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Application of TIMP-1 (tissue inhibitor of metalloproteinase-1) gene in preparation of product for treating diabetic skin ulcers

A TIMP-1, 1.TIMP-1 technology, applied in the field of application of TIMP-1 gene in the preparation of skin wound products for the treatment of diabetes, to achieve the effect of accelerated healing

Inactive Publication Date: 2015-03-25
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TIMP-1 encoded by tissue inhibitor of metalloproteinase-1 (tissue inhibitor of metalloproteinase-1, TIMP-1) gene (Genbank No.NC_000023.11) has attracted everyone's attention because of its extensive inhibitory effect on metalloproteinase activity, but There is no report that it acts on skin fibroblasts as a protein factor to play an anti-apoptotic role and promote the healing of chronic diabetic skin ulcers

Method used

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  • Application of TIMP-1 (tissue inhibitor of metalloproteinase-1) gene in preparation of product for treating diabetic skin ulcers
  • Application of TIMP-1 (tissue inhibitor of metalloproteinase-1) gene in preparation of product for treating diabetic skin ulcers
  • Application of TIMP-1 (tissue inhibitor of metalloproteinase-1) gene in preparation of product for treating diabetic skin ulcers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Detection of wound skin cell apoptosis and TIMP-1 expression in diabetic patients

[0054] Experimental objects and materials: skin samples

[0055] Diabetic foot group: from June 2011 to April 2013, 18 patients with type 2 diabetes hospitalized in our department due to diabetic foot, with an average age of 62±13.14 years; non-diabetic group: from June 2011 to April 2013 18 non-diabetic patients who were hospitalized for foot trauma in the orthopedics department of our hospital every month, with an average age of 55±15.45 years. The samples were obtained with the informed consent of the patients. All skin specimens were taken after local infection control, 0.5 cm from the edge of the foot wound. Cut the skin specimens and fix them with 4% paraformaldehyde solution to make paraffin sections with a thickness of 5 μm;

[0056] (1) Detection of apoptosis in wound skin cells of diabetic patients

[0057] ①Activated Caspase-3 (Cleaved Caspase-3) immunohistochemi...

Embodiment 2

[0070] Example 2 Effects of Glycosylation End Products (AGEs) on Human Skin Fibroblast Apoptosis and TIMP-1 Expression

[0071] (1) Cell source Surgical specimens after circumcision of normal children in the Department of Urology or Pediatric Surgery of our hospital were collected with the consent of the patient's family members. The experimental object is the 4th to 6th passage cells;

[0072] Human skin fibroblast culture: obtain human skin fibroblasts through primary culture, press 1×10 4 The density per well was inoculated into a 6-well plate, the cells grew to 60% confluence in the logarithmic growth phase, and the normal sugar medium with a volume fraction of 0.5% fetal bovine serum was replaced for 24 hours to make the cells enter a synchronized state; the cells were divided into 2 groups: one group is a normal control group (containing NG 5.6mmo / L, NG here refers to normal concentration of glucose, all groups of the following cell cultures contain NG, because NG is a ...

Embodiment 3

[0095] Example 3 Effect of exogenous rhTIMP-1 on AGEs-induced apoptosis of human skin fibroblasts

[0096] (1) Cell source: same as Example 2;

[0097] (2) Cell apoptosis detection by flow cytometry:

[0098] Human skin fibroblast culture: obtain human skin fibroblasts through primary culture, press 1×10 4 Inoculate into 6-well plate at the density of / well; the cells grow to 60% confluence in the logarithmic growth phase, replace the normal sugar medium with a volume fraction of 0.5% fetal bovine serum and starve for 24h, so that the cells enter a synchronized state; the cells are divided into 3 groups: normal control group, AGEs group and AGEs+rhTIMP-1 group, the culture medium of the above-mentioned different treatments was replaced according to the experimental groups, wherein, the culture medium of the normal control group contained NG 5.6mmo / L; the culture medium of the AGEs group contained NG 5.6mmo / L+AGEs 150ug / mL; AGEs+rhTIMP-1 group medium contained NG 5.6mmo / L+AGE...

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Abstract

The invention belongs to the technical field of genes, and particularly relates to an application of a TIMP-1 (tissue inhibitor of metalloproteinase-1) gene in preparation of a product for treating diabetic skin ulcers. The application comprises the following steps: building a recombinant expression vector with the TIMP-1 gene with the TIMP-1 gene or an adenovirus vector or a lentiviral vector; co-transfecting HEK293 cells with the recombinant expression vector with the TIMP-1 gene and adenovirus vector shuttle plasmids, and obtaining a recombinant adenovirus with the TIMP-1 gene; and transfecting the recombinant adenovirus with the TIMP-1 gene into skin cells of diabetes patients or animal models; and improving the TIMP-1 expression level. The application proves that the TIMP-1 is capable of inhibiting excessive fibroblast apoptosis caused by high glucose, in skin of the diabetes patients, thus healing of the diabetic skin ulcers is accelerated; and a basis is provided for inventing the TIMP-1 gene as a new therapeutic target and medicine for preventing and treating chronic diabetic foot ulcers.

Description

technical field [0001] The invention belongs to the field of gene technology, and in particular relates to the application of TIMP-1 gene in the preparation of skin wound products for treating diabetes. Background technique [0002] With the change of people's lifestyle and the extension of life expectancy, the incidence of diabetes has increased dramatically. Diabetes has become a major cause of chronic skin wounds in our country. Diabetic foot ulcer is the main manifestation of diabetic foot and an important cause of amputation in diabetic patients. The skin of diabetic patients is easily damaged, and once a foot ulcer occurs, it is very difficult to treat and heal. Moreover, the treatment cycle is long and the medical expenses are high, which seriously affects the quality of life of the patient. Therefore, the research on the prevention and treatment of diabetic skin lesions is one of the important clinical topics to be discussed urgently. [0003] Dysregulation of bio...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N15/74C12N15/66A61P3/10A61P17/02
Inventor 劳国娟严励任萌罗恒聪梁颖杨川
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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