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Method for performing Hpx protein induction and maintaining selective polarization of microglial cells, and applications thereof

A technology of microglia and protein, applied in the fields of bioengineering and medicine, can solve the problems that have not yet been reported on the polarization of Hpx protein

Active Publication Date: 2017-03-15
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the effect of Hpx protein on the polarization of microglia

Method used

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  • Method for performing Hpx protein induction and maintaining selective polarization of microglial cells, and applications thereof
  • Method for performing Hpx protein induction and maintaining selective polarization of microglial cells, and applications thereof
  • Method for performing Hpx protein induction and maintaining selective polarization of microglial cells, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Modeling of spinal cord injury, detection of correlation between Hpx and microglial cell polarization markers.

[0052] 1. Spinal cord injury modeling

[0053] After anesthetized by intraperitoneal injection of 4% chloral hydrate, the mice were placed prone on the foam board after skin preparation. The mouse spine was passively flexed, and the most obvious kyphosis of the thoracolumbar segment was calculated to the head side for 1-2 stages, and a longitudinal surgical incision was taken. Cut the skin, separate the bilateral musculature, reveal the spinous process and bilateral lamina, and locate the lamina stage. At chest 9-10, use a rongeur to bite off the 9th lamina of the chest to fully expose the spinal cord tissue. The spinal cord tissue can be seen to be bright white, and the longitudinal artery can be seen in the posterior center. If it is a control operation of laminectomy, the surgical incision can be sutured at this time; if it is a spinal cord c...

Embodiment 2

[0069] Example 2: Flow Cytometry Analysis

[0070] The microglial cells in the injured spinal cord of Hpx knockout mice were sorted by flow cytometry, and the polarization ratio was detected.

[0071] A. Collection of monocytes

[0072] The mice that have been modeled for a certain number of days were anesthetized by intraperitoneal injection of 4% chloral hydrate. After the anesthesia was complete, they were fixed on the foam board in a supine position, and the upper and lower limbs were pulled longitudinally as much as possible to fully stretch the spine structure. Make a "U"-shaped incision on the chest, cut the skin, cut the ribs, expose the heart, cut the right atrial appendage, and then puncture the left ventricle with an injection needle. First, use 1×PBS solution for continuous injection, about 40ml. Then the spinal cord tissue (including 1 mm above and below the spinal cord injury) was surgically removed under a microscope and placed in dissecting fluid at 4°C. The ...

Embodiment 3

[0076] Embodiment 3: histochemical experiment

[0077] Using immunohistochemical method, the expression level of M1 polarization marker molecule TNF-a in microglial cells of Hpx knockout mice with spinal cord injury was analyzed to further verify the regulation of Hpx on the polarization of microglial cells.

[0078] A. Histochemical staining steps

[0079] After placing the frozen sections in a 37°C oven for 1 hour, follow the steps below: wash the slices twice for 5 minutes with PBS; incubate overnight at 4°C with the anti-TNF-a primary antibody diluted in PBS; let the slices stand at room temperature for 40 minutes; wash with PBS at room temperature Slices, 5 minutes each time, three times in total; add secondary antibody, incubate at room temperature for 70 minutes; wash the slides with normal temperature PBS, 5 minutes each time, three times in total; then incubate with 1:1000 Hoechst for 7 minutes; wash with normal temperature PBS Films, 5 minutes each time, three times...

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Abstract

The invention belongs to the field of genetic engineering and medicines, and particularly relates to a method for performing Hemopexin protein specific induction and maintaining selective polarization of microglial cells, and applications thereof in treatment of spinal cord injury repair. According to the main technical scheme, the method comprises the following steps: (I) transplanting microglial cells into Hemopexin-knockout mice spinal cord to hinder the microglial cells to be selectively polarized; and (II) co-incubating the Hemopexin protein and microglial cells to be capable of inducing and maintaining the selective polarization of the microglial cells. The Hemopexin-induced microglial cells show an M2 phenotype, so that the repair of the spinal cord nerve injury can be effectively promoted.

Description

technical field [0001] The invention belongs to the fields of bioengineering and medicine, and in particular relates to a microglial cell under the action of plasma protein Hpx (Hemopexin) and its therapeutic application in spinal cord injury repair. Background technique [0002] The central nervous system injury, especially the spinal cord injury, has a high mortality and disability rate, and currently lacks an ideal treatment plan. The reasons for the poor prognosis of spinal cord injury include at least the neurodegeneration at the lesion, demyelination and remyelination disorder caused by the differentiation disorder of oligodendrocyte precursor cells. [0003] Microglia and macrophages play an important role in the repair of central injury, and their different polarization states are involved in the pathological process of secondary spinal cord injury. Polarization of microglia has now been found to play an important role in the regulation of neuronal and oligodendrocy...

Claims

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Application Information

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IPC IPC(8): A61K38/17C12N5/079A61K35/30A61P25/00
CPCA61K35/30A61K38/1709C12N5/0622C12N2501/998
Inventor 何成曹莉俞仲望
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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