CN1224630C - Targetted polypeptide for specificity of liver cancer blood vessel - Google Patents
Targetted polypeptide for specificity of liver cancer blood vessel Download PDFInfo
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- CN1224630C CN1224630C CN 200310122611 CN200310122611A CN1224630C CN 1224630 C CN1224630 C CN 1224630C CN 200310122611 CN200310122611 CN 200310122611 CN 200310122611 A CN200310122611 A CN 200310122611A CN 1224630 C CN1224630 C CN 1224630C
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Abstract
The present invention relates to the technical field of protein polypeptide biology, particularly to polypeptide for specificity combination of liver cancer blood vessel. Targeting polypeptide LCI-X7 peptide for specificity combination with endothelial cells of liver cancer blood vessel is screened out from a high diversion human liver cancer nude mouse model by adopting a display technique in a phage random peptide library body and a laser micro slitting technique by the present invention. The present invention can be specially combined on a liver tumor in situ of an orthotopic implantation human liver cancer nude mouse and on a tumor vessel of metastasis through the identification of experiments of the inside and the outside of animals. The present invention can be specially combined with most of human body liver cancer tumor vessels, and provides an important theory and practice foundation for diagnosis of liver cancer diversion relapse, targeted therapy, etc. The present invention has wide application prospects.
Description
Technical field
The invention belongs to biological technical field, relate to protein and peptide, be specifically related to a kind of can with liver cancer tumor vessel specificity bonded polypeptide.
Background technology
China is global liver cancer district occurred frequently, and according to statistics, annual about 200,000 people die from liver cancer, account for about half of global PLC mortality number.Modern medicine carried out scientific research over one hundred year to liver cancer, though obtained many achievements, but liver cancer crowd's overall 5 years survival rates still have only about 3% so far, and the biggest obstacle that improves prognosis in hcc is its high invasion and attack and rate of transform, even the radical surgery excision still has 50% patient can occur shifting in 5 years or recurrence.Therefore, the inherent mechanism of further investigation hepatoma Metastasis recurrence and corresponding therapeutic intervention thereof have become the further key of improving prognosis in hcc.There are some researches show that each link that tumor vessel is formed on liver cancer growth, transfer, recurrence all produces vital role.In recent years, though on the tumor vessel therapeutic intervention, get along with, but still lack special and effective treatment means.Up to now, can specificity at home and abroad not appear in the newspapers at the tumor vascular targeted molecular of liver cancer.
Summary of the invention
The purpose of this invention is to provide a kind of protein and peptide, particularly can be special at the tumor vascular target polypeptide of liver cancer.
The present invention adopt in the phage random peptide library body display technique and laser capture microdissection cutting technique on the high-transfer human liver cancer nude mice model, screen can with the target polypeptide of liver cancer tumor vascular endothelial cell specific combination, obtain 7 high peptides of specificity, has sequence " LPNISKP ", called after " LCI-X
7Peptide ", in body, reach experiment in vitro and confirm described 7 peptides and liver cancer tumor vascular endothelium bonded specificity height.
Experiment and tissue sample experiment in vitro are identified and are confirmed LCI-X of the present invention in original position plantation people liver cancer nude mouse
7Peptide has following characteristics:
1.LCI-X
7Peptide can combine with the liver tumor in situ blood vessel high special that original position is planted people's liver cancer nude mice, and tumor vessels such as mammary cancer, malignant melanoma, lung cancer are not had the specificity keying action;
2.LCI-X
7Peptide can be planted the outer metastasis tumor vessel specific combination of liver of people's liver cancer nude mice with original position, thereby has realized the spike to the hepatoma Metastasis kitchen range;
3.LCI-X
7Peptide can combine with the tumor vessel specificity in most of clinical patient liver cancer tissue, is presented at human hepatocellular tumor vessel surface and also has LCI-X
7The homology acceptor of peptide.
The result shows, LCI-X of the present invention
7But the peptide specificity is incorporated on the tumor vessel of the liver tumor in situ of original position plantation people liver cancer nude mice and metastasis, and can with most of human body liver cancer tumor vessel specific combination, LCI-X
7Peptide has application prospect extensively for hepatoma Metastasis relapse diagnosis, targeted therapy etc. provide important theory and practical basis.
Description of drawings
Fig. 1 is LCI-X
7Peptide binding specificity qualification result
A wherein: the anti-CD31 immunofluorescence dyeing of nude mice original position liver cancer, green fluorescence is the liver in situ cancerous tissue, red fluorescence is the tumor vessel that anti-D31 dyeing shows;
B: the LCI-X of yellow fluorescence mark
7The peptide specific combination is in liver in situ tumor blood vessel;
C:LCI-X
7The peptide specific combination is in the tumor vessel of liver cancer lung metastasis;
D:LCI-X
7The peptide specific combination is in people's liver cancer tumor vessel, fluorescent microscope, 200 *.
Embodiment
Embodiment 1
The present invention obtains LCI-X by following method and step screening
7Peptide.
1. set up people's liver cancer original position plantation nude mice model:
Adopt high-transfer human liver cancer cell line MHCC97-H (World J Gastroernterol2001,7:630-636) 1 * 10
7Become knurl in male BALB/c nu/nu nude mice (available from medicine institute of the Chinese Academy of Sciences) subcutaneous injection, take out subcutaneous tumors behind subcutaneous tumors length to 1～1.5cm, cut into 1mm * 1mm * 1mm, original position is planted in the nude mice liver, treats that tumor growth is to the 1cm;
2. screening in the body of random phage library:
Mouse tail vein injects f1 phage 10
12Pfu, inject phage peptide library behind the 5min, open chest behind the 5min, live body heart perfusion 20ml PBS wherein contains heparin anti-coagulating, and portal vein and infrahepatic vena cava intubate are replenished perfusion 20ml PBS, results tumor tissues and control tissue, bonded phage and measure titre, the phage that recovers in amplification, the purifying tumor tissues in the tissue that homogenate and repetitive scrubbing take off, recovery tissue, repetition obtains 100 phage clones that the left and right sides is special with screening step 10 time in the upper body.
Random phage of the present invention 7 peptide storehouses are available from PhD7, NEB company, and the f1 phage is available from Shanghai cell institute of the Chinese Academy of Sciences.
3, fusion gene order-checking:
Increase the respectively phage clone of above-mentioned acquisition, the extracting phage DNA, order-checking (Shanghai Shen You company) obtains 7 peptide sequences of showing in the phage, wherein LCI-X
7Peptide (sequence " LPNISKP ") occurrence frequency is 96%.
4, the synthetic and mark of artificial polypeptide:
The LCI-X that the synthetic occurrence frequency is the highest
7Peptide and control peptide thereof are at aminoterminal mark DNS yellow fluorescence.
Described synthetic LCI-X
7Peptide is given birth to worker company by Shanghai, and press currently known methods synthetic, and described control peptide is identical but 7 peptides that make up at random of amino acid composition.
5. specificity is identified:
1) combination experiment in the body:
With above-mentioned " LCI-X
7Phage " (show LCI-X
7Phage) and LCI-X
7Peptide inject respectively in the people's liver cancer original position plantation nude mouse and or contrast nude mices such as lotus mammary cancer, lung cancer, malignant melanoma, heart lavation behind the 3min, results tumor tissues and control tissue, fluorescent microscope is observed DNS yellow fluorescence distribution situation and phage-resistance immunohistochemical methods evaluation binding specificity down after the frozen section.The result shows: LCI-X
7But the peptide specificity is incorporated on the tumor vessel of the liver tumor in situ of original position plantation people liver cancer nude mice and metastasis, and tumor vessels such as mammary cancer, malignant melanoma and lung cancer are not had the specificity keying action;
2) external and body is interior in conjunction with suppressing experiment:
With LCI-X
7Phage and LCI-X
7Peptide is injected in the tumor bearing nude mice body successively, and whether observe both has competitive inhibitory effect.The result shows, LCI-X
7Phage and LCI-X
7Peptide can be competed in conjunction with the binding site on the liver cancer tumor vessel, and it has high degree of specificity with tumor vascular combination of liver cancer;
3) people's liver cancer tumor vessel is in conjunction with experiment:
Get the other normal liver tissue frozen section of people's liver cancer tumor tissues and cancer, phage-resistance immunohistochemical methods or LCI-X
7The peptide immunofluorescence experiment is identified its binding specificity.The result shows: LCI-X
7Peptide can with most people's liver cancer tumor vessel specific combination, combination rate reaches 85%, does not have the specificity keying action with the other normal liver tissue of cancer.
Table 1 is different tumour nude mice model " LCI-X
7Peptide " in conjunction with identifying (immunofluorescence dyeing) result.Table 2 is clinical patient liver cancer tissue sample " LCI-X
7Peptide " in conjunction with identifying (immunofluorescence dyeing) result.
Table 1
| Tumour | Hepatocellular carcinoma | Malignant melanoma | Adenocarcinoma of lung | Mammary cancer | Adenocarcinoma of colon | |||||
| Clone | D35 | D20 | SMMC 7721 | MHCC 97-H | MHCC 97-L | LM6 | A375 | A549 | Bcap -37 | Ls-17 4-T |
| Nude mice is counted positive rate | 8 8/8 | 8 7/8 | 8 6/8 | 8 8/8 | 8 7/8 | 8 8/8 | 8 0/8 | 8 0/8 | 8 0/8 | 8 3/8 |
Table 2
| Tissue slice No positive rate | Liver cancer 40 82.5% | The other liver 40 22.5% of cancer |
Claims (6)
1, the selectively targeted polypeptide of a kind of liver cancer tumor vessel is characterized in that being made up of amino acid " LPNISKP ", can with liver cancer tumor vascular endothelial cell specific combination.
2,, it is characterized in that described polypeptide is at aminoterminal mark DNS yellow fluorescence according to the selectively targeted polypeptide of liver cancer tumor vessel of claim 1.
3,, it is characterized in that described polypeptide does not have the specificity combination to mammary cancer, malignant melanoma and lung cancer tumor vessel according to the selectively targeted polypeptide of liver cancer tumor vessel of claim 1.
4,, it is characterized in that the other normal liver tissue of described polypeptide and people's liver cancer does not have specificity and combines according to the selectively targeted polypeptide of liver cancer tumor vessel of claim 1.
5, the purposes of the selectively targeted polypeptide of liver cancer tumor vessel of claim 1 in preparation diagnosing liver cancer transfer and relapse spike preparation.
6, the purposes of the selectively targeted polypeptide of liver cancer tumor vessel of claim 1 in preparation targeted therapy liver-cancer medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310122611 CN1224630C (en) | 2003-12-19 | 2003-12-19 | Targetted polypeptide for specificity of liver cancer blood vessel |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310122611 CN1224630C (en) | 2003-12-19 | 2003-12-19 | Targetted polypeptide for specificity of liver cancer blood vessel |
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| CN1552727A CN1552727A (en) | 2004-12-08 |
| CN1224630C true CN1224630C (en) | 2005-10-26 |
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Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113164B (en) * | 2007-07-12 | 2010-11-24 | 南开大学 | 12 peptide specifically combined with tumor metastasis related protein PRL-3 and uses thereof |
| CN101768210B (en) * | 2007-10-29 | 2012-02-29 | 昆明医学院第一附属医院 | Tumor targeting polypeptide and preparation method thereof |
| CN101143895B (en) * | 2007-10-29 | 2010-05-26 | 昆明医学院第一附属医院 | Polypeptide with tumour targeting effects and preparation method thereof |
| CN102060909B (en) * | 2009-11-11 | 2013-01-02 | 中国医学科学院放射医学研究所 | Tumor specific target polypeptide and application thereof |
| CN102807603A (en) * | 2011-06-03 | 2012-12-05 | 首都医科大学 | Aliphatic amine chain modified anti-tumor oligopeptide and synthetic method and application thereof |
| CN102432671A (en) * | 2011-11-30 | 2012-05-02 | 复旦大学附属中山医院 | Targeting polypeptide SPSCVLP capable of inhibiting growth and transfer of liver cancer and application thereof |
| CN103450340B (en) * | 2012-05-29 | 2015-08-05 | 首都医科大学 | The antineoplastic oligopeptide that heterocyclic carboxylic acid is modified, its synthesis, antitumor action and application |
| CN105218628A (en) * | 2014-06-10 | 2016-01-06 | 首都医科大学 | The β-carboline that tryptophan benzyl ester is modified, its synthesis, nanostructure, active and application |
| CN105218636A (en) * | 2014-06-11 | 2016-01-06 | 首都医科大学 | The β-carboline that LPNISKP modifies, its preparation, nanostructure, active and application |
| CN105218637A (en) * | 2014-06-11 | 2016-01-06 | 首都医科大学 | The indoles quinolizine that LPNISKP modifies, its preparation, nanostructure, active and application |
| CN105294836A (en) * | 2014-06-11 | 2016-02-03 | 首都医科大学 | LPNISKP modified 5-fluorouracil, preparation therefor, nanostructure thereof, activity thereof and application thereof |
| CN105039333B (en) * | 2015-08-21 | 2018-07-24 | 天津医科大学 | Hepatoma-targeting peptide and its application |
| JP6895460B2 (en) | 2016-06-03 | 2021-06-30 | アイメッド・バイオ・インコーポレイテッド | Antibody screening method using patient-derived tumor spheroids |
| CN107488214B (en) * | 2016-06-13 | 2021-03-30 | 首都医科大学 | warfarin-4-O-acetyl-LPNISKP and synthesis, activity and application thereof |
| CN107057683B (en) * | 2016-12-28 | 2019-04-12 | 浙江大学 | A kind of preparation method of fibroin fluorescence probe |
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