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Method for promoting epidermal cell proliferation

A kind of epidermal cells, low expression technology, applied in the field of stem cells and genetic engineering

Active Publication Date: 2014-12-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no literature report on the research and application of JAM1 gene modification of epidermal cells to promote their rapid proliferation

Method used

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  • Method for promoting epidermal cell proliferation
  • Method for promoting epidermal cell proliferation
  • Method for promoting epidermal cell proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Construction of the lentiviral overexpression plasmid pGC-FU-JAM1-GFP to infect human epidermal cells (hMSC).

[0078] 1. Separation and culture of epidermal cells

[0079] Adult body skin comes from Plastic Surgery of Changhai Hospital. Epidermal cells are obtained by primary culture.

[0080] 2. Preparation of total RNA of human epidermal cells

[0081] Using the total RNA extraction kit from Shanghai Huashun Biological Engineering Co., Ltd., the total RNA of human epidermal cells was extracted according to the conventional guanidine isothiocyanate method. Methods as below:

[0082] Take a monolayer of epidermal cells grown in a culture dish with a diameter of 3.5 cm, discard the medium directly, and add 1 ml of TRIzol to dissolve the cells. After the cells are fully dissolved, remove the cell lysate with a pipette. Incubate the cell lysate sample at 15~30°C for 5 minutes to completely decompose the ribosome. Add 0.2ml of chloroform per 1ml of TRIzol, close the c...

Embodiment 2

[0143] Example 2: Cell experiment (in vitro experiment)

[0144] Use fluorescence microscope to take pictures, draw growth curves, immunocytochemistry, western-blot and other biological experiment methods to analyze cell morphology changes, target gene expression and cell surface marker keratin expression from various aspects such as cell morphology and protein expression.

[0145] The specific method is as follows:

[0146] 1) Comparison of cell growth status

[0147] Observation of JAM1 infected by lentivirus with an inverted fluorescence microscope ov -EC, JAM1 kd -EC and GFP-EC. No obvious changes in cell morphology, such as figure 1 Shown.

[0148] 2) Draw a growth curve to detect cell proliferation

[0149] JAM1 ov -EC, JAM1 kd -EC and GFP-EC were seeded on a 24-well plate with 10,000 cells per well, cultured for 7 days, trypsinized into a single cell suspension every day, and carried out cell counting experiments with the help of a cell counting plate and a microscope, each ty...

Embodiment 3

[0191] Example 3: In vivo tumorigenicity test

[0192] The nude mouse BALB / c Nu strain used in the experiment, SPF grade. Weighing about 15~25g, 3 weeks old, purchased from Shanghai Experimental Animal Center. A total of 12 nude mice, divided into 4 groups: JAM1 ov -EC injection group, GFP-EC injection group, JAM1 kd -EC injection group, PBS injection group. Cell volume is 10 4 Use a 1ml syringe to draw 0.15ml of cell suspension and inject it under the skin of the back of the forelimb of nude mice. They were fed under SPF-level conditions, observed daily, and harvested 4 weeks after transplantation.

[0193] Animals in each group showed no difference in appearance and activity after experimental treatment. There was no difference in H-E staining, liver, spleen, kidney and other organs among nude mice in each group after sacrifice.

[0194] H-E staining was performed on the skin tissues of the injection sites of nude mice in each group. Observation under the microscope revealed t...

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Abstract

The invention relates to the technical fields of stem cells and gene engineering, and particularly provides a method for promoting epidermal cell proliferation based on gene modification and gene interference with epidermal cells by using a junctional adhesion molecule JAM1 (NM-016946) which is a transmembrane molecule and belongs to the immunoglobulin superfamily. The invention further provides a low expression epidermal cell of the junctional adhesion molecule JAM1 gene, i.e. JAM1kd-EC, and the proliferation ability of the epidermal cell, the properties and the biological safety of the epidermal cell and the like are detected. The invention further provides a method for promoting epidermal cell proliferation and an application of JAM1kd-EC in wound repair or preparation of skin grafts.

Description

Technical field [0001] The invention relates to the technical field of stem cells and genetic engineering, in particular to a method for promoting the proliferation of epidermal cells, and application of the method to the preparation of skin grafts. Background technique [0002] Cells have a certain lifespan, and their ability to divide is also limited. As the number of cell divisions increases, cell proliferation ability gradually weakens, and eventually stops proliferating. This is also the process of cells from stem cells to terminally differentiated cells after a brief expansion. This process is affected by many factors, and some factors can slow down or even reverse this process, allowing cells to regain the ability to differentiate and proliferate. [0003] At present, the strategy to make cells regain the ability of differentiation and proliferation is nuclear transplantation (Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH.. Viable offspring derived from fetal and ad...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/10C12N15/867A61L27/38A61L27/60
Inventor 刘厚奇仵敏娟周童郭晓灿
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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