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Method for detecting expression quantity of PDHB in neuronal cells and application of PDHB gene

A neuron cell and detection method technology, applied in the application field of PDHB gene, can solve problems such as unclear function and mechanism

Pending Publication Date: 2020-09-25
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the role and mechanism of PDHB in nerve injury and repair are still unclear

Method used

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  • Method for detecting expression quantity of PDHB in neuronal cells and application of PDHB gene
  • Method for detecting expression quantity of PDHB in neuronal cells and application of PDHB gene

Examples

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Effect test

Embodiment 1

[0019] Embodiment 1: A kit for detecting the expression level of PDHB in neuronal cells, which is characterized in that it includes the virus pAAV-CMV-bGlobin-eGFP-U6-shRNA containing PDHB interfering RNA and its control virus, and Tuj1 antibody.

Embodiment 2

[0020] Embodiment 2: A method for detecting the expression level of PDHB in neuron cells using a detection kit for the expression level of PDHB in neuron cells, characterized in that it comprises the following steps:

[0021] (2-1) By isolating DRG neurons at different time points after sciatic nerve injury in rats, real-time quantitative PCR was performed to detect PDHB mRNA, which increased rapidly within 3 hours after injury, and returned to normal levels in the next 3 days;

[0022] (2-2) Infect DRG neuron cells cultured in vitro with PDHB interference RNA virus and its control virus, and collect the infected DRG neuron cells after 5 days for real-time quantitative PCR to detect the interference efficiency of PDHB interference virus; at the same time, use 0.025 Digest the infected DRG neurons with % trypsin for 20s, add 10% FBS to terminate the reaction, centrifuge at 900rpm for 5min, discard the supernatant, add fresh Neurobasal medium to resuspend the infected DRG neurons...

Embodiment 3

[0041] Example 3: The present invention also provides an application of PDHB gene, which is used to prepare drugs for promoting nerve regeneration, and can also be used to study the role and mechanism of PDHB in nerve injury and repair, and to clarify whether intervening in the expression of PDHB will affect nerve regeneration and repair .

[0042] The specific experimental steps and results are as follows:

[0043] (3-1) SD rats were intrathecally injected with PDHB interference virus or its control virus, and infected rat DRG neurons. The results were as follows: figure 2 As shown in A, figure 2 The scale bar in A=50 μm;

[0044] (3-2) After 3 weeks, the sciatic nerve was crushed, and the sciatic nerve was perfused 3 days after the operation to measure the damaged sciatic nerve, and the regenerated axons were marked with SCG10 staining. The results are as follows figure 2 As shown in B, figure 2 Scale in B = 1 mm;

[0045] (3-3) Use Photoshop software to count the f...

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Abstract

The invention provides a method for detecting the expression quantity of PDHB in neuronal cells, which comprises the following steps: (1) separating DRG neurons at different time points after sciaticnerve injury of a rat, carrying out PCR to detect mRNA, and determining the change trend of the expression quantity of PDHB in a nerve regeneration process; (2) infecting DRG neuronal cells cultured in vitro with PDHB interference viruses or control viruses thereof, detecting the interference efficiency of PDHB through PCR, counting the longest length and the total length of DRG neuron axons afterresuspension, and prompting that axon regeneration of DRG neurons can be influenced by in vitro interference of PDHB expression; and (3) injecting PDHB interference viruses or control viruses thereofinto rat sheaths, and counting SCG10 fluorescence intensity change trends and axon regeneration indexes at different time points after sciatic nerve injury, which are consistent with result trends obtained by in-vitro experiments. According to the invention, the expression quantity change of PDHB in neuronal cells is clarified, and the regulation effect of PDHB on nerve regeneration is found.

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 PDHB in neuron cells and the application of the PDHB gene. Background technique [0002] Pyruvate dehydrogenase β (pyruvate dehydrogenase β, PDHB) is one of the essential subunits that constitute the pyruvate dehydrogenase complex (PDH). Participate in catalyzing the generation of acetyl-CoA from pyruvate derived from glycolysis or lactic acid metabolism, and acetyl-CoA enters the tricarboxylic acid cycle and is decomposed into CO 2 and H 2 O, and release ATP to provide energy for cell proliferation and differentiation. Extensively present in the cytoplasm, it can enter the nucleus under stimuli such as growth factors, oncogenic signals, and mitochondrial stress, and participates in promoting the acetylation of histone H3K9 and H3K18 to regulate gene expression. The expression of PDHB in tumor cells increase...

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N5/0793C12N15/86
CPCG01N33/56966C12N5/0619C12N15/86Y02A50/30
Inventor 于彬曹倩倩王东刘明稳刘炎顾晓松
Owner NANTONG UNIVERSITY
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