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Method for promoting survival of injured neurons by virtue of mitochondria transplantation

A neuron survival and mitochondrial technology, applied in the field of stem cell-based biotechnology, can solve problems such as immunogenicity, and achieve the effect of avoiding implantation, avoiding immunogenicity problems, and promoting neuronal cell regeneration

Inactive Publication Date: 2016-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, allogeneic exogenous stem cell transplantation will encounter immunogenicity problems

Method used

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  • Method for promoting survival of injured neurons by virtue of mitochondria transplantation
  • Method for promoting survival of injured neurons by virtue of mitochondria transplantation
  • Method for promoting survival of injured neurons by virtue of mitochondria transplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Mitochondria (BMSC-MitC) derived from bone marrow mesenchymal stem cells (BMSCs) increase ATP in damaged motor neurons.

[0027] VSC4.1 motor neurons 5 × 10 4 Each cell was planted in a 24-well plate, and oxygen-glucose deprivation (OGD 8h) induced neuronal injury model. There were three groups: OGD model group; OGD+BMSCs group (5×10 4 BMSCs / well, co-cultured in transwell for 24 h); OGD+BMSC-MitC group (mitochondrion was added to the medium, from 1.7×10 6 BMSCs / well, incubated for 4 h). After the time point, the ATP content in the neuron cells was measured using Beyontek's ATP kit. After 8 hours of OGD treatment, the ATP content of neurons decreased to 1 / 3 of the normal group, which was 1.75±0.08 nmol / mg protein; after 4 hours of mitochondrial co-culture, the ATP content increased significantly to 2.20±0.09 nmol / mg protein; BMSCs co-cultured for 24 hours, the ATP content increased significantly to 2.48±0.03 nmol / mg protein. There was no significant diff...

Embodiment 2

[0028] Example 2: Mitochondria derived from bone marrow mesenchymal stem cells (BMSC-MitC) promote the survival of injured motor neurons.

[0029] VSC4.1 motor neurons 5×10 4 Each cell was planted in a 24-well plate, and oxygen-glucose deprivation (OGD 8h) induced neuronal injury model. There were three groups: OGD model group; OGD+BMSCs group (5×10 4 BMSCs / well, co-cultured in transwell for 24h); OGD+BMSC-MitC group (mitochondrion was added to the medium, from 1.7×10 6 BMSCs / well, incubated for 4 h). After the time point, the LDH content in the cell supernatant was determined by using the LDH detection kit. After 8 hours of OGD treatment, the LDH content in the motor neuron supernatant increased significantly; after 4 hours of mitochondrial co-culture, the LDH content was significantly reduced to 63.40±5.61% of the OGD group; BMSCs co-cultured for 24 hours, the LDH content was significantly reduced to 75.52±1.83% of the OGD group. There was no significant difference in ...

Embodiment 3

[0030] Example 3: BMSC-MitC transplantation promotes structural regeneration and functional recovery after spinal cord injury in rats.

[0031] Establishment of spinal cord impact injury model in rats: 8-week-old female SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at 4 ml / kg after fasting for 7-12 hours before the experiment. After 1-2 min, the rat's muscles relax, the pain reflex disappears, and the anesthesia is successful. Rats were placed on an electric blanket in a prone position, the hair on the back was shaved, disinfected with iodine from the inside out, and a towel was spread. Separate and expose the T10 segment of the spinal cord. The spinal cord was impacted at a height of 5 cm using a spinal cord injury impactor (NYU ImpactorII). After the blow, the rat's spinal cord was congested, the hind limbs twitched, the tail flicked, and then completely relaxed. Divided into three groups: SCI+solvent group (H-DMEM); SCI+mitochondria group ...

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Abstract

The invention provides a method for promoting the survival of injured neurons by virtue of mitochondria transplantation. The method comprises the following steps: (1) carrying out trypsinization centrifuge on P1-P3-generation mesenchymal stem cells, and counting the number of the cells; (2) separating by virtue of a mitochondria separation agent to obtain mitochondria, and carrying out protein quantification and ATP content determination; and (3) preparing suspension from the mitochondria obtained in the step (2), and transplanting the suspension to a neuron injury region to contact with the neuron injury region. Compared with the transplantation of mesenchymal stem cells, the method has the advantages that the allogeneic immunogenicity problem is avoided, the implantation problem of chromosomes of allosome-derived stem cell nucleuses is avoided, and the regeneration of neuronal cells is promoted.

Description

technical field [0001] The invention relates to a stem cell-based biotechnology, which is a method for promoting the survival and recovery of damaged neurons by applying mitochondria transplantation derived from mesenchymal stem cells. Background technique [0002] Spinal cord injury undergoes primary and secondary injuries, resulting in damage and loss of nerve cells and disruption of nerve conduction. In recent years, with the development of stem cell biology and its regeneration technology, it has been found that exogenous stem cell transplantation can significantly improve the survival of injured neurons and promote the recovery of neuron function. However, allogeneic exogenous stem cell transplantation will encounter immunogenicity problems. Stem cells will eventually differentiate into mature cells in the body, and mature cells express (MHC / HLA) molecules, as long as they are stem cells of allogeneic origin, they will face the problem of immune rejection. Therefore, ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P25/00
CPCC12N5/0663A61K35/28
Inventor 张晓明王琳琳王超陈莹莹沈岳良
Owner ZHEJIANG UNIV
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