Method of modifying mesenchymal stem cell by JAM1 gene and application thereof

A technology of mesenchymal stem cells and gene modification, applied in the field of stem cells and genetic engineering

Inactive Publication Date: 2013-04-10
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

[0009] At present, there is no literature report about JAM1 gene modification of mesenchymal stem cells and its application in promoting local skin and hair regeneration

Method used

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  • Method of modifying mesenchymal stem cell by JAM1 gene and application thereof
  • Method of modifying mesenchymal stem cell by JAM1 gene and application thereof
  • Method of modifying mesenchymal stem cell by JAM1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: The lentiviral overexpression plasmid pGC-FU-JAM1-GFP was constructed to infect human embryo-derived mesenchymal stem cells (hMSC).

[0053] 1. Preparation of total RNA from human epidermal cells

[0054] Adult human skin was obtained from the Plastic Surgery Department of Changhai Hospital. Epidermal cells were obtained from primary culture. The total RNA was obtained by extracting the total RNA by the conventional guanidine isothiocyanate method with the total RNA extraction kit of Shanghai Huashun Bioengineering Co., Ltd. Methods as below:

[0055] 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 the sample tube tightly, shake the tube...

Embodiment 2

[0106] Example 2: Cell experiment (in vitro experiment)

[0107] Using fluorescent microscope photography, CCK-8 experiment, flow cytometry, western-blot and other biological experimental methods, analyze cell morphology changes from various aspects such as cell morphology and protein expression, target gene expression and cell surface markers CD29, CD44 and Expression of CD90.

[0108] The specific method is as follows:

[0109] 1) Comparison of cell growth status

[0110] Visualization of Lentivirally Infected JAM1 Using an Inverted Fluorescence Microscopy ov - hMSCs and hMSCs. Cell morphology did not change significantly, such as figure 1 shown.

[0111] 2) CCK-8 detects cell proliferation

[0112] JAM1 ov - MSCs and MSCs were seeded in 96-well plates with 5000 cells per well, and CCK-8 working solution was added after 1 day, 3 days, 5 days and 7 days after culture (10 μL / well, 37 degrees, 5% CO2 incubator and incubated for 4 hours , measure the A450 absorbance valu...

Embodiment 3

[0133] Example 3: In vivo differentiation assay

[0134] 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 45 nude mice were divided into 3 groups: JAM1 ov - hMSC injection group, hMSC 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. Feed under SPF conditions, observe daily, and collect samples 1, 3, 5, 7, and 14 days after transplantation. After 14 days, the cells injected with JAM1 overexpression had obvious hair formation.

[0135] 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.

[0136] H-E staining was performed on the skin ...

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Abstract

The invention relates to the technical field of stem cell and gene engineering, provides a method of modifying a mesenchymal stem cell by a link adhesion molecule JAM1 gene, and in particular relates to the design of a primer. JAM1cDNA is prepared by taking the total RNA (Ribonucleic Acid) of a human epidermal cell as a template and by amplifying RT-PCR; then, a recombinant human peGFP-N1-JAM1 eukaryotic expression vector is constructed. In order to improve transfection efficiency, a slow virus over-expression plasmid pGC-FU-JAM1-GFP is constructed, and the slow virus is infected with a human embryo mesenchymal stem cell (hMSC). The invention further provides an application of the method in the preparation of the stem cell promoting hair regeneration and promotion of partial skin hair regeneration in cell transplantation. JAM1ov-hMSC is intradermally transplanted to the skin tissue of a three-week nude mouse; under the induction of nude mouse skin microenvironment, the JAM1ov-hMSC is migrated to the hair follicle of the nude mouse, so that the growth period of the hair follicle of the nude mouse is prolonged, the hair follicle structure is improved and hair is regenerated.

Description

technical field [0001] The invention relates to the technical field of stem cells and genetic engineering, in particular to a method for JAM1 gene modification of mesenchymal stem cells, and its application in preparing stem cells for promoting hair regeneration and cell transplantation for promoting local skin and hair regeneration. Background technique [0002] Nowadays, more and more people will experience hair loss due to various factors in life. The hair follicle cycle is a series of cyclic activities that hair follicles undergo in adult mammals, which can be divided into anagen, catagen, and stagnation phases. Hair loss in both sexes manifests as progressive hair follicle miniaturization, resulting in a shortened hair growth cycle. Due to the shortened anagen period, the hair becomes thinner and shorter to the point where it does not grow at all. Since most hair loss develops gradually, the sooner treatment is started the better. [0003] With the deepening of stem ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867A61K35/54A61P17/14A61K35/28
Inventor 刘厚奇仵敏娟郭晓灿
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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