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Preparation and application of high expression TSP-1 stem cell for promoting regeneration of neuron axon

A technology of TSP-1, 1.TSP-1, applied in the field of neurorehabilitation tissue engineering, can solve the problems of unsatisfactory synapse reconstruction and axonal regeneration, and the microenvironment of axon growth inhibition is not solved, so as to avoid Overdose reactions and systemic toxicities, accelerated reconstitution, correct effects

Inactive Publication Date: 2018-03-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BMSCs transplantation for the treatment of SCI remains unsatisfactory in terms of synapse reconstruction and axon regeneration after transplantation due to the unresolved microenvironment of axon growth inhibition

Method used

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  • Preparation and application of high expression TSP-1 stem cell for promoting regeneration of neuron axon
  • Preparation and application of high expression TSP-1 stem cell for promoting regeneration of neuron axon
  • Preparation and application of high expression TSP-1 stem cell for promoting regeneration of neuron axon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Construction of adenovirus vector carrying TSP-1:

[0046] 1.1 Construction of adenovirus vector

[0047] 1.1.1 Synthetic target gene TSP-1 sequence:

[0048] The sequence is shown in SEQ ID NO:1.

[0049] 1.1.2 Purification of the target gene fragment

[0050] The target gene sequence synthesized in step 1.1.1 was digested with Not I and EcoR I to obtain TSP-1 fragment, and digested at 37°C for 3 hours. The digestion system is as follows:

[0051]

[0052]

[0053] After the digestion is completed, the gel is recovered, the target fragment is cut, and the target fragment is recovered using the DNA gel recovery and purification kit (column centrifugal type) (Gel-Spin DNA Extraction Kit). The specific method is as follows:

[0054] 1) After DNA electrophoresis (it is recommended to use TAE buffer for agarose gel electrophoresis), cut out the corresponding fragments with a clean blade under ultraviolet light. Carefully remove the DNA-free part of the gel block.

[0055] 2)...

Embodiment 2

[0123] Example 2. Transfection of bone marrow mesenchymal stem cells with adenovirus carrying TSP-1

[0124] 2.3 TSP-1 gene modified rat bone marrow mesenchymal stem cells

[0125] 2.3.1 Take the third generation rat bone marrow mesenchymal stem cells BMSCs, the cells are 1×10 6 / Well density inoculated in a 10cm petri dish, normal culture (medium is DMEM, double antibody, plus 10% FBS, culture conditions are 37℃, 5% CO2, saturated humidity culture conditions), 24h cells After the adherence is stable, prepare for transfection; dilute pHBAd-MCMV-GFP-TSP-1 and pHBAd-MCMV-GFP with 3ml high-sugar serum-free medium respectively, and calculate the amount of virus solution required with MOI of 50 as 5μL , Then add 5μL of Polybrene with a concentration of 2mg / ml, shake and mix well, add it to a petri dish with medium removed, and incubate in a 37℃, 5% CO2, saturated humidity incubator for 6 hours; after the incubation, aspirate the virus solution And serum-free medium, add 10ml of serum-c...

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Abstract

The invention relates to the technical field of neurological rehabilitation tissue engineering and specifically discloses a preparation method of a TSP-1 gene modified mesenchymal stem cell. The preparation method comprises the following steps: 1, constructing a TSP-1-carrying adenovirus vector: preparing a pHBAd-MCMV-GFP vector and a TSP-1 target fragment with a double digestion method, connecting a target gene and the pHBAd-MCMV-GFP vector, and identifying pHBAd-MCMV-GFP-TSP-1 with a sequencing method, so that the correctness of an exogenous gene sequence is ensured; 2, transfecting BMSCs with a TSP-1 adenovirus, so as to obtain the TSP-1 gene modified mesenchymal stem cell. In the preparation method, by selecting the TSP-1 gene modified mesenchymal stem cell, the recovery of transplanted neurons of the neurological rehabilitation tissue engineering and the regeneration of neurite can be accelerated, and the construction speed of a damaged local neuronal circuit is accelerated.

Description

Technical field [0001] The present invention relates to the technical field of nerve rehabilitation tissue engineering, in particular to the preparation and application of high-expressing TSP-1 stem cells for treating spinal cord injury and promoting axon regeneration. Background technique [0002] With the continuous development of my country’s economy, acute spinal cord injury (SCI) patients due to traffic accidents, falls from heights and other reasons are increasing year by year. For example, Tianjin, my country, has an incidence rate of 23.7 people per 1 million in recent years. Average age: 46.0±14.2 years old, showing a progressive increase trend. Spinal cord injury is more common in young adults, and often leads to patients with sensory, motor, reflex and sphincter dysfunction, especially cervical spinal cord injury, and even permanent disability. Most patients require long-term hospitalization and rehabilitation, bringing family and society A heavy burden. Therefore, fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10C12N5/0793
CPCC12N5/0619C12N5/0663C12N15/86C12N2501/998C12N2502/99C12N2510/00C12N2710/10343C12N2800/107
Inventor 张晓明王琳琳蒲玉洁孟珂林莉雅林和风
Owner ZHEJIANG UNIV
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