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Application of PDIA6 in preparation of medicine for treating and repairing spinal cord injury

A spinal cord injury and drug technology, applied in the field of biomedicine, can solve problems such as unclear, achieve obvious effects, promote repair, and promote the growth of neurites

Active Publication Date: 2022-06-28
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So whether spastin and PDIA6 have an interactive relationship, whether PDIA6 can regulate microtubule dynamics and promote nerve repair through spastin is still unclear

Method used

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  • Application of PDIA6 in preparation of medicine for treating and repairing spinal cord injury
  • Application of PDIA6 in preparation of medicine for treating and repairing spinal cord injury
  • Application of PDIA6 in preparation of medicine for treating and repairing spinal cord injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of plasmids and constructs

[0052] (1) PDIA6 and spastin sequences (available through commonly used databases such as Pub, ed or Uniprot) were obtained for cDNA preparation, respectively, which were then cloned into pEGFP-C1 (Clontech, CA, USA), pGEX-5x-3 (Amer - shamPharmacia Biotech, NJ, USA) and pCMV-Tag2 (Stratagene, CA, USA) vectors, and the constructed constructs were confirmed by DNA sequencing;

[0053] (2) Pull-down analysis using glutathione S-transferase (GST): Spinal cord tissue sections were ground and lysed, then GST-agarose beads (Invitrogen, CA, USA) were rinsed and mixed with lysis buffer at 4 After incubation for 1 h at 4°C, centrifugation was performed at 1000 g for 10 minutes. The supernatant was then collected and the above steps were repeated once. The appropriate bead fusion protein was then added to the spinal cord tissue and the samples were incubated overnight at 4°C. After centrifugation at 1000 g for 5 minutes at 4°C...

Embodiment 2

[0058] Example 2 Study on the role of PDIA6 in the repair of spinal cord injury

[0059] First, the construction of the rat SCI model includes the following steps:

[0060] The Sprague-Dawley (SD) rats used in the experiment were purchased from the Laboratory Animal Center of Sun Yat-Sen University. The rats were housed individually in a facility at 25 ± 3 °C and provided with regular food and water. Specifically, the following steps were included:

[0061] (1) SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35 mL / 100 g body weight), after which a 2.5 cm longitudinal dorsal incision was made to expose the T9-11 spinous process and lamina;

[0062] (2) The entire T10 lamina is removed, and the exposed area of ​​the spine is about 2.5mm × 3mm;

[0063] (3) Use a stabilizer to fix the T10 section bilaterally, and set the nitrogen tank to control the impact head to 18psi or 124kPa. Load a U-shaped stabilizer with a rat onto the platform of the Lo...

Embodiment 3

[0071] Example 3 Study on the interaction between PDIA6 and spastin

[0072] (1) Construct prokaryotic expression vector plasmids of GST-Spastin and GST-PDIA6 and transform them into BL21 competent cells;

[0073] (2) Pick a single clone and culture it in LB medium until the measured OD value is 0.4-0.6, add IPTG, and induce overnight in a shaker at 25°C;

[0074] (3) After the induction, the bacterial solution was centrifuged and lysed, and then the fusion protein was purified by GST beads, electrophoresed and stained with Coomassie brilliant blue.

[0075] Test results such as Figure 4 shown. The results showed that the molecular weight of the purified fusion protein was in line with expectations.

[0076] Then, using the prepared rat brain lysate, set aside an appropriate amount of Input as a positive control, and use GSTbeads to remove non-specific proteins, and add the successfully purified fusion expression protein of GST-Spastin and GST-PDIA6 to the brain lysate for...

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Abstract

The invention relates to application of PDIA6 protein or an active fragment thereof in preparation of drugs for treating and repairing spinal cord injury. A large number of researches prove that the PDIA6 plays a key role in SCI and interacts with the spastin to regulate and control outward growth of neurite, the expression level of the PDIA6 is up-regulated in the SCI process, and the PDIA6 can physically interact with the spastin in vivo and in vitro. Meanwhile, the effect of the PDIA6 and the spastin not only can promote the growth of neurite, but also can promote the repair of damaged neurons, and the promotion effect of co-transformation of the PDIA6 and the spastin is more obvious than that of single transformation of the spastin. According to the application disclosed by the invention, PDIA6 is a key target in spinal cord injury and repair processes and plays a role in regulating neurite growth and branching, and practical experimental evidence and scientific basis are provided for clinical treatment of spinal cord injury.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular, the present invention relates to the application of PDIA6 protein or its active fragment in preparing medicine for treating spinal cord injury and repairing. Background technique [0002] Spinal cord injury (SCI) is a serious injury to the central nervous system, which leads to motor and sensory dysfunction of the limbs below the level of injury. In severe cases, it can lead to permanent spinal cord dysfunction and seriously affect the quality of life of patients. The main cause of dysfunction after SCI is direct mechanical injury leading to neuronal damage, and subsequent secondary damage, including inflammatory response, oxidative stress response and excitatory injury, etc., resulting in neuronal axonal damage and glial Plasma cells proliferate, eventually leading to neuronal cell death. Neurological dysfunction caused by SCI is permanent because after spinal cord tissue damag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/52A61K38/46A61P25/00C12N9/90G01N33/573C12Q1/6883C12N15/11
CPCA61K38/52A61K38/46C12N9/90G01N33/573C12Q1/6883C12Y503/04001C12Y306/01003A61P25/00G01N2333/99G01N2800/28G01N2500/04C12Q2600/158C12Q2600/136A61K38/00A61K2300/00
Inventor 阳华纪志盛罗剑贤林宏生
Owner JINAN UNIVERSITY
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