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Method for maintaining and recovering primitiveness of human dermal papilla cell and application thereof

A dermal papilla cell, primitive technology, applied in the biological field

Active Publication Date: 2015-06-03
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 the research and application of JAM-A 3'UTR to modify dermal papilla cells to maintain and enhance their originality

Method used

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  • Method for maintaining and recovering primitiveness of human dermal papilla cell and application thereof
  • Method for maintaining and recovering primitiveness of human dermal papilla cell and application thereof
  • Method for maintaining and recovering primitiveness of human dermal papilla cell and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction of eukaryotic expression vector pcDNA3.1-JAM-A3'UTR, infecting human dermal papilla cells (DPC).

[0057] 1. Isolation and culture of hair papilla cells

[0058] Adult human skin was obtained from the Plastic Surgery Department of Changhai Hospital. Dermal papilla cells were obtained from primary culture. As the number of passages increases, the morphology of dermal papilla cells and the expression of some marker molecules will change. In order to ensure the accuracy and reliability of the experiment, all our in vitro and in vivo experiments select the fifth generation of dermal papilla cells.

[0059] 2. Preparation of human genomic DNA

[0060] Use Promega Genomic DNA purification kit, the method is as follows:

[0061] [1]. Collect human cells into a clean 1.5ml OD tube;

[0062] [2]. Add 600 microliters of nuclear lysate, repeatedly pipette with a pipette gun to lyse the tissue until the visible tissue blocks disappear, and let it st...

Embodiment 2

[0098] Embodiment 2: cell experiment (in vitro experiment)

[0099] Using fluorescent microscope photography, immunocytochemistry and other biological experimental methods, the morphological changes of dermal papilla cells, the expression of target genes and the expression of cell surface marker keratin were analyzed from the aspects of cell morphology and protein expression.

[0100] The specific method is as follows:

[0101] 1) Comparison of cell growth status

[0102] Infected JAM-A 3'UTR observed with an inverted microscope oe DPC, JAM-A OE DPC and CON-DPC. JAM-A 3'UTR oe DPC cells can maintain the form of cohesive growth for a long number of generations, and there is no obvious change in the form of CON-DPC, such as figure 1 shown. JAM-A OE The algebra of the condensation-like growth of DPC has not improved significantly, so the follow-up experiments will focus on JAM-A OE DPC.

[0103] 2) Real-time PCR detection of the expression of JAM-A 3'UTR after trans...

Embodiment 3

[0137] Embodiment 3: test for promoting hair growth

[0138] The nude mouse BALB / c Nu strain used in the experiment, SPF grade. Weight about 15-25g, 3 weeks old, purchased from Shanghai Experimental Animal Center. A total of 9 nude mice were divided into 2 groups: JAM-A 3'UTR oe DPC injection group and CON-DPC injection group, PBS injection group. Cell volume is 5ⅹ10 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.

[0139] There was no difference in the size, activity, etc. of the animals in each group after the experimental treatment. JAM-A3'UTR oe Visible hair growth can be seen near the DPC transplantation site, such as Figure 4 shown.

[0140] Compared with nude mice in each group after sacrifice, there was no difference in H-E staining, liver, spleen, kidney and...

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Abstract

The invention discloses a new application of 3'UTR of JAM-A. The JAM-A3'UTRoeDPC is obtained by successfully constructing an expression plasmid of the 3'UTR of JAM-A and performing human dermal papilla cell (DPC) plasmid transfection. Compared with the DPC, the JAM-A3'UTRoeDPC can keep the primitiveness of the papilla in vitro for a long time and can express the specific marked molecules of DPC on a high level, such as versican and ALPL after growth of agglomeration samples thereof for multiple generations. Each group of the cell is transported to the three-week-old nude mice skin tissue, and the conditions that the nude mice hair growth can be obviously promoted and the new hair can be thickened compared with the matched group by using the plasmid transfected DPC are found. A new concept and mode can be provided for culturing the human dermal papilla cell in vitro, maintaining and recovering the primitiveness of the DPC cell. A new concept and method for transplanting the DPC to induce the hair regeneration also can be increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for maintaining and restoring the originality of human dermal papilla cells and its application. Background technique [0002] Hair has important physiological and social functions, but now with the increasing pressure of life, the pace of hair loss is becoming more and more serious. In the process of hair regeneration and periodic replacement, dermal papilla cells (dermal papilla cell, DPC) play a very important role (see literature: Enshell-Seijffers D, Lindon C, Kashiwagi M, Morgan BA.beta-catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair. Dev Cell. 2010 Apr 20; 18(4):633-42). DPCs are a special group of mesoderm-derived mesenchymal-like cells that can send signals to initiate hair follicle development and hair cycle turnover. Among them, the periodic replacement of hair, that is, the hair follicle cycle, is a series of periodic act...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N5/10A61K35/36A61P17/14A61L27/38A61L27/60
Inventor 刘厚奇仵敏娟王越徐辰夏照帆徐莎
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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