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Method for detecting expression quantity of HOXD9 after peripheral nerve injury, and application of HOXD9 gene

A peripheral nerve injury, HOXD9 technology, applied in the field of neuroscience basic medicine, can solve the problems of hypermethylation, affecting tumor prognosis, and unclear role and mechanism

Active Publication Date: 2020-03-24
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Downregulation of HOXD9 expression leads to hypermethylation of DNA promoters in various cancers and affects tumor prognosis
However, the role and mechanism of HOXD9 in nerve injury and repair are still unclear

Method used

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  • Method for detecting expression quantity of HOXD9 after peripheral nerve injury, and application of HOXD9 gene
  • Method for detecting expression quantity of HOXD9 after peripheral nerve injury, and application of HOXD9 gene

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Embodiment 1

[0019] The present invention provides a method for detecting the expression level of HOXD9 after peripheral nerve injury, comprising the following steps:

[0020] (1) By taking different time points (0h, 15min, 30min, 3h, 12h, 1d, 3d) of SD rats (180-220g) with sciatic nerve crushing, the L4-6 dorsal root ganglion was placed in a Ca-free 2 + / Mg 2 +- In Hibernate A, after cutting off excess tissue, transfer it to 1mg / mL collagenase, digest at 37°C for 90min, then digest with 0.25% trypsin at 37°C for 15min (blow gently every 5min to fully digest the tissue), add Stop the reaction with stop solution (PBS containing 10% FBS), pipette with a 1 mL pipette tip, mix thoroughly, centrifuge, 900rpm×5min; discard the supernatant, and centrifuge twice with 15%BSA, 900rpm×5min;

[0021] Discard the supernatant, add the medium, mix the cells, pass through a 200-mesh sieve, and finally obtain relatively pure DRG neurons, and quickly freeze the cells;

[0022] After all the time points are...

Embodiment 2

[0029] The present invention also provides an application of the HOXD9 gene, which can be used to prepare nerve regeneration-promoting drugs, and can also be used to study the function and mechanism of HOXD9 in nerve injury and repair, and determine whether interfering with the expression of HOXD9 will affect nerve regeneration and repair.

[0030] The specific experimental steps and results are as follows:

[0031] The Institute of Model Animals of Nanjing University was commissioned to construct HOXD9 Cas9-CKO mice, and the virus PFD-rAAV-CMV-Cre-WPRE-pA (AAV2 / 9 type) and its control virus purchased from Wuhan Privy Brain Science and Technology Co., Ltd. were injected into the sheath Injected into homozygous Hoxd9 Cas9-CKO mice, the sciatic nerve was crushed 3 weeks later, the sciatic nerve was perfused 3 days later, and the sciatic nerve was cleared, and the regenerated axons were marked by STMN2 staining (such as figure 2 ). It was found that the length of axon regenerat...

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Abstract

The invention provides a method for detecting the expression quantity of HOXD9 after peripheral nerve injury. The method comprises the following steps: (1) separating dorsal root ganglion neuron nucleoproteins at different time points after sciatic nerve injury of a rat, performing transcription factor activity detection, and determining the expression quantity change trend of a transcription factor HOXD9 in the nerve regeneration process; (2) observing the expression quantity and the expression trend of related messenger RNA results of HOXD9 by applying a PCR technology; (3) displaying intracellular localization of the HOXD9 in an DRG neuron by adopting an immunofluorescence staining technology; and (4) counting the HOXD9 fluorescence intensity change trends at different time points afterthe sciatic nerve injury, and enabling the fluorescence intensity to be the highest on the third day and to be consistent with a result trend obtained by a PCR experiment. The expression quantity change of HOXD9 after peripheral nerve injury is clarified, and the regulation effect of THE HOXD9 on neuron axon regeneration is discovered.

Description

technical field [0001] The invention belongs to the field of neuroscience basic medicine, and in particular relates to a method for detecting the expression level of HOXD9 after peripheral nerve injury and the application of the HOXD9 gene. Background technique [0002] The HOX (homebox, homeobox gene) gene family includes four gene clusters: HOXA, HOXB, HOXC, and HOXD. Among them, the HOXD9 gene plays an important role in the development of the spine and upper limbs, and is highly expressed in tissues such as the kidney, testis, colon, spleen, placenta and bladder. HOXD9 acts as a proto-oncogene in the study of hepatocellular carcinoma and promotes the migration and invasion of cancer cells. Downregulation of HOXD9 expression can lead to hypermethylation of DNA promoters in various cancers and affect tumor prognosis. However, the role and mechanism of HOXD9 in nerve injury and repair are still unclear. Contents of the invention [0003] The technical problem to be solv...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851A61K38/17A61P25/00G01N21/64
CPCC12Q1/6851A61P25/00G01N21/6428A61K38/00C12Q2531/113
Inventor 周松林徐灵驰秦婧于彬顾晓松
Owner NANTONG UNIVERSITY
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