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Protein and pharmaceutical composition for treating ischemic diseases

An ischemic disease and protein-binding technology, which is applied in the field of proteins and pharmaceutical compositions for the treatment of ischemic diseases, and can solve the problems of a small number of organ donations, a limited number of mature cardiomyocytes, and inability to repair diseased myocardium.

Inactive Publication Date: 2016-12-14
同济大学附属东方医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At the present stage, heart transplantation is the main treatment for these patients. However, the number of organ donations is extremely small and is also subject to ethical restrictions. A promising alternative to heart transplantation is stem cell therapy, which has emerged in recent years. Replacing necrotic cardiomyocytes to achieve the purpose of cell regeneration, the existing experimental results show that transplanted cells, including bone marrow stem cells and embryonic stem cells, can develop into mature cardiomyocytes in the myocardium and form intercalated disc connections with host cells, preventing ventricular expansion and improving Cardiac function, but the number of mature cardiomyocytes developed from stem cells is limited, and cannot fundamentally play a role in repairing diseased myocardium
[0004] There is currently no effective drug for treating ischemic diseases in this field, so there is an urgent need to develop new drugs that have excellent effects, no toxic side effects, and can replace organ transplantation and stem cells

Method used

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  • Protein and pharmaceutical composition for treating ischemic diseases
  • Protein and pharmaceutical composition for treating ischemic diseases
  • Protein and pharmaceutical composition for treating ischemic diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Preparation of IGFBP-4 protein

[0086] 1. Cloning the IGFBP-4 gene:

[0087] cDNA was obtained by reverse transcription of human mRNA extracted by conventional methods. Using the cDNA as a template, using primers corresponding to both sides of the ORF frame of the IGFBP-4 gene (SEQ ID NO.: 1), the amplification product of human IGFBP-4 was obtained by RT-PCR amplification, and the length of the product was consistent with the predicted value .

[0088] 2. Construction of IGFBP-4 eukaryotic expression vector:

[0089] The PCR amplification product obtained above was subcloned by TA into the pcDNA3.1-TOPO-6XHis eukaryotic cell expression vector (purchased from Invitrogen).

[0090] 3. Transfection:

[0091] The host cells were transfected into HEK293 cells by conventional methods, and cultured under the condition of 500 μg / ml G418 to obtain multiple growing HEK293 cell lines, namely transformants.

[0092] Select the transformant with high expression level and culti...

Embodiment 2

[0096] Preparation of Animal Lower Limb Ischemia-Ischemia Model

[0097] 1. Model Establishment and Specimen Collection

[0098] Ten 8-week-old male C57BL / 6 mice (The Jackson Laboratory, USA) were randomly divided into an experimental group and a control group, with 5 mice in each group. Directly inject IGFBP-4 (5μg / 50μl) into the ischemic lower limbs at several points, and inject the same volume of normal saline to the mice in the control group. Laser Doppler imaging shows ischemic parts in blue and bloody parts in red.

[0099] 2. When the mice were 12 weeks old, the left femoral artery was ligated and resected to establish the mouse lower limb ischemia model. Before operation, immediately after operation, and 7, 14, 21, 28 days after operation, the blood perfusion of lower limbs of mice was monitored by laser Doppler ultrasound scanner.

Embodiment 3

[0101] Animal lower limb ischemia model treated with drugs

[0102] After the mouse lower limb ischemia model was established (embodiment 2), the blood perfusion situation of the mouse lower limbs was monitored with a laser Doppler ultrasonic scanner; the mouse tail vein was utilized to inject recombinant IGFBP-4 protein (5 μg / 25g) or Placebo (PBS) was used as a control; after 6 days, the blood perfusion of lower limbs of mice was also monitored by laser Doppler ultrasound scanner.

[0103] Observation and evaluation of therapeutic effects: the results showed that the lower limb ischemia of mice in the one-time injection of recombinant IGFBP-4 protein group was significantly improved, Figure 4 shows the results of IGFBP-4 protein for animal treatment, wherein, Figure 4 A is the direct observation result of anatomy; Figure 4 B is the infrared image of blood vessels on day 0 after treatment; Figure 4 C is the vascular infrared imaging image 6 days after treatment, the lef...

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Abstract

The invention relates to a protein and a pharmaceutical composition for treating ischemic diseases. Specifically, it is found by the inventor that insulin-like growth factor binding protein, especially insulin like growth factor binding protein-4 (IGFBP-4) has an excellent effect on treating ischemic diseases. IGFBP-4 protein can promote vascular proliferation and the differentiation of stem cells into myocardial cells. IGFBP-4 protein also has good therapeutic and preventive effects on ischemic diseases, especially on myocardial ischemia and lower limb ischemia and has no toxic or side-effect.

Description

technical field [0001] The invention relates to the fields of bioengineering and biomedicine, in particular, the invention relates to proteins and pharmaceutical compositions for treating ischemic diseases. Background technique [0002] Ischemic diseases have become the main cause of human death, and the number of patients with myocardial ischemia and lower limb ischemia is increasing. At present, for every 3 deaths in the world, 1 person dies from myocardial ischemia diseases. In cardiovascular diseases, various reasons can lead to the death of heart cells, mainly because one of the biggest characteristics of mature cardiomyocytes is that these cells lose the ability to divide and proliferate shortly after birth. In this way, the dead cells are eventually replaced by non-functional scar tissue, which eventually leads to heart failure. The prognosis of such patients with extremely low cardiac function is also extremely poor. [0003] At the present stage, heart transplant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/30A61K48/00A61P9/10C12N15/85C12N5/078
Inventor 朱伟东
Owner 同济大学附属东方医院
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