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Heterogenous skin transfected by recombined genes

A skin and wound technology, applied in the field of isolated pig skin, can solve problems such as difficulty, no application, limited effect, etc., achieve good growth conditions, reduce wound scar formation, and promote healing

Inactive Publication Date: 2008-05-14
CHONGQING ZONGSHEN JUNHUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no application of it in burn wound skin grafting
At the same time, due to the difficulty in the preparation and purification of CTLA4, the cost of systemic or even local application is expensive, and studies have found that only when CTLA4 in the body is maintained at a certain level, its ability to induce graft-specific immune tolerance is most obvious
It shows that direct application of CTLA4 pure product is not only difficult, but also has limited effect

Method used

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  • Heterogenous skin transfected by recombined genes
  • Heterogenous skin transfected by recombined genes
  • Heterogenous skin transfected by recombined genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Construction of Ig fusion protein expression vector ( figure 1 )

[0023] 1. Isolation and activation of peripheral blood mononuclear cells (PBMC)

[0024] Routine isolation of peripheral blood mononuclear cells from healthy people, RPMI1640 adjusted cell density to 1×10 6 Cells / ml, add PMA (15nM) and anti-CD3 monoclonal antibody (10 μg / ml), 37°C 5% CO 2 After culturing for 6 hours, the lymphocytes were activated and CTLA-4mRNA was highly expressed.

[0025] 2. Extraction and identification of total cellular RNA

[0026] 1) Extraction: Collect activated PBMCs by centrifugation, wash cells with PBS (DEPC) × 2, and use 1 × 10 7 Add 1 ml of Tripure to PBMC, mix well, RT for 5 minutes, add 0.2 ml of chloroform, shake vigorously for 15 seconds, RT for 5 minutes, centrifuge at 10,000 rpm for 15 minutes, transfer the upper colorless aqueous phase to another centrifuge tube, add 0.5 ml of isopropanol, mix well, -20°C for 10 minutes, centrifuge at 10,000 rpm for 15 minutes...

Embodiment 2

[0053] The construction of embodiment 2CTLA-4Ig retrovirus expression vector

[0054] pLXSN (Clontech, USA) and pUC19-CTLA-4Ig were digested with EcoRI and HindIII, respectively, and then filled with dATP, dNTP and DNA E. coli polymerase Klenow fragments, and then digested with BamHI, and the electrophoresis The large fragment of pLXSN was recovered and the small fragment of pUC19-CTLA-4Ig was ligated to obtain the expression pLXCTLA-4Ig of CTLA-4Ig.

Embodiment 3

[0055] Example 3 Construction of CTLA4Ig Adenovirus Expression Vector

[0056] 1. Blunt the ends of the cDNA fragments.

[0057] Add 1 μl of 10× buffer solution to the ethanol-precipitated cDNA (0.5 pmol) centrifuge tube, add sterile double distilled water to 9 μl, 70°C for 5 minutes, add 1 μl T4 DNA polymerase, mix gently, do not shake vigorously, and incubate at 37°C for 5 minutes Minutes, adjust the DNA concentration to 1 μg and 50 μl with DNA Dilition buffer, and mix well. Transfer to an ice bath to inactivate the polymerase, which can be used directly in the ligation reaction (if it cannot be used immediately, it should be extracted and precipitated with alcohol immediately, then dissolved in DNA Dilition buffer and stored at -20°C).

[0058] 2. The blunt-ended cDNA was inserted into pAxCAwt (TakaRa, Japan) Koshi plasmid.

[0059] Linearize pAxCAwt: pAxCAwt 5 μl, h buffer (high salt buffer) 5 μl, Swa I 2 μl, ddH 2 O 38μl, mix well and incubate at 25°C for 2 hours; add ...

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Abstract

A heterogenous skin transfected by recombined gene CTLA4Ig and able to express the recombined protein CTLA4Ig is disclosed. This invented heterogenous skin can be used to cover and seal the burn wound, promote wound heeling and reduce the scar formation.

Description

technical field [0001] The present invention relates to a kind of isolated pig skin used to cover people's burn wounds, specifically, the present invention relates to a kind of isolated pig skin tissue transfected with CTLA4Ig recombinant gene, which is used to cover wounds such as burns, thereby promoting Wound healing, reducing scar formation. Background technique [0002] The research on allogeneic skin (species) transplantation of burn wounds is an urgent need for modern burn treatment: burns are a special type of trauma with a high incidence and disability rate, and the fundamental problem is the problem of the wound surface. Timely and effective coverage and sealing will inevitably lead to disturbance of the internal environment of the patient, infection and multi-organ dysfunction; in addition, non-physiological healing of the wound will occur, which will increase the formation of scars and aggravate the degree of disability. Therefore, the research on burn wounds ha...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61F2/10C12N15/09C12N15/12
Inventor 吴军魏泓易绍萱罗高兴贺伟峰周立新陈烯炜张宁
Owner CHONGQING ZONGSHEN JUNHUI BIOTECH
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