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A kind of biological scaffold loaded with t-pa gene and preparation method thereof

A biological scaffold and gene technology, applied in the field of medical devices, can solve the problems of reduced thrombus formation rate, trauma, and accelerated atherosclerotic plaque formation, and achieve the effects of inhibiting SMC proliferation, preventing thrombosis, and preventing thrombosis

Active Publication Date: 2016-06-01
深圳市贝安特医疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, delayed arterial local healing due to vascular hypersensitivity to polymers and chronic inflammatory stimuli, especially the obstruction of the endothelialization process, accelerates new atherosclerotic plaque formation and increases the chance of stent thrombosis, especially at later stages (1 months to 1 year) and later (more than 1 year) thrombosis, the duration of clinical antithrombotic therapy is significantly prolonged
The second-generation and third-generation DESs mainly address the above-mentioned deficiencies. The design uses a thinner support framework, more durable and better biocompatibility, and even bioabsorbable polymers to reduce inflammation and reduce allergic reactions. Anti-proliferative drugs can significantly reduce the rate of stent thrombosis, and its effect needs to be clinically verified
Although this carrier is safe, convenient, and effective, surgical sutures are foreign bodies and invasive procedures, making them unsuitable for medicinal use

Method used

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  • A kind of biological scaffold loaded with t-pa gene and preparation method thereof
  • A kind of biological scaffold loaded with t-pa gene and preparation method thereof
  • A kind of biological scaffold loaded with t-pa gene and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The invention provides a method for preparing a biological scaffold loaded with t-PA gene, comprising the steps of:

[0038]Step 1: Preparation of albumin nano solution: prepare albumin nano solution of t-PA gene plasmid with albumin and t-PA gene plasmid as raw materials;

[0039] Preparation of the coated stent: a. Prepare the stent surface coating liquid according to the ratio of 200 μg, 100 μg and 50 μg of type IV collagen, laminin and fibronectin per milliliter of hyaluronic acid;

[0040] b. After aseptic treatment, the stent is placed in the surface coating solution of the stent treated by ultrasonic oscillation. The conditions of the ultrasonic oscillation treatment are 40KHz, 50W, 40-60 minutes; after incubation at 25°C for 1 hour, no Bacteria were air-dried, and molded on a balloon with a diameter of 3 mm to obtain a coated stent;

[0041] Step 2: soak the coated stent obtained in step 1 in the albumin nano solution, incubate for 30-40min, and dry;

[0042] ...

Embodiment 2

[0052] The invention provides a method for preparing a biological scaffold loaded with t-PA gene, comprising the steps of:

[0053] Construction and expression of gene plasmids: Design 3 pairs of primers based on the three EST sequences and the t-PA gene sequence to amplify the three-break sequence of t-PA from the three EST cloning plasmids, and at the ends of the three pairs of primers respectively Introduced restriction sites. Three EST clones were amplified and cultured in LB medium containing ampicillin and chloramphenicol, and the three EST clone plasmids were extracted as templates for PCR amplification and three t-PA fragments were amplified, and the amplified products were recovered and sequenced for identification. Plasmid pSecTag2B and three t-PA fragments t-PA-1, t-PA-2, t-PA-3 were digested by HindIII and XhoI, HindIII and KpnI, KpnI and BamHI, BamHI and XhoI respectively, and purified by QIAGENPCRProductPurificationKit , T4 DNA ligase was ligated overnight at 14...

Embodiment 3

[0061] Embodiment 3 animal experiments

[0062] 1. Materials

[0063] 1.1 Animals: 18 purebred New Zealand white rabbits, male, weighing 2.3-2.5kg, provided by the Animal Center of Southern Medical University.

[0064] 1.2 Main reagents and instruments: plasmid pSecTag2B, competent cells E.ColiJM109, and Chinese hamster ovary cell lines were provided by Yaneng Biotechnology (Shenzhen) Co., Ltd.; restriction enzymes HindIII, KpnI, BamHI and XhoI were purchased from Bao Bioengineering Ltd.; VentDNA polymerase and T4DNA ligase were purchased from NewEngland Biolabs; rabbit anti-human t-PA polyclonal antibody, mouse anti-smooth muscle α-actin and PCNA monoclonal antibody, bovine serum albumin, t-PA ELISA detection kit Provided by Jingmei Bioengineering Co., Ltd. The ultrasonic generator is a product of Ningbo Xinzhi Biotechnology Co., Ltd.

[0065] 2 Animal models and scaffolding

[0066] 2.1 18 rabbits were randomly divided into two groups: experimental group (n=10) and contr...

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Abstract

The present invention relates to the technical field of medical appliances and particularly relates to a biological scaffold for loading a t-PA gene and a preparation method thereof. The biological scaffold for loading the t-PA gene comprises a scaffold, a substrate layer covering the surface of the scaffold, a gene layer covering the surface of the substrate layer and seal coating on the outer surface of the scaffold. A stainless steel scaffold surface layer of the scaffold simulates a vascular basement membrane material to induce a blood vessel endothelium to quickly creep onto the surface of the scaffold, in order to prevent thrombus; a nano gene layer plays the functions of slow releasing and controlled releasing gene drugs; a hyaluronic acid layer between two layers of nanoparticles serves as a connecting layer, since the hyaluronic acid layer has charges, the surfaces of the nanoparticles are positively charged in an acid environment, and the two layers of nanoparticles are firmly adhered together by charge attraction. The outermost layer provides a good micro environment for nano gene transfection.

Description

technical field [0001] The invention relates to the technical field of medical devices, in particular to a biological scaffold loaded with t-PA gene and a preparation method thereof. Background technique [0002] Currently, there are mainly two types of stents in clinical use: bare metal stents (BMSs) and drug-eluting stents (drugeluting stents, DESs). The main components of drug-eluting stents include: bare metal framework, macromolecular polymers, and anti-proliferative drugs such as everolimus, biolimus, or sirolimus. A number of randomized investigations have shown that compared with balloon angioplasty alone, BMSs can significantly reduce the complications of interventional therapy and the occurrence of late restenosis; compared with BMSs, DESs can significantly reduce restenosis, but stent thrombosis is worrying. The first-generation DESs contain sirolimusorpaclitaxel, which can significantly reduce intimal hyperplasia in the stent and significantly reduce the inciden...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L31/16A61L31/12
Inventor 季军屠洪吴大方何霞刘强陈小玲令文萍
Owner 深圳市贝安特医疗技术有限公司