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Use of human midkine protein blocking peptide in preparation of antitumor medicament

An anti-tumor drug and midkine technology, which is applied in the fields of molecular biology and biochemistry of biotechnology, can solve the problems of liver and kidney function damage, poor specificity of tumor cells, etc., achieves significant clinical significance, and inhibits tumor angiogenesis. , the effect of inhibiting the growth of malignant tumor cells

Inactive Publication Date: 2008-09-03
HUZHOU CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current traditional tumor chemotherapy drugs include alkylating agents, anti-metabolite drugs, anti-tumor antibiotics, platinum-based anti-tumor drugs, etc. The mechanism of action is mostly to inhibit the synthesis of DNA, RNA and protein of cells, and the specificity of the effect on tumor cells is poor. Therefore, it often has relatively large side effects, such as bone marrow suppression, liver and kidney damage

Method used

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  • Use of human midkine protein blocking peptide in preparation of antitumor medicament
  • Use of human midkine protein blocking peptide in preparation of antitumor medicament
  • Use of human midkine protein blocking peptide in preparation of antitumor medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Screening of Human Midkine Protein Blocking Peptides

[0028] (1) Expression and purification of human midkine protein: soluble expression and purification of human midkine protein according to the human midkine mRNA sequence.

[0029] (2) Screening of phage polypeptide library and identification and sequence analysis of positive clones Screening of phage 7-peptide library and loop 7-peptide library (solid coating method):

[0030] The MK recombinant protein was used to coat the plate with a coating amount of 15 μg / well, and the plate was washed after being blocked with the blocking solution. Add 10 μl of the original library / well (100 μl diluted with 0.1% TBST), shake gently at room temperature for 10-60 minutes, wash the plate, and elute the bound phage with 100 μl of acidic eluent [0.2M glycine-hydrochloric acid (pH2.2), 1mg / ml BSA] After centrifugation, the eluate was neutralized with 150 μl of 1M Tris-HCl (PH 9.1). Collect the eluate, take a small amou...

Embodiment 2

[0056] Example 2: MTT method detects the inhibitory effect of blocking peptide on the growth of human HepG2 liver cancer cells and human umbilical vein endothelial cells (HUVEC)

[0057] Experimental group:

[0058] In the experiment, the screened polypeptide MK-P3 acted on HepG2 and HUVEC cells, each of which was divided into three concentration gradient groups of 15 μmol / L, 50 μmol / L and 150 μmol / L.

[0059] MTT detection of cell proliferation:

[0060] Human liver cancer cell line (HepG2) and human umbilical vein endothelial cells (HUVEC) were provided by the Institute of Infectious Diseases, Zhejiang University, with DMEM medium (Gibco company), set at 37°C, 5% CO 2 cultured in an incubator. Cells in the logarithmic growth phase were inoculated into a 96-well plate, 200 μl / well, 3×10 3 cells / well, 3 wells per group, repeated 3 times, 37°C, 5% CO 2 Cultivate in an incubator. After the cells are 60-80% fused, replace the serum-free medium and culture for 24 hours. Then ...

Embodiment 3

[0064] Example 3: Preparation of BALB / c mouse xenograft tumor model, analysis of anti-angiogenesis and anti-tumor activity of MK blocking peptide in mice

[0065] Animal model establishment

[0066] H22 cells were provided by the Institute of Infectious Diseases, Zhejiang University; BALB / c mice, 18-20g, male and female, were provided by Shanghai Bikai Experimental Center. The H22 cells were injected into the peritoneal cavity of BALB / c mice, and the ascites was collected around 10-14 days to collect tumor cells. Use 1640 medium to adjust the concentration of cell suspension to 5×10 6 / ml, subcutaneously take a little seeding cell suspension 0.1ml from the back of each BALB / c mouse, and observe the tumor formation on the back of the mouse and whether there is redness, swelling and ulceration on the surface.

[0067] Experimental grouping and processing:

[0068] 96 BALB / c mice were randomly divided into 8 groups, 12 in each group:

[0069] (1) MK-P3 group: intraperitoneal ...

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Abstract

The invention relates to an application of human midkine protein blocking peptide for preparing antineoplastic medicine, wherein the human midkine protein blocking peptide comprises the following amino acid sequence: CTSTAMDNC. The invention provides an application of human midkine protein blocking peptide for preparing antineoplastic medicine, the polypeptide can inhibit the growth of malignant tumor cells, particularly for inhibiting the generation of malignant tumor neovascularization, provides foundation for screening new medicines, and has great clinical significance.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biochemistry of biotechnology, and specifically relates to the application of a human midkine protein blocking peptide in the preparation of antitumor drugs. Background technique [0002] Malignant tumor is an important disease that seriously endangers human health. The prevention and treatment of tumor is a global problem, which has attracted extensive attention from all over the world. The development of practical and effective anti-tumor drugs is a major issue to be solved urgently. [0003] The current traditional tumor chemotherapy drugs include alkylating agents, anti-metabolite drugs, anti-tumor antibiotics, platinum-based anti-tumor drugs, etc. The mechanism of action is mostly to inhibit the synthesis of DNA, RNA and protein of cells, and the specificity of the effect on tumor cells is poor. Therefore, it often has greater side effects, such as bone marrow suppression, liver and kid...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K38/08A61P35/00
Inventor 戴利成沈建芬
Owner HUZHOU CENT HOSPITAL
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