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 foun

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

Example Embodiment

[0021] Example 1

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

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

[0024] Routinely isolate mononuclear cells from healthy human peripheral blood, RPMI1640 adjusts the cell density to 1×10 6 Cells / ml, add PMA (15nM) and anti-CD3 monoclonal antibody (10μg / ml), 37℃ 5% CO 2 Cultivate for 6 hours to activate lymphocytes and highly express CTLA-4mRNA.

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

[0026] 1) Extraction: collect activated PBMC by centrifugation, wash the cells with PBS (DEPC) × 2, press each 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, 10,000 rpm / centrifuge for 15 minutes, transfer the upper colorless water phase to another centrifuge tube, add Mix 0.5 ml of isopropanol, -20°C for 10 minutes, 10,000 rpm / centre for 15 minutes, add 1 ml of 75% ethanol to...

Example Embodiment

[0053] Example 2 Construction of CTLA-4Ig retroviral expression vector

[0054] The 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 fragment respectively, and then digested with BamHI, and electrophoresed The large fragment of pLXSN is recovered and the small fragment of pUC19-CTLA-4Ig is connected to obtain the expression pLXCTLA-4Ig of CTLA-4Ig.

Example Embodiment

[0055] Example 3 Construction of CTLA4Ig adenovirus expression vector

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

[0057] Add 1μl of 10× buffer solution to the alcohol precipitation cDNA (0.5pmol) centrifuge tube, then add sterile double distilled water to 9μl, 70℃ for 5 minutes, add 1μl T4DNA polymerase, mix gently, do not shake vigorously, incubate at 37℃5 After 10 minutes, adjust the DNA concentration to 1μg and 50μl 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 can not be used immediately, it should be extracted and precipitated immediately, dissolved in DNA Dilition buffer and stored at -20°C).

[0058] 2. Insert the blunt-ended cDNA into pAxCAwt (TakaRa, Japan) Korotkoff plasmid.

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

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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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