＹＯＮＧ Ｓ． ＣＨＵＮＧら“Ｔｈｅ Ｄｅｔｅｃｔｉｏｎ ｏｆ Ｈｕｍａｎ Ｐａｎｃｒｅａｔｉｃ Ｃａｎｃｅｒ−Ａｓｓｏｃｉａｔｅｄ Ａｎｔｉｇｅｎ ｉｎ ｔｈｅ Ｓｅｒｕｍ ｏｆ Ｃａｎｃｅｒ Ｐａｔｉｅｎｔｓ”Ｃａｎｃｅｒ、１９８７年、６０巻、１６３６−１６４３頁 河上浩康ら“各種消化器癌における血清ＳＬＸ（ｓｉａｌｙｌ ＳＳＥＡ−１）測定の臨床的有用性”日本消化器病学会雑誌、１９８９年、８６巻、１１４１−１１４８頁 On the other hand, various tumor markers such as CA19-9, DUPAN-2, SPAN-1, and SLX are used for detection of tumors of pancreatic cancer and contribute to the diagnosis of pancreatic cancer (for example, Non-Patent Documents 1 to 4 below). reference).
Masakazu Zhang et al., “Clinical Significance of New Tumor Marker CA19-9 Focusing on Pancreatic Cancer Cases” Bile and Pancreas, 1985, Vol. 6, pp. 1129-1135 TAKASAKI et al., “Correlative Study on Expression of CA19-9 and DU-PAN-2 in Tumor Tissue and in Serum of Pancreatic Cancer Patenties, Vol.14, pp.35, 1988. YONG S. CHUNG et al., “The Detection of Human Pancreatic Cancer-Associated Antigen in the Serum of Cancer Pattens” Cancer, 1987, 60, 1636-1643. Hiroyasu Kawakami et al. “Clinical usefulness of serum SLX (sialyl SSEA-1) measurement in various gastrointestinal cancers” Journal of Japanese Society of Gastroenterology, 1989, 86, 1141-1148

しかしながら、上記腫瘍マーカーのうち最も特異性が高く感度も良好で、広く用いられているＣＡ１９−９であっても早期膵癌の陽性率は低く、病期Ｉの膵癌の検索は当該検査では不可能である。さらに、Ｌｅｗｉｓ抗原陰性患者ではＣＡ１９−９は仮に進行膵癌でも陰性となってしまうことがあるという問題点を有する（Ｈｉｒａｎｏら、“Ｌｏｓｓ ｏｆ Ｌｅｗｉｓ ａｎｔｉｇｅｎ ｅｘｐｒｅｓｓｉｏｎ ｏｎ ｅｒｙｔｈｒｏｃｙｔｅｓ ｉｎ ｓｏｍｅ ｃａｎｃｅｒ ｐａｔｉｅｎｔｓ ｗｉｔｈ ｈｉｇｈ ｓｅｒｕｍ ＣＡ１９−９ ｌｅｖｅｌｓ”、Ｊ Ｎａｔｌ Ｃａｎｃｅｒ Ｉｎｓｔ、１９８７年、７９巻、１２６１〜１２６８頁）。更に、慢性膵炎や胆管炎、胆石症の患者でも高値を示してしまい、膵癌を確実に検出する腫瘍マーカーとしては課題が残っている。   However, among the above tumor markers, it has the highest specificity and good sensitivity, and even the widely used CA19-9 has a low positive rate of early pancreatic cancer, and the search for stage I pancreatic cancer is impossible by this test It is. In addition, CA19-9 may be negative even in advanced pancreatic cancer in Lewis antigen-negative patients (Hirano et al., “Loss of Lewis antigen expression in some cancer patients with the United States 19th. levels ", J Natl Cancer Inst, 1987, 79, 1261-1268). Furthermore, patients with chronic pancreatitis, cholangitis, and cholelithiasis show high values, and there remains a problem as a tumor marker for reliably detecting pancreatic cancer.

そこで、本発明は、上記課題を鑑み、より早期膵癌の陽性率が高く、膵癌をより確実に検出する腫瘍マーカー及びそれを用いた膵癌検出キットを提供することを目的とする。   In view of the above problems, an object of the present invention is to provide a tumor marker that has a higher positive rate of early pancreatic cancer and more reliably detects pancreatic cancer, and a pancreatic cancer detection kit using the same.

本発明者らは、膵癌患者の手術前と外科的切除が行われ膵癌が切除された手術後の血清（各２０件）を比較検討していくうちに、ＡｐｏＣ−１蛋白が手術前の膵癌患者には発現しているのに対し、切除後の患者には殆ど発現されていないことを発見し、この蛋白が膵癌において腫瘍マーカーとして用いることが出来ることを見いだし、本発明を完成するに至った。この蛋白は膵癌の早期の患者でも発現しており、従来の腫瘍マーカーよりより鋭敏に広範囲に膵癌患者を検出することが可能である。   While the present inventors have comparatively examined serum (20 cases each) before and after surgery for pancreatic cancer patients after surgical excision and excision of pancreatic cancer, the ApoC-1 protein is preoperative pancreatic cancer. It was found that it was expressed in patients but rarely expressed in patients after resection, and found that this protein could be used as a tumor marker in pancreatic cancer, leading to the completion of the present invention. It was. This protein is also expressed in patients with early pancreatic cancer, and it is possible to detect pancreatic cancer patients more extensively and more sensitively than conventional tumor markers.

即ち、本発明の一形態に係る膵癌検出用腫瘍マーカーは、ＡｐｏＣ−１蛋白からなることを特徴とする。これにより、従来の腫瘍マーカーより早期膵癌の陽性率が高く、膵癌をより確実に検出することが可能となる。これは例えば、プロテインチップシステム、ELISA法などを用いて検出することができる。ELISA法としては、例えば（Micheal D. Curry, Walter J McConathy, Jim D. Fesmire, and Peter
Alaupovic. “Quantitative Determination of Apolipoproteins C−1 and C−II in Human Plasma by Separate
Electroimmunoassays.”, Clin.Chem. １９８１年、２７巻、５４３−５４８頁、Caroline
Petit−Turcotte, Sheldon M. Stohl, et al.”Apolipoprotein
C−1 Expression in the Brain in Alzheimer’s Disease.”
Neurobiology of Disease ２００１年、８巻、９５３−９６３頁）等に記載の常法を用いることができる。That is, the tumor marker for detecting pancreatic cancer according to one embodiment of the present invention is characterized by comprising ApoC-1 protein. As a result, the positive rate of early pancreatic cancer is higher than that of conventional tumor markers, and pancreatic cancer can be detected more reliably. This can be detected using, for example, a protein chip system, ELISA method or the like. Examples of ELISA methods include (Micheal D. Curry, Walter J McConathy, Jim D. Fesmire, and Peter
Alaupovic. “Quantitative Determination of Apolipoproteins C−1 and C−II in Human Plasma by Separate
Electroimmunoassays. ”, Clin. Chem. 1981, 27, 543-548, Caroline
Petit-Turcotte, Sheldon M. Stohl, et al. “Apolipoprotein
C-1 Expression in the Brain in Alzheimer's Disease. ”
Neurobiology of Disease 2001, Vol. 8, pp. 953-963) can be used.

また、本発明の他の一形態に係る膵癌検出キットは、プロテインチップシステムにおいて用いられ、膵癌のより確実な検出に寄与することができる。 Moreover, the pancreatic cancer detection kit according to another embodiment of the present invention is used in a protein chip system, and can contribute to more reliable detection of pancreatic cancer.

プロテインチップシステムとは、蛋白質の発現、相互作用、翻訳後修飾などの機能解析や、目的蛋白質の精製・同定などを効率的に行うことを目的として開発されたシステムのことをいい、蛋白質解析に適した様々な化学的性質を表面にもたせたプロテインチップと、測定に用いられるプロテインチップリ−ダ−と、測定・解析に使用するソフトウェアがインスト−ルされたコンピュ−タ−を有して構成される。 A protein chip system is a system developed for the purpose of efficiently performing functional analysis such as protein expression, interaction, post-translational modification, and purification and identification of the target protein. It consists of a protein chip with various suitable chemical properties on the surface, a protein chip reader used for measurement, and a computer installed with software used for measurement and analysis. The

プロテインチップシステムは、血清や尿、培養液細胞破砕液などクル−ドなサンプルから、プロテインチップに対するアフィニティ−を利用して目的蛋白質を捕捉し、その質量数を測定することができる。ラベルやタグを使わず、プロテインチップ上で簡便に蛋白質の解析ができ、少量のサンプルから短時間に結果を得ることができる利点がある。プロテインチップリ−ダ−には限定されるわけではないが、例えば時間飛行型質量分析計（ＳＥＬＤＩ−ＴＯＦ−ＭＳ）が好適である。時間非行型質量分析計は、プロテインチップ表面に捕捉されている物質にレ−ザ−を照射し、イオン化させる。そのイオン化した物質は電場の中で加速され、飛行管の中を検出器に向かって飛行していく。このとき飛行する速度は分子量（正確には分子量をイオン化された電荷数で割った値）に比例する。従って、レ−ザ−が照射されてから検出器に到達するまでの時間を測定することにより、プロテインチップ表面に存在する物質の分子量を知ることができ、目的蛋白質を同定することができる。 The protein chip system can capture the target protein from a sample sample such as serum, urine, and culture cell disruption liquid using affinity for the protein chip, and measure the mass number. There is an advantage that a protein can be easily analyzed on a protein chip without using a label or a tag, and a result can be obtained in a short time from a small amount of sample. Although not limited to a protein chip reader, for example, a time-of-flight mass spectrometer (SELDI-TOF-MS) is suitable. In the time delinquent mass spectrometer, a substance captured on the surface of a protein chip is irradiated with a laser to be ionized. The ionized material is accelerated in the electric field and flies through the flight tube toward the detector. The flying speed at this time is proportional to the molecular weight (more precisely, the value obtained by dividing the molecular weight by the number of ionized charges). Therefore, by measuring the time from the irradiation of the laser until reaching the detector, the molecular weight of the substance present on the surface of the protein chip can be known, and the target protein can be identified.

以上、本発明により、より早期膵癌の陽性率が高く、膵癌をより確実に検出する腫瘍マーカー及びそれを用いた膵癌検出キットを提供することができる。   As described above, according to the present invention, it is possible to provide a tumor marker with a higher positive rate of early pancreatic cancer and more reliably detecting pancreatic cancer, and a pancreatic cancer detection kit using the same.

以下、本発明に係る膵癌検出用腫瘍マーカー、検出キットとしての効果について、実際の患者を対象とした具体的な例を用いて説明する。   Hereinafter, the effect as a tumor marker for pancreatic cancer detection and a detection kit according to the present invention will be described using a specific example for an actual patient.

２００１年６月から２００３年１０月までに治癒手術が施行され、浸潤性膵管癌の病理組織診断のついた２０症例を対象症例とし、これらの術前、術後の血清をサンプルとして用いた。この２０症例を表１に示す。なお、この症例全てにおいて、術前の血清は補助療法（化学療法や放射線療法等）を施行しなかった血清を、術後の血清は手術の影響を考慮し、術後状態が安定した後、少なくとも術後３、４週間後の術後補助療法を行う前に採取した血清を用いた。なお、各血清は、採血後速やかに５分間、３０００回転数で遠心分離を行い、その上清を分注し−３０℃にて保存した。
Healing surgery was performed from June 2001 to October 2003, and 20 cases with histopathological diagnosis of invasive pancreatic duct cancer were used as target cases, and these pre- and post-operative sera were used as samples. These 20 cases are shown in Table 1. In all cases, preoperative serum was serum that did not receive adjuvant therapy (chemotherapy, radiation therapy, etc.), postoperative serum was considered after surgery, and the postoperative condition was stable. Serum collected before postoperative adjuvant therapy at least 3 and 4 weeks after surgery was used. Each serum was centrifuged immediately at 3000 rpm for 5 minutes after blood collection, and the supernatant was dispensed and stored at -30 ° C.

プロテインチップシステムにおけるプロテインチップリ−ダ−としては時間飛行型質量分析計（ＳＥＬＤＩ−ＴＯＦ−ＭＳ）を用いた。プロテインチップとしてはＷＣＸ２（陽イオン交換チップ）（ＣＩＰＨＥＲＧＥＮ社製）を用い、ＢｕｆｆｅｒとしてはｐＨ６．５（５０ｍＭ ｓｏｄｉｕｍ ｐｈｏｓｐｈａｔｅ）ｕｒｅａを用いた。サンプルの組成はｕｒｅａ;患者血清２０μｌ＋変性ｂｕｆｆｅｒ ２０μｌ＋ｐＨ ｂｕｆｆｅｒ １６０μｌ（１／１０希釈）とし、プロテインチップの各スポットに１００μｌずつ投与し、それぞれ同一サンプルを２スポットずつ行った。そして各スポットに対しシナピン酸をＥＡＭ溶解液（５０％アセトノトリル／０．５％ＴＦＡ）に溶かして塗布した。これをプロテインチップ検出器であるＳＥＬＤＩ−ＴＯＦ−ＭＳを用いて血清中にある蛋白を検出し、術前術後の蛋白比較（Serum protein profiling）を行い、同一個体間で術前上昇し術後低下するｐｅａｋ（蛋白）を見つけ、その候補蛋白として質量数およそ６６３０Ｄａのｐｅａｋを検出した。この代表的なｐｅａｋ例を図１に、この蛋白の術前、術後peak intensityを図２に示す。   A time-flight mass spectrometer (SELDI-TOF-MS) was used as a protein chip leader in the protein chip system. WCX2 (cation exchange chip) (manufactured by CIPHERGEN) was used as the protein chip, and pH 6.5 (50 mM sodium phosphate) urea was used as the buffer. The composition of the sample was urea; 20 μl of patient serum + 20 μl of denatured buffer + 160 μl of pH buffer (diluted 1/10), 100 μl was administered to each spot of the protein chip, and 2 spots of the same sample were performed. Then, sinapinic acid was dissolved in EAM solution (50% acetonotolyl / 0.5% TFA) and applied to each spot. SELDI-TOF-MS, a protein chip detector, is used to detect the protein in the serum, and protein comparison is performed before and after surgery (Serum protein profiling). A decreasing peak (protein) was found, and a peak having a mass number of about 6630 Da was detected as a candidate protein. FIG. 1 shows a typical peak example, and FIG. 2 shows pre- and post-operative peak intensity of this protein.

図１によると、ｐＨ６．５の条件下、ｕｒｅａ ｂｕｆｆｅｒにおいて術前から術後に有意にｐｅａｋが低下する質量がみいだされ、その質量数は６６３０Ｄａ（p＝０．００３８）であった。ｐｅａｋを有する患者を陽性と判定すると２０例中１８例（９０％）が膵癌陽性であると判定できた。なお従来の膵癌に対する特異的な腫瘍マーカーであるＣＡ１９−９は２０例中１５例（７５％）の陽性にすぎず、また、ＣＥＡの陽性率は２例（１０％）にすぎなかった。その中で、症例２０はＬｅｗｉｓ抗原（ａ−，ｂ+）患者でＣＡ１９−９正常値であったが、この蛋白では陽性であり、更に、症例２及び３は早期の膵癌である病期Ｉで、両者ともこの蛋白では陽性であったが、症例３ではＣＡ１９−９は陰性であった。（表１参照）すなわち、この蛋白によると、早期の膵癌においてもより高い頻度で検出が可能で、従来のＣＡ１９−９に比しても鋭敏であり、しかもＬｅｗｉｓ抗原（ａ−，ｂ+）患者に対しても検出可能な膵癌検出用腫瘍マーカーとして用いることができることがわかった。   According to FIG. 1, under the condition of pH 6.5, a mass in which peak was significantly decreased from the pre-operation to the post-operation was found in the urea buffer, and the mass number was 6630 Da (p = 0.0038). When a patient having a peak was determined to be positive, 18 out of 20 cases (90%) could be determined to be positive for pancreatic cancer. CA19-9, which is a specific tumor marker for conventional pancreatic cancer, was only positive in 15 cases (75%) of 20 cases, and the positive rate of CEA was only 2 cases (10%). Among them, case 20 was a Lewis antigen (a−, b +) patient who had a normal value of CA19-9, but was positive for this protein, and cases 2 and 3 were stage I, an early pancreatic cancer. Both were positive for this protein, but in case 3, CA19-9 was negative. (See Table 1) That is, according to this protein, it can be detected at a higher frequency even in early pancreatic cancer, is more sensitive than conventional CA19-9, and Lewis antigen (a-, b +). It was found that it can be used as a tumor marker for detection of pancreatic cancer that can be detected for patients.

そこで、上記の結果を踏まえ、血清から特異的蛋白質の精製を行い、膵癌検出用のマーカーと期待される蛋白質についての特定を行った。なおこの特異的蛋白質の精製は、至適ｐＨ（ｐＩ）の検討及び至適塩（ＮａＣｌ）濃度の検討を通じて行った。   Therefore, based on the above results, a specific protein was purified from serum, and a protein expected to be a marker for detecting pancreatic cancer was identified. The specific protein was purified through examination of optimum pH (pI) and examination of optimum salt (NaCl) concentration.

まず、ｕｒｅａ ｂｕｆｆｅｒにおいてｐＨを４．５，５．０，５．５，６．０と段階的にｐＨを上げていき、最も６６３０Ｄａのｐｅａｋが高くなる至適ｐＨ、すなわち最も単離、精製するのに適したｐＨを調べた。その結果、ｐＨ４．５の条件下で最もｐｅａｋが高かった。次に塩濃度を０、５０，１００，１５０，２００，３００，４００，５００ｍＭと段階的にあげていき、６６３０Ｄａのｐｅａｋが下がり始める塩濃度を調べた。その結果、５００ｍＭまで塩濃度をあげていってもｐｅａｋが下がり始めることはなかった。   First, in the urea buffer, gradually increase the pH to 4.5, 5.0, 5.5, 6.0, and the optimum pH at which the peak of 6630 Da is the highest, that is, the most isolated and purified A suitable pH was investigated. As a result, the peak was the highest under the condition of pH 4.5. Next, the salt concentration was gradually increased to 0, 50, 100, 150, 200, 300, 400, and 500 mM, and the salt concentration at which the peak of 6630 Da began to decrease was examined. As a result, the peak did not begin to fall even when the salt concentration was increased to 500 mM.

そして、上記至適な条件を踏まえてＦＰＬＣを行い、ｐｅａｋの高いＦＰＬＣ ｆｒａｃｔｉｏｎをＨＰＬＣにかけた。ＦＰＬＣの条件は、ＮａＣｌを１５ｍＭずつ分離したｆｒａｃｔｉｏｎを作製し、１６５ｍＭ〜１０００ｍＭまで３０ｍＭごとのｆｒａｃｔｉｏｎを用い、ＮＰ ２０ ｃｈｉｐにてＳＥＬＤＩ−ＴＯＦ−ＭＳ解析を行った。ＨＰＬＣの条件はリニア−グラディエントにてＢｕｆｆｅｒ;０．１％（Ａ）〜８０％（Ｂ）アセトニトリル、流量；２００μｌ/ｍｉｎで行い、分離カラムはＣ−１８を用いた。これにより精製された目的蛋白を、Ｎ末端アミノ酸シ−クエンスにてアミノ酸１５残基目までを解析し同定を行った結果、２つの蛋白はそれぞれ６６３０Ｄａの本体がｍａｔｕｒｅ型のＡｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｃ−１（５７アミノ酸）（成熟型ＡｐｏＣ−１蛋白）、６４２０Ｄａの本体はｍａｔｕｒｅ型のＮ末端側のアミノ酸が２つ欠失したＡｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｃ−１（５５アミノ酸）であることが判明した。この結果を図３〜９に示す。即ち、上記膵癌検出用腫瘍マーカーとして使用可能な蛋白は、ＡｐｏＣ−１蛋白であることが判明した。   And FPLC was performed based on the said optimal conditions, and FPLC fraction with high peak was applied to HPLC. The FPLC was prepared by preparing fractions in which NaCl was separated by 15 mM and performing SELDI-TOF-MS analysis using NP 20 chips using fractions of 30 mM from 165 mM to 1000 mM. The HPLC conditions were linear-gradient Buffer: 0.1% (A) to 80% (B) acetonitrile, flow rate: 200 μl / min, and C-18 was used as the separation column. As a result of analyzing and identifying the purified target protein up to the 15th amino acid residue with the N-terminal amino acid sequence, the two proteins were each a 6630 Da body type apolipoprotein C-1 (57 Amino acid) (mature ApoC-1 protein), the body of 6420 Da was found to be Apolipoprotein C-1 (55 amino acids) from which two amino acids on the N-terminal side of the shape type were deleted. The results are shown in FIGS. That is, it was found that the protein that can be used as the tumor marker for detecting pancreatic cancer is ApoC-1 protein.

次に、この蛋白の血清値と臨床病理学的因子及び生存期間との関連を検討することにより、この蛋白と膵癌悪性度との相関がないかを検討した。ＡｐｏＣ−１蛋白のプロテインチップシステムによる解析で得られたpeak intensityのデ−タ−（図２参照）の術前値の中央値で血清ＡｐｏＣ−１高値群、低値群を２群にわけ、２群間をＫａｐｌａｎ−Ｍｅｉｅｒ法にて統計解析を行ったところ、ＡｐｏＣ−１血中濃度が高値の患者は無再発生存期間（p＝０．００１１；Logrank）及び生存期間（p＝０．００６２；Logrank）が共に有意に短いことが判明した（図１０参照）。   Next, by examining the relationship between the serum level of this protein and clinicopathological factors and survival time, it was examined whether there was a correlation between this protein and the malignancy of pancreatic cancer. The median preoperative value of peak intensity data (see FIG. 2) obtained by analysis with the protein chip system of ApoC-1 protein is divided into two groups, the serum ApoC-1 high value group and the low value group, When statistical analysis was performed between the two groups using the Kaplan-Meier method, patients with a high ApoC-1 blood concentration were found to have a recurrence-free survival period (p = 0.0011; Logrank) and a survival period (p = 0.0062). ; Logrank) was found to be significantly shorter (see FIG. 10).

以上、本膵癌検出用腫瘍マーカーによって、より早期膵癌の陽性率が高く、膵癌をより確実に検出する腫瘍マーカー及びそれを用いた膵癌検出キットを実現することができる。さらに、この腫瘍マーカーの値は患者の生存期間と関連があり、膵癌悪性度にも密接な関係を認めることから、予後の指標となる可能性も併せ持つ既存の膵癌検出用腫瘍マーカーにはない特徴を有する。本発明の実施には少量の患者血清で十分であり、また−３０℃にて保存し、いつでも測定可能である。従って、プロテインチップ法とそのチップに用いること、より好ましくはＷＣＸ２（陽イオン交換チップ）を用いれば、陽性率も高く、早期の病期Ｉでも陽性で膵癌を検出可能である。なお膵癌検出用腫瘍マーカーとしてＡｐｏＣ−１を検出する時の好ましい条件は、ｐＨ３以上７以下であり、より好ましくはｐＨ６以上７以下である。   As described above, this tumor marker for detecting pancreatic cancer can realize a tumor marker that has a higher positive rate of early pancreatic cancer and more reliably detects pancreatic cancer, and a pancreatic cancer detection kit using the same. In addition, the value of this tumor marker is related to the patient's survival time, and is closely related to the malignancy of pancreatic cancer. Have A small amount of patient serum is sufficient for the practice of the present invention and can be stored at -30 ° C and measured at any time. Therefore, when the protein chip method and its chip are used, and more preferably WCX2 (cation exchange chip) is used, the positive rate is high, and pancreatic cancer can be detected positively even in the early stage I. A preferable condition for detecting ApoC-1 as a tumor marker for detecting pancreatic cancer is pH 3 or more and 7 or less, more preferably pH 6 or more and 7 or less.

なお、健常人の血清中にもＡｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｃ−１は存在しており、現在まででは脂質代謝関与し、トリグリセリド転送リポ蛋白であるカイロミクロンやVLDLに多く認められ、肝臓の粗面小胞体内で合成され、血中平均濃度は約６mg/dlといわれている。ＡｐｏＣ−１の脂質関連の機能は他のアポリポ蛋白に比べ未解明の部分が非常に多く、（１）ApoE蛋白の形態を変化させLDL receptor(LDLR)やLDLR related proteinとリポ蛋白の結合を阻害すること、（２）LCAT活性を促進させる働きをもつこと等が示唆されている。（The interaction of human apolipoprotein C−I
with sub−micellar phospholipid. Benjamin W.
Atcliffe et al. Eur. J. Biochem. 2001;268: 2838−2846.）Apolipoprotein C-1 is also present in the serum of healthy individuals, and until now, it is involved in lipid metabolism and is frequently found in chylomicron and VLDL, which are triglyceride transfer lipoproteins, in the rough endoplasmic reticulum of the liver. It is synthesized and the average blood concentration is said to be about 6 mg / dl. ApoC-1 has many lipid-related functions that are still unclear compared to other apolipoproteins. (1) Alters the form of ApoE protein and inhibits the binding of LDL receptor (LDLR) and LDLR related protein to lipoproteins. (2) It has been suggested that it has a function of promoting LCAT activity. (The interaction of human apolipoprotein C-I
with sub−micellar phospholipid. Benjamin W.
(Atcliffe et al. Eur. J. Biochem. 2001; 268: 2838-2846.)

最近、ApoC−1遺伝子の癌への関連が大規模な遺伝子クラスタ−解析により１００種近くの候補の１つとしてあげられ、特に膵癌組織で発現が増強されるとの報告があり（Byungwoo et al. Cancer Research ２００２年、６１巻、１８３３〜１８３８頁）、他の臓器での正常部、癌部での遺伝子発現の相違についてNational Cancer Instituteのデ−タ−ベ−ス内 (http://cgap.nci.nih.gov/SAGE/Viewer)
に登録されている。これらのことからもＡｐｏＣ−１は膵癌検出用腫瘍マーカーとなりうる可能性が非常に高く、さらに、膵癌患者血清中でのＡｐｏＣ−１値増加の原因として膵癌組織中での発現増強が遺伝子レベルに留まらず、蛋白レベルでも同様に認められればさらに膵癌特異的なマーカーとしての信憑性が揺るぎないものとなる。そこで、本発明者は実際にＡｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｃ−１が膵癌の組織中で発現されているか否かを確認するために手術標本より得られた膵癌の癌部、非癌部の凍結標本を用いて膵癌組織及び正常膵組織のＡｐｏＣ−１遺伝子発現をRT−PCR、Real−time RT−PCRならびにＡｐｏＣ−１蛋白発現をWestern blot法、さらに今回プロテインチップ解析を行った全２０症例のホルマリン固定されたパラフィン包埋標本を用いて免疫染色法でＡｐｏＣ−１蛋白の腫瘍内局在を確認した。Recently, the association of the ApoC-1 gene with cancer has been listed as one of nearly 100 candidates by large-scale gene cluster analysis, and it has been reported that its expression is particularly enhanced in pancreatic cancer tissues (Byungwoo et al. Cancer Research 2002, 61, 1833-1838), within the National Cancer Institute database of differences in gene expression in normal and cancerous parts of other organs (http: // cgap .nci.nih.gov / SAGE / Viewer)
It is registered in. From these facts, ApoC-1 is very likely to be a tumor marker for detection of pancreatic cancer. Furthermore, as a cause of an increase in ApoC-1 value in serum of pancreatic cancer patients, enhanced expression in pancreatic cancer tissue is at the gene level. If it is also observed at the protein level, the credibility as a marker specific to pancreatic cancer will not be shaken. Therefore, the present inventor used pancreatic cancer using frozen specimens of cancerous and non-cancerous parts of pancreatic cancer obtained from surgical specimens to confirm whether Apolipoprotein C-1 is actually expressed in the tissue of pancreatic cancer. ApoC-1 gene expression in tissues and normal pancreatic tissues was RT-PCR, Real-time RT-PCR and ApoC-1 protein expression was Western blotted, and all 20 cases were subjected to protein chip analysis in formalin-fixed paraffin. Using the embedded specimen, the localization of ApoC-1 protein in the tumor was confirmed by immunostaining.

まず、膵癌組織、正常膵組織および膵癌細胞株のＡｐｏＣ−１遺伝子発現を確認するためにRT−PCRを行った。まず、凍結標本組織を粉砕しRNeasy Mini
Kit (キアゲン, Tokyo,
Japan)を用いtotal RNAを作成、その後T−Primed
First−Strand Kit
for reverse transcription−PCR (アマシャムバイオサイエンス, USA社)を使用しcDNAを作成した。さらにcDNAをAmpliTaq
Gold PCR Master Mix (バイオシステム, Foster City, CA社)を用い、３７サイクル、アニ−リング温度５８℃の条件下でRT−PCRを行った。得られたPCR産物を２％アガロ−スゲルにて電気泳動をおこなった。ＡｐｏＣ−１primerとしてForward−primer, 5’−CTCCAGTGCCTTGGATAAGC
−3’
Reverse−primer, 5’−TTGAGTTTCTCCTTCACTTTCTGA
−3’の配列を用い、ＡｐｏＣ−１PCR産物の長さは150bpとなるようデザインした。その結果、癌部では非癌部より明らかにＡｐｏＣ−１遺伝子が強発現していた。その結果を図１１に示す。First, RT-PCR was performed to confirm ApoC-1 gene expression in pancreatic cancer tissues, normal pancreatic tissues, and pancreatic cancer cell lines. First, the frozen specimen tissue is crushed and RNeasy Mini
Kit (Qiagen, Tokyo,
Japan) to make total RNA, then T-Primed
First-Strand Kit
cDNA was prepared using for reverse transcription-PCR (Amersham Bioscience, USA). In addition, the AmpliTaq cDNA
RT-PCR was performed using Gold PCR Master Mix (Biosystem, Foster City, CA) under conditions of 37 cycles and an annealing temperature of 58 ° C. The obtained PCR product was electrophoresed on a 2% agarose gel. Forward-primer, 5'-CTCCAGTGCCTTGGATAAGC as ApoC-1primer
−3 '
Reverse-primer, 5'-TTGAGTTTCTCCTTCACTTTCTGA
Using the −3 ′ sequence, the length of the ApoC-1 PCR product was designed to be 150 bp. As a result, the ApoC-1 gene was clearly expressed more strongly in the cancerous part than in the non-cancerous part. The result is shown in FIG.

次に手術で切除された膵癌の癌部、非癌部各１６検体の凍結標本を用いて上記と同様の過程でそれぞれcDNAを作成。その後、ＡｐｏＣ−１のmRNAレベルを定量化するためにLightCycler 装置とLightCycler−Fast Start DNA Master SYBR Green I kit and PCR amplifications (ロッシュダイアグノスティック, Tokyo, Japan社)を用いReal−time RT−PCRを行った。組織間のmRNA量を同一にするためにそれぞれのGAPDHのmRNA値比を算出した。その結果、すべての検体において癌部では非癌部よりＡｐｏＣ−１遺伝子の発現が増強しており（p＞０．０００１）（図１２参照）、同一個体内での癌部、非癌部１３対においては癌部では非癌部より平均値２５２倍、中央値３９．１倍の発現量の差を認めた。   Next, cDNA was prepared in the same process as above using frozen specimens of 16 specimens of cancerous and non-cancerous pancreatic cancers resected by surgery. Then, to quantify the ApoC-1 mRNA level, Real-time RT-PCR was performed using the LightCycler instrument and LightCycler-Fast Start DNA Master SYBR Green I kit and PCR amplifications (Roche Diagnostics, Tokyo, Japan). went. In order to make the amount of mRNA between tissues the same, the mRNA value ratio of each GAPDH was calculated. As a result, in all the specimens, the expression of ApoC-1 gene was enhanced in the cancerous part compared to the non-cancerous part (p> 0.0001) (see FIG. 12), and the cancerous part and non-cancerous part 13 in the same individual. In the pair, the difference in the expression level was found in the cancerous part with an average value 252 times and a median value of 39.1 times in the cancerous part.

さらに、上記の検体のうち癌部、非癌部が同一個体からの対となる１３ペアを癌部のmRNA値(Tumor)を非癌部のＡｐｏＣ−１mRNA値(Normal)でわり、その値の中央値で高値群、低値群の２群に分け膵癌の悪性度との相関関係を検討したところ、高値群では低値群に比べ有意に生存期間が短く、前述のプロテインチップシステムによる血清中ＡｐｏＣ−１と膵癌患者の生存期間の関係と相関する結果となった（図１３参照）。ちなみにこの１３対の患者はプロテインチップシステム解析に用いた患者グル−プとは異なる群である。   Furthermore, among the above specimens, the cancer part and the non-cancer part are paired from the same individual, and the cancer part mRNA value (Tumor) is replaced with the non-cancer part ApoC-1 mRNA value (Normal). We examined the correlation with malignancy of pancreatic cancer by dividing the median into two groups, the high group and the low group. As a result, the high group had a significantly shorter survival time than the low group. The results correlated with the relationship between ApoC-1 and the survival time of pancreatic cancer patients (see FIG. 13). Incidentally, these 13 pairs of patients are a different group from the patient group used for the protein chip system analysis.

続いて、膵癌組織の癌部、非癌部および４種類の膵癌細胞株（MIA
PaCaII、PanC−1、CFPAC−1、AsPC−1）のＡｐｏＣ−１蛋白レベルを確認するためにＷｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇを行った。１次抗体としてMouse−ａｎｔｉ−ｈｕｍａｎ−Apolipoprotein C−1 monoｃｌｏｎａｌ ａｎｔｉｂｏｄｙ（CHEMICON INTERNATIONAL社）を用いた。コントロ−ルとしてｇｏａｔ ａｎｔｉ−β−ａｃｔｉｎ ａｎｔｉｂｏｄｙ（Ｓａｎｔａ Ｃｒｕｚ社）を用いた。Subsequently, cancerous and non-cancerous parts of pancreatic cancer tissue and four types of pancreatic cancer cell lines (MIA)
Western blotting was performed to confirm the ApoC-1 protein levels of PaCaII, PanC-1, CFPAC-1, AsPC-1). Mouse-anti-human-Apolipoprotein C-1 monoclonal antibody (CHEMICON INTERNATIONAL) was used as the primary antibody. As a control, goat anti-β-actin antibody (Santa Cruz) was used.

膵癌、膵正常組織および培養細胞より蛋白を抽出した後、それぞれ２０μｇの蛋白量をアプライし１０〜２０％、および１５−２５％グラディエントゲル（DRC社）を用いＳＤＳ−ＰＡＧＥを行った。その後ＰＶＤＦ膜（Ｍｉｌｌｉｐｏｒｅ社）に転写し、０.５％スキムミルクにて非特異的反応をブロッキングした後１：５００に希釈した抗ApoC−1抗体を１次抗体として用いた。２次抗体として１：３０００に希釈したＧｏａｔ−ａｎｔｉMouse−ＩｇＧ ａｎｔｉｂｏｄｙ（ＨＲＰ）（Ｂｉｏ−Ｒａｄ社）を使用した。発色液としてＥＣＬ液（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社）を使用した。その結果、ＡｐｏＣ−１蛋白発現は膵正常組織においては認められず、膵癌組織で発現しており、さらに４つの膵癌細胞株すべてにおいて発現を確認した（図１４参照）。   After extracting proteins from pancreatic cancer, normal pancreatic tissue and cultured cells, 20 μg of protein was applied, and SDS-PAGE was performed using 10-20% and 15-25% gradient gels (DRC). Thereafter, the resultant was transferred to a PVDF membrane (Millipore), non-specific reaction was blocked with 0.5% skim milk, and then diluted to 1: 500, an anti-ApoC-1 antibody was used as a primary antibody. As a secondary antibody, Goat-antiMouse-IgG antibody (HRP) (Bio-Rad) diluted 1: 3000 was used. An ECL solution (Amersham Pharmacia Biotech) was used as a color developing solution. As a result, ApoC-1 protein expression was not observed in pancreatic normal tissues, but was expressed in pancreatic cancer tissues. Furthermore, expression was confirmed in all four pancreatic cancer cell lines (see FIG. 14).

次に、今回プロテインチップ解析を行った全２０症例を対象とし、免疫組織学的染色を行い、ＡｐｏＣ−１蛋白発現の膵癌組織中の発現及び局在を確認した。免疫染色は１０％ホルマリン固定され、パラフィン包理された組織標本を用い、ストレプトアビジン−ビオチンペルオキシダ−ゼ法（Ｄａｋｏ ＬＳＡＢ２ ｋｉｔ， Ｄａｋｏ Ｊａｐａｎ社）にて行った。その際、抗ＡｐｏＣ−１抗体の希釈濃度は１：１００で行った。その結果、非癌部特に正常膵管においてＡｐｏＣ−１蛋白は発現を認めず、癌部においては膵管癌の細胞質に特異的に発現していることが確認された（図１５参照）。   Next, immunohistological staining was performed on all 20 cases subjected to protein chip analysis this time, and the expression and localization of ApoC-1 protein expression in pancreatic cancer tissues were confirmed. Immunostaining was performed by a streptavidin-biotin peroxidase method (Dako LSAB2 kit, Dako Japan) using a tissue specimen fixed with 10% formalin and embedded in paraffin. At that time, the dilution concentration of the anti-ApoC-1 antibody was 1: 100. As a result, it was confirmed that ApoC-1 protein was not expressed in the non-cancerous part, particularly in the normal pancreatic duct, and was specifically expressed in the cytoplasm of pancreatic ductal cancer in the cancerous part (see FIG. 15).

以上より、本研究者は初めて膵癌組織中及び膵癌細胞株のＡｐｏＣ−１遺伝子および蛋白発現を実証することに成功し、さらに膵癌組織におけるＡｐｏＣ−１蛋白発現の局在も明らかとした。これらの研究結果はＡｐｏＣ−１蛋白が膵臓の癌化と密接に関係し、発現量の増大に伴い血中に逸脱し、その血清中の濃度を測定することにより鋭敏なかつ確実な膵癌の診断に大きく寄与できることとなる。さらに、膵癌の悪性度の指標としてもＡｐｏＣ−１蛋白を活用することができる。   Based on the above, the present investigator succeeded in demonstrating the expression of ApoC-1 gene and protein in pancreatic cancer tissues and pancreatic cancer cell lines for the first time, and further revealed the localization of ApoC-1 protein expression in pancreatic cancer tissues. The results of these studies indicate that ApoC-1 protein is closely related to pancreatic canceration, deviates into the blood as the expression level increases, and is sensitive and reliable for diagnosis of pancreatic cancer by measuring its serum concentration. It can greatly contribute. Furthermore, ApoC-1 protein can also be used as an index of malignancy of pancreatic cancer.

次に、実際にＡｐｏＣ−１蛋白が膵癌の癌化作用にどのように携わっているかを解明するために４種類の膵癌細胞株（MIA PaCaII、PanC−1、CFPAC−1、AsPC−1）で、RNAi(RNA interference)を用いＡｐｏＣ−１遺伝子を特異的に抑制するsiRNAを細胞株にtransfectionし検討した。ＡｐｏＣ−１遺伝子が真に抑制されているかを確認するために、Real−time RT−PCRを用いＡｐｏＣ−１RNAレベルを定量化し、ＡｐｏＣ−１蛋白レベルをWestern blotにて検討した。   Next, four types of pancreatic cancer cell lines (MIA PaCaII, PanC-1, CFPAC-1, AsPC-1) were used to elucidate how ApoC-1 protein is actually involved in the carcinogenic action of pancreatic cancer. Then, siRNA that specifically suppresses the ApoC-1 gene using RNAi (RNA interference) was transfected into a cell line and examined. In order to confirm whether the ApoC-1 gene was truly suppressed, Real-time RT-PCR was used to quantify the ApoC-1 RNA level, and the ApoC-1 protein level was examined by Western blot.

まず、RNAiを用いたＡｐｏＣ−１遺伝子の特異的な抑制を起こすためのsiRNAを２種類デザインし、より効果的にＡｐｏＣ−１遺伝子を抑制することのできるsiRNAを選択した。このOligonucleotideの配列は以下に示す通りである、Target sequence, siRNA−2; CTG GAG GAC AAG GCT CGG
GAA。First, two types of siRNA for causing specific suppression of the ApoC-1 gene using RNAi were designed, and siRNA capable of suppressing the ApoC-1 gene more effectively was selected. The sequence of this Oligonucleotide is as shown below, Target sequence, siRNA-2; CTG GAG GAC AAG GCT CGG
GAA.

ここで、Real−time RT−PCRおよびWestern blot法は前述と同様の過程でおこなった。癌細胞で特異的に発現が認められるＡｐｏＣ−１にどのような癌化への作用が認められるかを検討する。RNAiを用いることにより特異的にＡｐｏＣ−１mRNAの発現を抑制することで、膵癌細胞株におけるＡｐｏＣ−１の機能解析を行った。   Here, Real-time RT-PCR and Western blot method were performed in the same process as described above. We examine what effect on canceration is observed in ApoC-1, which is specifically expressed in cancer cells. Functional analysis of ApoC-1 in pancreatic cancer cell lines was performed by specifically suppressing the expression of ApoC-1 mRNA by using RNAi.

まず、１０％FBS入りの培養液で１０x１０4 cells/ml の細胞浮遊液を作る。６well plateに２ml（２０x１０4cells/well）ずつ細胞を撒き、顕微鏡で確認、翌日５０％confluentを目標にする。３７℃、５％CO2インキュベ−タ−にて２４時間培養する。
続いて、２４時間後以下の手順でsiRNA Transfectionを行う。
1. Oligoを解凍
2. Transfection
Reagentの調整
Lipofectamine2000（Invitrogen
life
technologies社）6μlをOpti−MEM medium(GIBCO, Invitrogen Corporation) 244μlで希釈し、室温で５分から１０分放置
3. Oligo
dilutionの調整
20nMのannilingしたOligo 10μl＜(200nM
final conc.の場合)、濃度により適宜調整する＞をOpti−MEM medium 240μlで希釈する。
4. Transfection
Reagent 250μlをoligo dilutionに加え、軽く混合しtotal 500μlとする。室温で１５分から２０分放置。
5. Opti−MEMを５0０μl加えtotal 1000μlにする。
6. plate培養液を吸引し、Opti−MEM medium 2mlで洗浄。
7. ５のTransfection
mixtureを細胞にふりかける。
8. 37℃、５％CO2インキュベ−タ−で４時間培養後、FBS1５０μl、DMEM350μl加え、total 1500μl(10%血清)とする。
その後適宜、細胞からcell lysateを用いmRNA、proteinを得、その後の実験に使用する。First, a cell suspension of 10 × 10 4 cells / ml is prepared with a culture solution containing 10% FBS. Seed 2 ml (20x10 4 cells / well) each in a 6-well plate, confirmed with a microscope, and targeted 50% confluent the next day. Incubate for 24 hours at 37 ° C., 5% CO 2 incubator.
Subsequently, siRNA transfection is performed 24 hours later according to the following procedure.
1. Unzip Oligo
2. Transfection
Reagent adjustment
Lipofectamine2000 (Invitrogen
life
technologies) 6μl diluted with 244μl Opti-MEM medium (GIBCO, Invitrogen Corporation) and left at room temperature for 5-10 minutes
3. Oligo
Adjustment of dilution
20nM anniling Oligo 10μl <(200nM
In the case of final conc.), adjust appropriately according to the concentration> is diluted with 240 μl of Opti-MEM medium.
4. Transfection
Add 250 μl of Reagent to the oligo dilution and mix gently to make a total of 500 μl. Leave at room temperature for 15-20 minutes.
5. Add 500 μl of Opti-MEM to a total of 1000 μl.
6. Aspirate the plate culture and wash with 2 ml of Opti-MEM medium.
7. Transfection of 5
Sprinkle the mixture onto the cells.
8. After culturing at 37 ° C in a 5% CO2 incubator for 4 hours, add 150 µl of FBS and 350 µl of DMEM to make a total of 1500 µl (10% serum).
Thereafter, mRNA and protein are obtained from the cells using cell lysate as appropriate, and used in subsequent experiments.

膵癌細胞株のＡｐｏＣ−１遺伝子がＡｐｏＣ−１siRNAにより特異的に抑制されているかを確認するために、上記の手順で行って得られたcDNAを用いReal−time RT−PCRを行った。その結果、４種類の膵癌細胞株すべてにおいて９０％前後の遺伝子抑制が認められた。さらにＡｐｏＣ−１蛋白レベルをWestern blotにて検討したところ、MIA PaCa IIにＡｐｏＣ−１siRNAをtransfectionし４８時間後の蛋白レベルは、siRNA oligo最終濃度２０ｎＭより発現が低下していることが確認できた（図１６参照）。   In order to confirm whether the ApoC-1 gene of the pancreatic cancer cell line was specifically suppressed by ApoC-1 siRNA, Real-time RT-PCR was performed using the cDNA obtained by the above procedure. As a result, gene suppression of about 90% was observed in all four types of pancreatic cancer cell lines. Furthermore, when the ApoC-1 protein level was examined by Western blot, it was confirmed that the expression of the protein level 48 hours after transfection of ApoC-1 siRNA into MIA PaCa II was lower than the final siRNA oligo concentration of 20 nM. (See FIG. 16).

ＡｐｏＣ−１の膵癌細胞株における細胞増殖への関わりを調べる目的でCell
proliferation assayを行った。９６ｗｅｌｌ ｍｉｃｒｏｐｌａｔｅに０．５ｘ１０４個/１ｗｅｌｌの細胞を入れ、２４時間３７℃、５％ＣＯ２条件下でインキュベ−トし、その後特異的ＡｐｏＣ−１siRNAを最終濃度２０nMとなるように、transfection用培養液に混入し、４時間後培養液を交換し、同条件下でインキュベ−トし細胞培養を行い、２４、４８、７２、９６、１２０、１４４時間後にＣｅｌｌ Counting Ｋｉｔ−８ ［CCK−８］ （ DOJINDO ,JAPAN社）を用いCell proliferation assayを行った。吸光度４５０ｎｍにて測定し、培養細胞に特異的ＡｐｏＣ−１siRNAを投与した細胞とコントロ−ルsiRNAを投与した細胞との増殖能を比較したところ特異的ＡｐｏＣ−１siRNAを投与した細胞の方がコントロ−ルの細胞より細胞増殖能が有意に抑制されるという結果を得た（図１７参照）。ネガティブコントロ−ルとしてnon−silencing siRNA (Luciferase;GL2)（キアゲン、Ｊａｐａｎ社）を使用した。Cell for the purpose of investigating the involvement of ApoC-1 in cell proliferation in pancreatic cancer cell lines
A proliferation assay was performed. Place 0.5 × 10 4 cells / well in 96-well microplate, incubate for 24 hours at 37 ° C. under 5% CO 2 , and then culture for transfection so that specific ApoC-1 siRNA has a final concentration of 20 nM. 4 hours later, the culture medium was changed, and the cells were incubated under the same conditions for cell culture. After 24, 48, 72, 96, 120, and 144 hours, Cell Counting Kit-8 [CCK-8] Cell proliferation assay was performed using DOJINDO (Japan). Absorbance was measured at 450 nm, and the proliferation ability was compared between the cells administered with specific ApoC-1 siRNA and the cells administered with control siRNA. The cells administered with specific ApoC-1 siRNA were more controlled. As a result, the cell proliferation ability was significantly suppressed compared to the cells in the cell (see FIG. 17). Non-silencing siRNA (Luciferase; GL2) (Qiagen, Japan) was used as a negative control.

プロテインチップシステムを用いた膵癌術前術後血清の典型的なprotein peak（質量６０００〜７０００Da）を示す図。上が術前、下が術後の結果を示す。The figure which shows the typical protein peak (mass 6000-7000 Da) of the blood serum before and after pancreatic cancer surgery using a protein chip system. Top shows pre-operative results and bottom shows post-operative results. 質量数６６３０Ｄａの蛋白の術前術後における発現を検討した結果を示す図。The figure which shows the result of having examined the expression before and after the operation of the protein of mass number 6630Da. プロテインチップシステムを用いた解析において、ｐＨを変化させた場合における結果を示す図。The figure which shows the result in the case of changing pH in the analysis using a protein chip system. プロテインチップシステムを用いた解析において、ＮａＣｌ濃度を０ｍＭ〜５００ｍＭまで段階的に変化させた場合における結果を示す図。The figure which shows the result in the case of changing NaCl concentration in steps from 0 mM to 500 mM in the analysis using a protein chip system. ＨＰＬＣのｐｅａｋを示す図。The figure which shows the peak of HPLC. ｐｅａｋの高いＨＰＬＣ ｆｒａｃｔｉｏｎに対しＳＥＬＤＩ−ＴＯＦ−ＭＳを用いて解析を行った結果を示す図。The figure which shows the result of having analyzed using SELDI-TOF-MS with respect to HPLC fraction with high peak. ２回目のＨＰＬＣのｐｅａｋを示す図。The figure which shows the peak of HPLC of the 2nd time. ｐｅａｋの高いＨＰＬＣ ｆｒａｃｔｉｏｎに対しＳＥＬＤＩ−ＴＯＦ−ＭＳを用いて解析を行った結果を示す図。The figure which shows the result of having analyzed using SELDI-TOF-MS with respect to HPLC fraction with high peak. Ｎ末端アミノ酸配列の分析結果を示す図。The figure which shows the analysis result of an N terminal amino acid sequence. プロテインチップシステム解析で得られた術前血清におけるＡｐｏＣ−１蛋白のpeakintensity中央値で分けた２群間をＫａｐｌａｎ−Ｍｅｉｅｒ法により無再発生存期間及び生存期間の解析を行った図。The figure which analyzed the recurrence-free survival period and survival period by Kaplan-Meier method between two groups divided by the median peak intensity of ApoC-1 protein in the preoperative serum obtained by protein chip system analysis. 膵癌組織、正常膵組織および膵癌細胞株のＡｐｏＣ−１遺伝子発現をRT−PCRで検討した図。The figure which examined the ApoC-1 gene expression of a pancreatic cancer tissue, a normal pancreatic tissue, and a pancreatic cancer cell line by RT-PCR. 膵癌の癌部、非癌部各１６検体の凍結標本を用いてReal−time RT−PCRを行い、ＡｐｏＣ−１mRNAを定量化した図。The figure which quantified ApoC-1 mRNA by performing Real-time RT-PCR using the frozen specimen of each 16 specimens of the cancer part of pancreatic cancer, and a non-cancer part. ＡｐｏＣ−１mRNA値の中央値で２群間に分けたときの悪性度との相関を示す図。The figure which shows the correlation with a malignancy when dividing between two groups by the median value of ApoC-1 mRNA value. ＡｐｏＣ−１蛋白発現を膵正常および癌組織、４つの膵癌細胞株においてWesternblot法にて確認した図。The figure which confirmed ApoC-1 protein expression by Westernblot method in a pancreatic normal and cancer tissue, and four pancreatic cancer cell lines. 、免疫組織学的染色を行い、ＡｐｏＣ−１蛋白発現の膵癌組織中の発現及び局在を確認した図。The figure which performed the immunohistological dyeing | staining and confirmed the expression and localization in the pancreatic cancer tissue of ApoC-1 protein expression. 膵癌細胞株のＡｐｏＣ−１遺伝子をＡｐｏＣ−１siRNAにより特異的に抑制し、それをReal−time RT−PCRとWestern blot法を用いて確認した図。The figure which confirmed the ApoC-1 gene of a pancreatic cancer cell line specifically by ApoC-1 siRNA, and confirmed it using Real-time RT-PCR and Western blot method. ＡｐｏＣ−１の膵癌細胞株における細胞増殖への関わりをCell proliferationassayにて検討した図。The figure which examined the relation to cell proliferation in the pancreatic cancer cell line of ApoC-1 by Cell proliferationassay.

Claims (3)

ＡｐｏＣ−１蛋白からなる膵癌検出用腫瘍マーカー。   A tumor marker for detection of pancreatic cancer comprising ApoC-1 protein. ＡｐｏＣ−１蛋白を至適条件下で検出するためのプロテインチップを含む膵癌検出用キット。   A pancreatic cancer detection kit comprising a protein chip for detecting ApoC-1 protein under optimal conditions. 前記プロテインチップは、請求項２記載の膵癌検出用キット。
The kit for detecting pancreatic cancer according to claim 2, wherein the protein chip is used.
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