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Recombinant vector, fibroin film modified by transgenic marrow stromal cells and application thereof

A recombinant plasmid and homologous recombination technology, which is applied in the direction of introducing foreign genetic material, cytokines/lymphokines/interferon, application, etc., can solve the problem of no progress in small-caliber artificial blood vessels, and achieve the promotion of wound repair, The effect of promoting angiogenesis

Inactive Publication Date: 2011-11-16
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still no progress in how to make the scaffold material fully vascularized, and further develop and prepare angiogenesis-inducing silk fibroin-based scaffold materials combined with MSCs for small-caliber artificial blood vessels.

Method used

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  • Recombinant vector, fibroin film modified by transgenic marrow stromal cells and application thereof
  • Recombinant vector, fibroin film modified by transgenic marrow stromal cells and application thereof
  • Recombinant vector, fibroin film modified by transgenic marrow stromal cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Map of pAdTrack-CMV-Ang-1-PolyA-promoter-VEGF 165 Double Gene Recombination Transfer Plasmid

[0055] The polyAΔ296-298-promoter (referred to as PolyA-promoter) and the IRES sequence are disclosed in the application number 200810244301.3 Chinese Invention Patent Application Specification. The double-gene recombination transfer plasmid of the present invention is based on the pAdTrack-CMV-PolyA-promoter transfer plasmid, inserting the Ang-1 fragment between the Bgl II and Sal I restriction sites, and inserting the Ang-1 fragment at the Not I and Xho I restriction sites Insert the VEGF165 fragment between the points, and the plasmid map is as follows figure 1 shown.

Embodiment 2

[0056] Embodiment 2: Cloning of target gene

[0057]Compare the conserved sequence of Ang-1, design primers P1 and P2 (as shown in Table 1), and introduce Bgl II and Sal I restriction sites at both ends; pAdTrack-CMV-CMV- The Ang-1-IRES-VEGF165 plasmid was used as a template, and the PCR product of the Ang-1 target fragment obtained by PCR amplification was identified by agarose electrophoresis.

[0058] Compare the conserved sequence of VEGF165, design primers P3 and P4 (as shown in Table 1), and introduce Not I and Xho I restriction sites at both ends respectively, and use the pAdTrack-CMV-Ang-1- The IRES-VEGF165 plasmid was used as a template, and the PCR product of the VEGF165 target fragment obtained by PCR amplification was identified by agarose electrophoresis.

[0059] Table 1PCR primers

[0060]

[0061] The PCR conditions were: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 50 s, annealing at 58°C for 50 s, extension at 72°C for 50 s, a total of 30...

Embodiment 3

[0073] Example 3: Construction of pAdTrack-CMV-Ang-1-PolyA-promoter single gene recombination transfer plasmid

[0074] The PCR product of Ang-1 gene purified by the DNA cleaning kit and the transfer plasmid pAdTrack-CMV-PolyA-promoter extracted by the mini-plasmid extraction kit were digested with Bgl II and Sal I at 37°C for 5 hours, and recovered by tapping rubber For the target fragment, follow the gel recovery method and steps of the kit, and then use T4 DNA ligase to ligate it overnight at 4°C, and then transform the ligated product into Escherichia coli DH5α, and select it on a Kana (50 μg / ml) resistant plate Positive single clones were identified by PCR, double enzyme digestion and DNA sequence determination, and the positive clones were stored in a -20°C refrigerator for later use.

[0075] The double enzyme digestion system is as follows:

[0076] pAdTrack-CMV-PolyA-promoter Plasmid Double Digestion System Ang-1PCR Product Double Digestion System

[0077] 10×Buffer...

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Abstract

The invention relates to the biology field, and concretely discloses a recombinant plasmid pAdTrack-CMV-VEGF165-PolyA-promoter-Ang of VEGF165 and Ang-1 as well as a recombinant plasmid pAdTrack-CMV-VEGF165-IRES- Ang-1 of VEGF165 and Ang-1, wherein the preservation numbers are CCTCC No: M 2010221, CCTCC NO: M 2010220. The invention also discloses a homologous recombination of the plasmid and adenovirus and a recombinant adenovirus incased and generated from a QBI-293A cell. The invention also provides a fibroin film modified by transgenic marrow stromal cells. Rabbit corneal limbus injected with the recombination adenovirus provided in the invention is capable of successfully inducing the formation of corneal neovascularization; after the fibroin film modified by transgenic cells is implanted in a rabbit cornea layer, the neovascularization can be grown in a cornea of a fibroin film implanted area and the density of blood vessels is obviously increased, therefore, VEGF165, Ang-1, CD34 can be successfully expressed in a rabbit cornea layer. Wound repairing experiments of rats prove that the fibroin film modified by transgenic marrow stromal cells of the invention is capable of promoting the blood vessel generation of the wound organization and repairing wound, so that the recombinant adenovirus and the fibroin film modified by transgenic marrow stromal cells of the invention have medical application value.

Description

technical field [0001] The invention relates to the biological field, in particular to a recombinant carrier and a silk membrane modified by transgenic bone marrow stromal cells and an application thereof. Background technique [0002] The mechanism of body damage repair is that there must be the formation of microvessels first, followed by the formation of new granulation tissue, and then the required various cells and nutrients can be input to promote the repair of damaged tissues. Angiogenesis is a complex, step-by-step, and orderly process that not only requires the stimulation of stimulating factors and the participation of many cells, but also the participation of various cytokines and growth factors and the cooperation of extracellular matrix. Ang-1, VEGF, FGF, PDGF play an important role in wound granulation tissue formation, angiogenesis and collagen fiber synthesis. [0003] VEGF is a dimeric glycoprotein composed of two identical subunits cross-linked by disulfid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N7/01A61K48/00A61P9/00A61P27/02A61L27/38A61L27/22A61L27/54C12R1/93
CPCA61L27/54C12N15/86C07K14/52C07K14/515A61L27/3834A61L27/3604A61K48/005A61L2300/258C12N2840/203C07K14/43586C12N2710/10043A61P9/00A61P27/02
Inventor 杨吉成缪竞诚盛伟华韩亚丽刘铁连张晓峰
Owner SUZHOU UNIV
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