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Improved fat stem cell for epidermis damage repair

A technology of adipose stem cells and human adipose stem cells, which is applied in the field of cell and genetic engineering and epidermal damage repair, can solve the problems of burn patients such as lack of sufficient autologous skin, immune rejection, poor treatment effect, etc. Effect of cultivation, rapid growth

Inactive Publication Date: 2015-03-11
XINJIANG MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the objective existence of many shortcomings in the current domestic and foreign repair methods for deep burns or large-area skin defect wounds, such as damage to the donor site, lack of sufficient autologous skin in patients with large-area burns, and allogeneic skin transplantation and Research status of skin tissue engineering products with practical problems such as immune rejection and poor therapeutic effect

Method used

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  • Improved fat stem cell for epidermis damage repair
  • Improved fat stem cell for epidermis damage repair
  • Improved fat stem cell for epidermis damage repair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Preparation of Improved Adipose Stem Cells for Epidermal Damage Repair

[0066] 1. Isolation and in vitro culture identification of human adipose stem cells:

[0067] Isolation and culture of human adipose stem cells: adipose tissue was cut into mince, washed several times with PBS, digested with 0.1% collagenase I on a shaker at 37°C, centrifuged for 30 min to remove the supernatant, and the pellet was resuspended in 10% fetal bovine serum In the DMEM culture medium, transfer it into a culture dish; replace the culture medium every other day; after reaching the logarithmic growth phase, digest with 0.25% trypsin, and pass passage at a ratio of 1:3. For the results, see the attached figure 1 .

[0068] Detection of growth curve: Take cells in logarithmic growth phase, digest with trypsin, wash and centrifuge, prepare single cell suspension, and inoculate into 24-well plate. Randomly pick cells from 4 wells every day to digest, count, take the average val...

Embodiment 2

[0126] Example 2: The method for promoting the differentiation of human adipose stem cells to the epidermis

[0127] 1. Isolation, culture and identification of human adipose stem cells.

[0128] 1.1. Cut the adipose tissue into 0.3cm×0.3cm×0.3cm size, rinse with 0.1mmol / L phosphate buffered saline (PBS), transfer it to a bottle, digest with 0.1% collagenase I, cut it into paste repeatedly Set it on a shaker at 37°C, centrifuge at 1000 r / min for 30 min to remove the supernatant, resuspend the pellet in 0.075 mmol / L potassium chloride, let it stand for 10 min, then centrifuge to remove the supernatant, and resuspend the pellet in 10% fetal bovine serum DMEM culture medium, transfer to a Petri dish; replace the culture medium every other day. After reaching the logarithmic growth phase, it was digested with 0.25% trypsin at 37°C, and passaged at a ratio of 1:3. See the attached figure 1 .

[0129] 1.2. Draw the cell growth curve, see the attached Figure 4 , flow cytometr...

Embodiment 3

[0185] Embodiment three: Q-PCR detection test

[0186] 1. RNA extraction.

[0187] 1.1. Aspirate the culture medium, add 1 mL Trizol, keep at room temperature for 5 minutes, pipette the cells repeatedly, and transfer the cell lysate into a 1.5 mL EP tube.

[0188] 1.2. Shake vigorously for 15 seconds, add 0.2 times the volume of chloroform, shake for 15 seconds, and place at room temperature for 2 to 3 minutes.

[0189] 1.3. Centrifuge at 12000 rpm for 10 minutes.

[0190] 1.4. Aspirate the upper aqueous phase, transfer it to another new EP tube, add 1.1 times the volume of isopropanol, mix well, and place at room temperature for 10 minutes.

[0191] 1.5.12000 rpm, centrifuge for 10 minutes, discard the supernatant.

[0192] 1.6. Add 1 mL of absolute ethanol to wash the RNA pellet.

[0193] 1.7. Centrifuge at 7500 rpm for 5 minutes and discard the supernatant.

[0194] 1.8. Fully absorb the residual ethanol solution, let it dry for 2~3 minutes, and add 20 uL DEPC-treate...

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Abstract

The invention discloses an improved fat stem cell for epidermis damage repair. The fat stem cell is subjected to separation and in-vitro culture to construct the slow virus vector containing p63 gene. The recombinant vector containing p63 gene is pLV.EX3d.P / neo-EF1A>TP63 / flag>IRES / eGFP. When the p63 gene is transformed to the fat stem cell by the slow virus, the Q-PCR (quantitative polymerase chain reaction) detection indicates that the expression does not exceed the untransfected fat stem cell, and the differentiation capacity of the improved fat stem cell to the epidermis cell does not exceed the untransfected fat stem cell, thereby achieving the effect of quickly and effectively covering and repairing the wound surface. The improved fat stem cell for epidermis damage repair has the characteristic of high transformation efficiency of the target gene, can conveniently and quickly implement the long-term stable expression of the target gene, has the advantages of accessible material, high growth speed, no rejection of the autologous tissue and the like, and is widely used in the fields of burns, orthopedics and the like.

Description

technical field [0001] The invention belongs to the technical field of cell and genetic engineering. Specifically, the present invention relates to a technical field in which genetic engineering technology is used to modify adipose stem cells to affect their differentiation direction, and the modified adipose stem cells are applied to the repair of epidermal damage. Background technique [0002] The skin is the largest organ of the human or animal body, covering the whole body, protecting various tissues and organs in the body from physical, mechanical, chemical and pathogenic microorganisms, and having important physiological functions. Skin damage caused by inflammation, ulcers, burns and other factors can be life-threatening in severe cases. Clinical treatment methods include autologous skin transplantation, allogeneic skin transplantation and xenogeneic skin transplantation. Skin grafts can save lives and help wounds heal. Stem cell transplantation treatment such as e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61L27/38A61L27/60
Inventor 冯树梅王玮谭周赖成霞李甜白生宾郭琼钟近洁秦纹
Owner XINJIANG MEDICAL UNIV
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