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Method for promoting epidermal cell proliferation

A technology of epidermal cells and epidermal stem cells, applied in applications, animal cells, vertebrate cells, etc.

Active Publication Date: 2013-07-10
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: The lentiviral overexpression plasmid pGC-FU-JAM1-GFP was constructed to infect human epidermal cells (hMSC).

[0078] 1. Isolation and culture of epidermal cells

[0079] Adult human skin was obtained from the Plastic Surgery Department of Changhai Hospital. Epidermal cells were obtained from primary culture.

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

[0081] The total RNA of human epidermal cells was extracted by the conventional guanidine isothiocyanate method with the total RNA extraction kit of Shanghai Huashun Bioengineering Co., Ltd. Methods as below:

[0082]Take a 3.5cm-diameter dish of epidermal cells growing in a single layer, discard the medium directly, add 1ml of TRIzol to dissolve the cells, and remove the cell lysate with a pipette after the cells are fully dissolved. Incubate the cell lysate sample at 15-30°C for 5 minutes to completely decompose the ribosomes. Add 0.2ml chloroform per 1ml TRIzol, close the cap of t...

Embodiment 2

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

[0144] Using fluorescent microscope to take photos, draw growth curves, immunocytochemistry, western-blot and other biological experimental methods, 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] Visualization of Lentivirally Infected JAM1 Using an Inverted Fluorescence Microscopy ov -EC,JAM1 kd -EC and GFP-EC. Cell morphology did not change significantly, 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 in 24-well plates with 10,000 cells per well, cultured for 7 days, and digested with trypsin every day to form a single-cell suspension. Cells were replicated in triplicate wells, and the experiment was r...

Embodiment 3

[0191] Example 3: In vivo tumorigenicity test

[0192] Nude mice BALB / c Nu strain, SPF grade, were used in the experiment. Weight about 15 ~ 25g, 3 weeks old, purchased from Shanghai Experimental Animal Center. A total of 12 nude mice were divided into 4 groups: JAM1 ov -EC injection group, GFP-EC injection group, JAM1 kd - EC injection group, PBS injection group. 10 cells 4 0.15ml of the cell suspension was extracted with a 1ml syringe and injected subcutaneously into the back of the forelimb of the nude mouse. They were fed under SPF conditions, observed daily, and samples were collected 4 weeks after transplantation.

[0193] Animals in each group had no difference in appearance, activity, etc. after the experimental treatment. Compared with nude mice in each group after sacrifice, there was no difference in H-E staining, liver, spleen, kidney and other organs.

[0194] H-E staining was done on the skin tissue of the injection site of the nude mice in each group, and...

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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 its application to the preparation of skin grafts. Background technique [0002] Cells have a certain lifespan, and the ability of cells to divide is also limited. With the increase of the number of cell divisions, the ability of cell proliferation gradually weakens, and finally stops proliferation. This is also the process by which cells go from stem cells through transient expansion cells to terminally differentiated cells. 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 differentiation and proliferation ability is nuclear transfer (Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH..Viable offspring derive...

Claims

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Application Information

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Patent Type & Authority Applications(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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