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Skin repair related miRNA marker and application thereof

A skin repair and marker technology, applied in the field of kits and miRNA detection reagents, to achieve the effect of great practical significance and practical value, high accuracy and high degree of automation

Pending Publication Date: 2022-08-09
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So as to provide solutions for solving problems related to skin repair, skin wound healing process monitoring, related drug development, skin inflammation and apoptosis diagnosis in this field

Method used

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  • Skin repair related miRNA marker and application thereof
  • Skin repair related miRNA marker and application thereof
  • Skin repair related miRNA marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Skin injury modeling and skin collection

[0080] Nine adult female mice with similar body weights were selected and divided into 3 groups. Using a hole puncher to create wounds on the back skin of the mice respectively, the skin was collected as a 0-day sample. Then, the wound edge skins of the three groups of mice were collected on the 7th day, 14th day and 21st day of wound modeling respectively. After the skin was collected, it was quick-frozen in liquid nitrogen and transferred to -80°C for storage until RNA extraction.

Embodiment 2

[0081] Example 2: Extraction of total RNA from skin

[0082] Take 50-100 mg of skin samples, add 6 metal balls with a diameter of 2 mm and 2 metal balls with a diameter of 5 mm and 1 mL of Trizol reagent from Invitrogen Company, fasten the cap of the centrifuge tube, and use the Jingxin tissue grinder to grind the sample at 70 Hz and 90 s. Cryogenic grinding; after placing the ground sample tubes in a refrigerator at 4°C for 5 min, add 200 μL of chloroform to each tube, fasten the cap of the centrifuge tube, shake by hand, place in a refrigerator at 4°C for 5 min, and centrifuge at 12000 rpm for 15 min at 4°C The liquid in the tube is divided into 3 clear layers; prepare a new 1.5 mL centrifuge tube without nuclease and number it, add 500 μL of isopropanol to each tube, take out the centrifuged specimen, and carefully pipette 500 μL of the upper transparent liquid to the corresponding numbered tube. In the centrifuge tube, fasten the cap of the centrifuge tube, vortex for 5s, ...

Embodiment 3

[0083] Example 3: High-throughput sequencing of miRNAs

[0084] The miRNA library was constructed and sequenced, followed by the identification of differentially expressed genes, and the P value was adjusted using the Benjamini & Hochberg method. By default, a corrected P-value of 0.05 or 0.01 was set as the threshold for significant differential expression. On the target gene candidates of differentially expressed miRNAs, Gene Ontology (GO) enrichment analysis was used. GOseq-based Wallenius non-central hypergeometric distribution can adjust gene length bias for GO enrichment analysis. Potential functions of predicted miRNAs by KEEG pathway enrichment. Statistical enrichment of target gene candidates in the KEGG pathway was tested using KOBAS software. The candidate miRNAs related to the wound repair process were determined, and the final screening found that miRNAs miR-7a-5p, miR-335-5p and miR-501-3p played a key role in the wound repair process.

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Abstract

The invention belongs to the technical field of biology, particularly discloses a miRNA marker related to skin repair and application of the miRNA marker, and develops a two-step detection reagent, a kit and a detection method for tissue miRNA based on a real-time quantitative PCR (polymerase chain reaction) technology. The miRNA provided by the invention comprises one or more of miR-7a-5p, miR-335-5p and miR-501-3p, and the miRNA comprises one or more of miR-7a-5p, miR-335- The kit contains a probe combination for detecting the miRNA, and the miRNA can be used as a marker for skin repair process research and skin modification related drug screening, and can also be used as a marker for detecting skin inflammation and cell apoptosis. The detection kit and the detection method provided by the invention have the advantages of small sample size, high automation degree and good specificity, can significantly improve the detection efficiency in combination with other detection indexes and clinical symptoms, and are convenient for batch screening and clinical detection use of skin trauma repair drugs.

Description

technical field [0001] The present invention belongs to the field of biotechnology, in particular, the present invention provides miRNA markers related to skin repair, their application in skin repair process detection, related drug screening, and related miRNA detection reagents and kits. Background technique [0002] The skin is composed of four parts: epidermis, dermis, subcutaneous tissue and subcutaneous appendages. It is a natural barrier to protect the human body from harmful external factors, and it is also the first line of defense for the human body. The weight of the skin reaches 5% to 15% of the body weight of the human body, and the entire area of ​​the skin can reach 1.5 to 2 square meters. In daily life, skin trauma, skin diseases, large-area burns, chronic ulcers, or skin defects from surgical trauma often lead to suppuration, pain, swelling and pain, etc., which affect the patient's work and quality of life. In severe cases, it can lead to sepsis, Tetanus a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6806C12Q1/6851C12N15/113C12N15/11
CPCC12Q1/6883C12Q1/6806C12Q1/6851C12N15/113C12Q2600/136C12Q2600/158C12Q2600/166C12Q2600/178C12N2310/141C12Q2527/125C12Q2531/113C12Q2563/107C12Q2525/207C12Q2525/307C12Q2545/113C12Q2561/101C12Q2521/107
Inventor 曹宇浩宋雪梅竺亚斌赵枫庞倩
Owner NINGBO UNIV
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