CN110904200B - Method for detecting expression level of HOXD9 after peripheral nerve injury and application of HOXD9 gene - Google Patents
Method for detecting expression level of HOXD9 after peripheral nerve injury and application of HOXD9 gene Download PDFInfo
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- CN110904200B CN110904200B CN201911236200.6A CN201911236200A CN110904200B CN 110904200 B CN110904200 B CN 110904200B CN 201911236200 A CN201911236200 A CN 201911236200A CN 110904200 B CN110904200 B CN 110904200B
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Abstract
The invention provides a method for detecting the expression level of HOXD9 after peripheral nerve injury, which 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 result expression quantity and expression trend of the related messenger RNA of the HOXD9 by applying a PCR technology; (3) Displaying intracellular localization of HOXD9 in DRG neurons by immunofluorescence staining; (4) And (3) counting the change trend of the fluorescence intensity of the HOXD9 at different time points after sciatic nerve injury, wherein the fluorescence intensity reaches the brightest in 3 days and is consistent with the result trend obtained by a PCR (polymerase chain reaction) experiment. The invention defines the expression quantity change of the HOXD9 after peripheral nerve injury, and discovers the regulating effect of the HOXD9 on the regeneration of neuron axons.
Description
Technical Field
The invention belongs to the field of basic medicine of neuroscience, and particularly relates to a method for detecting the expression quantity of HOXD9 after peripheral nerve injury and application of a HOXD9 gene.
Background
The HOX (homeobox gene) gene family includes four gene clusters: HOXA, HOXB, HOXC, HOXD. Among them, the HOXD9 gene plays an important role in the development of the spine and upper limbs, and has high expression in tissues such as kidney, testis, colon, spleen, placenta, and bladder. HOXD9 has been shown to act as a protooncogene in hepatocellular carcinoma studies, and has a promoting effect on migration and invasion of cancer cells. Down-regulation of HOXD9 expression may lead to hypermethylation of DNA promoters in a variety of cancers, affecting tumor prognosis. However, the role and mechanism of HOXD9 in nerve injury and repair is not well-defined.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for detecting the expression level of HOXD9 after peripheral nerve injury and an application of the HOXD9 gene, to clarify the expression level change of HOXD9 after peripheral nerve injury and to discover the regulating effect of HOXD9 on the regeneration of neuron axons.
In order to solve the above technical problems, an embodiment of the present invention provides a method for detecting an expression level of HOXD9 after peripheral nerve injury, including the following steps:
(1) SD rat L4-6 dorsal root ganglion injured by sciatic nerve clamping at different time points is placed in a Ca-free position 2 +/Mg 2 Cutting off redundant tissues in Hibernate A, transferring the cut redundant tissues into 1mg/mL collagenase, digesting for 90min at 37 ℃, then digesting for 15min at 37 ℃ by using 0.25% pancreatin, adding a stop solution to stop the reaction, blowing and beating by using a 1mL gun head, fully and uniformly mixing, centrifuging at 900rpm multiplied by 5min; discard the supernatant, centrifuge BSA 2 times at 15% concentration, 900rpm × 5min; discarding the supernatant, adding culture medium, mixing the cells uniformly, and sieving with a 200-mesh sieve to obtain relatively pure DRG neurons; the cells are frozen and stored quickly, after all time points are collected, the nucleoprotein separation and the activity detection of the transcription factor are carried out, and the expression quantity change trend of the transcription factor HOXD9 in the nerve regeneration process is determined;
(2) Observing the result expression quantity and expression trend of the related messenger RNA of the HOXD9 by applying a PCR technology;
(3) Displaying intracellular localization of HOXD9 in DRG neurons by immunofluorescence staining;
(4) And (4) counting the change trend of the fluorescence intensity at different time points after sciatic nerve injury, wherein the fluorescence intensity reaches the brightest in 3 days and is consistent with the result trend obtained by a PCR (polymerase chain reaction) experiment.
Wherein the time points in the step (1) comprise 0h,15min,30min,3h,12h,1d and 3d.
Wherein the stop solution used in the step (1) is a PBS solution containing 10% FBS.
Wherein the weight of the SD rat in the step (1) is 180-220 g.
The invention also provides an application of the HOXD9 gene in preparing a nerve regeneration promoting medicine.
The technical scheme of the invention has the following beneficial effects:
the invention defines the expression quantity change of the HOXD9 after peripheral nerve injury, and discovers the regulating effect of the HOXD9 on the regeneration of neuron axons.
Drawings
FIG. 1 is a graph showing the change in expression level of HOXD9 at different time points after injury caused by pinching sciatic nerve according to the present invention;
FIG. 2 is a graph showing the result of transparentization and staining of sciatic nerve clamp injury after homozygote injection of virus into HOXD9 Cas9-CKO mice in the invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
Example 1
The invention provides a method for detecting the expression level of HOXD9 after peripheral nerve injury, which comprises the following steps:
(1) SD rats (180-220 g) with sciatic nerve injured by taking different time points (0h, 15min,30min,3h,12h,1d, 3d) and placing the SD rats in the back root ganglia of L4-6 without Ca 2 +/Mg 2 In the positive-Hibernate A, cutting off redundant tissues, transferring the cut redundant tissues into 1mg/mL collagenase, digesting the collagenase for 90min at 37 ℃, then digesting the collagenase for 15min at 37 ℃ by using 0.25 percent pancreatin (gently blowing and beating the tissues every 5min to ensure the tissues to be fully digested), adding a stop solution (PBS containing 10 percent FBS) to stop the reaction, blowing and beating the collagenase by using a 1mL gun head, fully and uniformly mixing the solution, centrifuging the solution, and multiplying the reaction time by 900rpm for 5min; discard the supernatant, centrifuge BSA 2 times at 15% concentration, 900rpm × 5min;
discarding the supernatant, adding culture medium, mixing the cells, sieving with 200 mesh sieve to obtain relatively pure DRG neuron, and rapidly freezing the cells;
after all time points are collected, delivering the collected data to Wuhan Banner biology Limited company for nucleoprotein separation and transcription factor activity detection, and determining the expression quantity change trend of the transcription factor HOXD9 in the nerve regeneration process;
(2) Observing the result expression quantity and expression trend of related messenger RNA (mRNA) of the HOXD9 by applying a PCR (Polymerase Chain Reaction) technology;
(3) Displaying intracellular localization of HOXD9 in DRG neurons by immunofluorescence staining;
(4) And (4) counting the change trend of the fluorescence intensity at different time points after sciatic nerve injury, wherein the fluorescence intensity reaches the brightest in 3 days and is consistent with the result trend obtained by a PCR (polymerase chain reaction) experiment.
The specific experimental procedures and results are as follows:
through separating Dorsal Root Ganglion (DRG) neuron nucleoproteins at different time points after rat sciatic nerve injury, transcription factor activity detection is carried out, and the expression quantity of a transcription factor HOXD9 is gradually increased in the nerve regeneration process, reaches a peak value in 3 days and then is reduced (as shown in figure 1A); the protein level expression changes were substantially consistent with messenger RNA (mRNA) results (FIG. 1B). Immunofluorescent staining showed that HOXD9 was mainly localized in the cytoplasm of DRG neurons. The trend of the fluorescence intensity at different time points after the injury was also consistent with the results obtained from our previous experiment, and the fluorescence intensity reached the brightest at 3 days (see FIG. 1C). In fig. 1, scale =50 μm.
Example 2
The invention also provides an application of the HOXD9 gene, which can be used for preparing a nerve regeneration promoting medicine and researching the function and mechanism of the HOXD9 in nerve injury and repair, and clearly indicates whether the expression of the HOXD9 influences the nerve regeneration repair.
The specific experimental procedures and results are as follows:
HOXD9 Cas9-CKO mice constructed by entrusted Nanjing university model animals, virus PFD-rAAV-CMV-Cre-WPRE-pA (AAV 2/9 type) purchased from Wuhan Shu Mikan scientific and technological Limited company and control virus thereof were intrathecally injected into homozygous Hoxd9 Cas9-CKO mice, sciatic nerve injury was performed after 3 weeks, sciatic nerve transparentization was performed by perfusion after 3 days, and axon regeneration was marked by STMN2 staining (see FIG. 2). The length of regenerated axons after sciatic nerve injury of mice in an experimental group with HOXD9 selectively knocked out is obviously shortened compared with that of a control group, and the suggestion that the axons after sciatic nerve injury can be influenced by interfering with the expression of HOXD 9. In FIG. 2, FIG. 2A is a schematic view showing the intrathecal injection of the virus, and FIG. 2B is a schematic view showing the growth of regenerated axons after 3 days of sciatic nerve dissection and staining with STMN2 (i.e., SCG 10). In fig. 2, scale =500 μm.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. The application of the HOXD9 gene is characterized in that the application is used for preparing a nerve regeneration promoting medicine.
2. The use according to claim 1, wherein the method for detecting the expression level of HOXD9 after peripheral nerve injury comprises the following steps:
(1) SD rat L4-6 dorsal root ganglion injured by sciatic nerve at different time points is placed in a Ca-free position 2 +/Mg 2 Cutting off redundant tissues in Hibernate A, transferring the cut redundant tissues into 1mg/mL collagenase, digesting for 90min at 37 ℃, then digesting for 15min at 37 ℃ by using 0.25% pancreatin, adding a stop solution to stop the reaction, blowing and beating by using a 1mL gun head, fully and uniformly mixing, centrifuging at 900rpm multiplied by 5min; discard the supernatant, centrifuge 2 times in 15% BSA at 900rpm × 5min; discarding the supernatant, adding the culture medium, uniformly mixing the cells, and screening the cells by a 200-mesh screen to obtain relatively pure DRG neurons; the cells are frozen and stored quickly, after all time points are collected, the nucleoprotein separation and the activity detection of the transcription factor are carried out, and the expression quantity change trend of the transcription factor HOXD9 in the nerve regeneration process is determined;
(2) Observing the result expression quantity and expression trend of the related messenger RNA of the HOXD9 by applying a PCR technology;
(3) Displaying intracellular localization of HOXD9 in DRG neurons by immunofluorescence staining;
(4) And (3) counting the change trend of the fluorescence intensity of the rat at different time points after sciatic nerve injury, wherein the fluorescence intensity reaches the brightest in 3 days and is consistent with the result trend obtained by a PCR experiment.
3. The use according to claim 2, wherein the time points in step (1) comprise 0h,15min,30min,3h,12h,1d and 3d.
4. The use according to claim 2, wherein the stop solution used in step (1) is 10% FBS in PBS.
5. The use according to claim 2, wherein the weight of the SD rat in the step (1) is 180-220 g.
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| WO2003074736A1 (en) * | 2002-03-04 | 2003-09-12 | Institute Of Gene And Brain Science | Diagnostics and remedies for malignant brain tumor |
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| WO2003074736A1 (en) * | 2002-03-04 | 2003-09-12 | Institute Of Gene And Brain Science | Diagnostics and remedies for malignant brain tumor |
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| Title |
|---|
| Hox Genes and Spinal Cord Development;Ellen M. Carpenter;《Dev Neurosci》;20020731;第24卷(第1期);24-34 * |
| Targeted Disruption of Hoxd9 and Hoxd10 Alters Locomotor Behavior, Vertebral Identity, and Peripheral Nervous System Development;Cecile C. de la Cruz等;《Developmental Biology》;19991215;第216卷(第2期);595-610 * |
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