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Liver cancer targeted peptide and application thereof

A technology of use and selection, applied in the direction of peptides, medical preparations with non-active ingredients, radioactive carriers, etc., can solve the problems of large differences in clinical applications, inability to effectively simulate the tumor growth environment of patients with liver cancer, etc., and achieve specific liver cancer targeting. Effect

Active Publication Date: 2015-11-11
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The currently published research on liver cancer targeting peptide technology is mainly aimed at a single liver cancer cell. For example, the patent CN101918433B of the Academia Sinica discloses a peptide that specifically binds to HCC cells; the patent CN201110451767.2 of Shaanxi Normal University discloses a specific HepG2 cell line-binding peptides; some are targeting peptides targeting liver cancer tumor blood vessels. For example, the patent CN1224630C of Zhongshan Hospital Affiliated to Fudan University discloses the use of phage random peptide library in vivo display technology and laser microdissection technology in highly metastatic human The target peptides that can specifically bind to liver cancer tumor vascular endothelial cells are screened on the nude mouse model of liver cancer; in the actual screening process of target peptides, some receptor cells are synthesized in vitro or liver cancer target peptides are screened using tumors in vivo in model mice. It cannot effectively simulate the tumor growth environment in patients with liver cancer, and the difference from clinical application will be relatively large

Method used

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  • Liver cancer targeted peptide and application thereof
  • Liver cancer targeted peptide and application thereof
  • Liver cancer targeted peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the preparation method of polypeptide

[0040] The polypeptides HCC-1 (SEQ ID NO: 8), HCC-5 (SEQ ID NO: 10), HCC-20 (SEQ ID NO: 12), HCC-46 (SEQ ID NO: 2), HCC-47 (SEQ ID NO: 4), HCC-48 (SEQ ID NO: 6) was produced by Chinapeptide Company. The peptide synthesis steps are as follows:

[0041] (1) Resin swelling. Put 2-ChlorotritylChlorideResin into the reaction tube, add dichloromethane DCM (15ml / g), shake for 30min;

[0042] (2) Filter out the solution through a sand core, add Fmoc-Leu-OH amino acid with a molar excess of 3 times, add dimethylformamide DMF to dissolve, then add DIEA with a molar excess of 10 times, shake for 60 minutes, and seal with methanol;

[0043] (3) Deprotection. Remove DMF, add 20% piperidine DMF solution (15ml / g), 5min, remove and add 20% piperidine DMF solution (15ml / g), 15min;

[0044] (4) Detection. Take out the piperidine solution, take more than a dozen resins, wash them three times with ethanol, add detection reagents f...

Embodiment 2

[0055] Example 2: Liver biopsy slice culture and activity identification method

[0056] 1. Preparation and culture of live liver slices

[0057] 1) C57BL / 6 mice aged 6-8 weeks were killed by decapitation, and the complete liver lobe was taken out, immediately placed in 4°C pre-cooled and pre-oxygenated KHB buffer, and the liver lobe was fixed with tissue adhesive. Fixed on the sample holder glued to the agar sheet.

[0058] 2) The biotome cuts liver slices with a thickness of 250 μm. In order to better ensure the integrity of the tissue and reduce the damage to the liver tissue caused by cutting, the slice is performed at a speed not higher than 0.05mm / s, and the blade amplitude is 1.75mm. The vibration frequency is 75Hz.

[0059] 3) After rinsing the collected slices in DPBS with 1% P / S for 3 times, place them on a culture plate with liver biopsy medium, and place them in a culture plate containing 5% CO 2 cultured in a constant temperature incubator at 37°C.

[0060] 2....

Embodiment 3

[0090] Embodiment 3: the screening method of polypeptide

[0091] Utilizing the liver slice culture and activity identification method established in Example 2, fresh tumor specimens from patients with clinical hepatocellular carcinoma were taken for biopsy, and the liver cancer tumor tissue was cut into 250 μm thick slices, cultured in an incubator for 1 hour, and then phage Biopanning, after 4 rounds of screening, the final round of recovered products were collected for next-generation sequencing, and the sequence with the largest number of clones was selected as a candidate liver cancer tissue-targeting peptide. The specific process is as follows:

[0092] 1) The tissue specimens of the clinical liver cancer patients just removed were sliced ​​in vivo according to the above method and cultured, and the titer was 1×10 11 The pfu of phage was mixed with the culture medium, added to the culture plate, and incubated with the live slices for 2 hours.

[0093] 2) Aspirate the c...

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Abstract

The invention discloses a targeted polypeptide capable of being specifically combined with tumors, particularly a targeted peptide capable of being specifically combined with liver cancer tissues and application thereof in diagnosis and treatment of liver cancer. The liver cancer targeted peptide is preferably HCC-47 of which the amino acid sequence is SQDIRTWNGTRS; and the liver cancer targeted peptide is specifically combined with the liver cancer tissues, and can not be specifically combined with cervical carcinoma cells Hela, mammary cancer cells MDA-MB231, kidney cancer cells CRL-1932 and lung cancer cells A549. The polypeptide is obtained by in-vitro biological elutriation by combining a bacteriophage display library and a living body cross sectioning technique. The polypeptide can be used in a molecular imaging preparation for early diagnosis of liver cancer. The polypeptide can also be used in targeted modification and preparation of drugs for treating liver cancer. The polypeptide can also be used for targeted modification on drug transport carriers, thereby providing a new way for diagnosing or treating patients with liver cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a targeting polypeptide, especially a targeting peptide capable of specifically binding to liver cancer tissue and its application in the diagnosis and treatment of liver cancer. Background technique [0002] Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of the common malignant tumors in my country, with a high mortality rate. The mortality rate in malignant tumors is second only to gastric cancer and esophageal cancer, ranking third. Among all treatment measures, surgical treatment is still the first choice for long-term survival. After decades of hard work, the 10-year survival rate after liver cancer surgery has increased by nearly 6 times, but the 5-year survival rate still does not exceed 5%. Traditional chemotherapeutic drugs have very limited efficacy in HCC and may have serious side effects. Therefore, the targeted delivery of therapeutic drugs for liver c...

Claims

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Application Information

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IPC IPC(8): C12N15/11C07K7/08A61K47/48A61K9/127A61K51/08A61P35/00A61K103/34
Inventor 尹海芳高先军赵静雯荆韧威左冰峰
Owner TIANJIN MEDICAL UNIV
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