CN114099642A - Application of gene Apln in preparation of drugs for treating diseases related to cell angiogenesis - Google Patents
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
Abstract
The invention belongs to the technical field of biomedicine, and discloses application of a gene Apln in preparation of a medicine for treating a cell-angiogenesis related disease, wherein the cell-angiogenesis related disease is nerve injury. The cell-angiogenic disease is nerve injury, preferably spinal cord injury. The invention also discloses a medicine for treating diseases related to cell angiogenesis, a medicine target gene Apln, and a detection reagent for the condition after repairing the diseases related to cell angiogenesis, which comprises a tissue immunofluorescence staining reagent for detecting the protein level of Apln. The invention provides the application of the gene Apln in the preparation of the medicine for treating the diseases related to the cell hemangioblast, provides a new treatment direction for treating the diseases related to the cell hemangioblast, and provides a theoretical basis for the medicine research by taking the gene Apln as a molecular intervention target.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of a gene Apln in preparation of a medicine for treating a cell-angiogenesis related disease, a medicine for treating the cell-angiogenesis related disease and a reagent for detecting the condition of the cell-angiogenesis related disease after repair.
Background
Spinal cord injury is a serious devastating disease that causes severe neurological dysfunction below the level of injury in patients, placing a burden on the patient and on the patient's family. Spinal cord injury can result in primary and secondary injuries. Primary lesions include loss of blood vessels, transection of axons, and necrosis of cells. Secondary injuries include apoptosis/death of cells spreading from the site of injury, degeneration and demyelination of axons. The abundant blood vessels in the spinal cord transport nutrients and associated regenerative factors to the spinal cord, providing nutrients and support for the injured, regenerated axons. The loss of local blood vessels resulting from spinal cord injury disrupts the blood-spinal cord barrier, causing inflammation and ischemia, exacerbating the impairment of neural function. Current research on post-spinal cord injury focuses primarily on glial scarring, inflammatory responses, and the like, but little attention has been paid to the regulatory role of blood vessels in spinal cord injury.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides an application of a gene Apln in preparing a medicine for treating diseases related to cell angiogenesis, a medicine for treating diseases related to cell angiogenesis and a reagent for detecting the condition after repairing the diseases related to cell angiogenesis.
In order to achieve the above object, the embodiment of the present invention provides an application of a gene Apln in preparing a medicament for treating a disease associated with cell angiogenesis.
Furthermore, the medicine takes the gene Apln as a molecular intervention target.
Further, the cell-angiogenesis-related disease is nerve injury.
Preferably, the nerve injury is spinal cord injury.
Preferably, the medicament is used for treating secondary injury caused by blood vessel loss and blood vessel loss after spinal cord injury, and the medicament is used for promoting angiogenesis and axon growth.
Preferably, the medicament reduces the protein level of the gene Apln after spinal cord injury.
The embodiment of the invention also provides a medicament for treating diseases related to cell angiogenesis, which is characterized by comprising an Apln medicament, wherein the Apln medicament is Apelin TFA 13.
Further, the medicine also comprises medically acceptable auxiliary agents.
The embodiment of the invention also provides a reagent for detecting the condition of the repaired cell-angioblasts-related diseases, which is characterized by comprising a tissue immunofluorescent staining reagent for detecting the level of the Apln protein.
Preferably, the detection reagent further comprises CD31 labeled blood vessels and Tuj1 labeled axons to detect blood vessel and axon status.
The technical scheme of the invention has the following beneficial effects:
(1) the invention provides the application of the gene Apln in the preparation of the medicine for treating the diseases related to the cell hemangioblast, provides a new treatment direction for treating the diseases related to the cell hemangioblast, and provides a theoretical basis for the medicine research by taking the gene Apln as a molecular intervention target.
(2) A spinal cord injury model is constructed in the embodiment of the invention, and the results of qRT-PCR verification of the differential genes at different time points after spinal cord injury show that the expression of Apln is obviously increased at 1d, and then the expression levels of 3d and 7d are reduced, which are consistent with the sequencing result. And performing tissue immunofluorescence verification on the Apln protein at different time points after spinal cord injury. CD31 labeled blood vessels, Apln labeled target protein, DAPI labeled nucleus. The histochemical results show that Apln protein levels after spinal cord injury are elevated at 3d expression levels after injury and subsequently decreased by 7 d. Meanwhile, an SD rat spinal cord hemisection injury model is constructed, Apln medicament (Apelin TFA 13) treatment is carried out, and a tissue immunostaining result shows that the areas of blood vessels and axons (Tuj 1 positive) of rats at the injury position after the Apelin TFA13 treatment are more than those of a PBS treatment group; the reliability of the invention is verified through the experiments, and a theoretical basis is provided for further research.
Drawings
FIG. 1 is a graph showing the change in the expression level of the key gene Apln mRNA, which was examined in example 1 of the present invention.
FIG. 2 is a photograph of immunofluorescence staining of Apln protein tissue at a lesion site at various time points after spinal cord lesion in example 2 of the present invention.
FIG. 3 is a graph of immunofluorescence staining of tissue of axons (Tuj 1) and blood vessels (CD 31) in spinal cord injury region after Apelin/PBS treatment after spinal cord injury in SD rats in example 3 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.
Example 1 mRNA expression of Apln after spinal cord injury in SD rats
1. Construction of spinal cord hemisection injury model of SD rat
Adult female Sprague-Dawley rats (200-. Rats were anesthetized by intraperitoneal injection of 85 mg/kg chloral monohydrate, 42 mg/kg magnesium sulfate, and 17 mg/kg sodium pentobarbital) mixed anesthetic. Following induction of anesthesia, laminectomy was performed at T9-T10. After exposing the spinal cord, the spinal cord was hemisected by an ophthalmic iris knife on the right side of T9 and the hemisection was ensured to be complete. Sporoxylum is sprinkled on the wound to prevent infection, fascia, muscle tissue and skin are sutured, and iodophors are wiped. Body temperature was maintained at 37 ℃ with a heating blanket throughout the procedure. And (3) giving the experiment animal drinking water with the cephalosporin after the operation to prevent wound infection.
2. Sequencing of RNA after sampling
Segments of 5 mm length were taken at each time point (0 h, 0.5h, 3h, 6h, 12h, 1d, 3d, 7d, 14d, 21d and 28 d) after spinal cord injury in 18 rats, distal and proximal to the site of injury at T9. Segments from 6 rats at each time point were mixed together. The tissue was placed in a centrifuge tube of RNasefree, 1ml Trizol lysate was added, ground electrokinetically and left at room temperature for 5 min. Adding 0.2 ml chloroform, shaking vigorously for 15s, standing at room temperature for 3 min, centrifuging at 4 deg.C for 15 min, and 12000 Xg. Taking the upper water phase in a new centrifuge tube, adding 0.5 ml isopropanol, standing for 10 min, centrifuging at 4 deg.C for 10 min, 12000 Xg. The supernatant was removed, 1ml of 75% ethanol was added to wash the RNA pellet, shaken and centrifuged at 4 ℃ at 7500 Xg. Removing supernatant, opening the centrifuge tube containing the RNA precipitate, putting into a fume hood for air drying, adding a proper amount of RNase-free water after the precipitate becomes semitransparent, and determining the concentration of the RNA by using Onedrop. The RNA was then sequenced, three replicates per time point.
3. Verification of Gene expression by qRT-PCR
After extracting RNA according to the steps, performing reverse transcription by using a TaKaRa kit, performing reverse transcription on 500 ng of RNA to cDNA, and performing operation on ice, wherein each reaction system is 10 mu l, and the reaction conditions in a PCR instrument are as follows: 45 min at 37 ℃, 5 min at 85 ℃ and infinity at 4 ℃. The cDNA obtained by reverse transcription was diluted 10-fold with RNase-free water. PCR was performed in ABI Stepone system by adding corresponding reagents, templates and primers according to SYBR Premix Ex Taq system. The Real-time PCR instrument program is: stage 195 deg.C, 2 min; stage 2 cycle 35, 95 ℃ for 15s, 60 ℃ for 1 min; stage 395 ℃ for 15s, 60 ℃ for 1 min and 95 ℃ for 15 s. Each sample was 3 replicates with GAPDH as internal control. And (4) carrying out statistical analysis on the experiment according to the experiment requirement.
qRT-PCR verification is carried out on Apln mRNA at different time points after spinal cord injury, the result is shown in figure 1, the qRT-PCR detection shows the expression change of Apln mRNA at different time points after rat hemisection injury, a bar chart represents the relative expression quantity of mRNA, a broken line represents the FPKM expression quantity of RNA-Seq, and R represents the FPKM expression quantity of RNA-Seq2Represents the correlation of the results of qRT-PCR and RNA-Seq (with GAPDH as internal reference, p)<0.05, **p<0.01, ***p<0.001，****p<0.0001); as can be seen in FIG. 1, Apln expressed significantly at 1d mRNA levels, followed by decreased 3d and 7d expression levels after spinal cord injuryThe results were consistent with the sequencing results.
Example 2: apln protein expression verification by tissue immunofluorescence after spinal cord injury
1. Perfused material selection and frozen sectioning
Rats were anesthetized by intraperitoneal injection and perfused with 4% paraformaldehyde via the heart. Spinal cord samples 10 mm long from the proximal region of the injury site to the distal region of the injury site were taken at designated times (n =3 per time point) after surgery. All tissues were fixed for 6 hours before transfer to 30% sucrose and sections frozen after the tissues settled in 30% sucrose solution. 20 μm rows were frozen longitudinally and attached directly to the slide.
2. Tissue immunofluorescence staining verification of Apln protein expression
Slides were rewarmed at room temperature and washed three times with PBS for 10 min each. The tissue was circled with a pen, added with immunostaining blocking solution, and incubated at 37 ℃ for 1 h. Removing the sealing solution, adding the primary antibody Apln diluted according to a certain proportion, and incubating overnight at 4 ℃. Discard primary antibody, wash the slide three times with PBS for 10 min each time, and then react with secondary antibody for 2h at room temperature. The secondary antibody is discarded, the PBS is washed for three times, each time for 10 min, and the mounting is carried out by using an anti-fluorescence quenching mounting agent. Finally, the sections were observed and photographed under a fluorescent microscope (axioiimager M2, Zeiss).
Tissue immunofluorescence was performed on Apln protein levels at different time points in the damaged area after spinal cord injury, as shown in fig. 2, Apln labeled target protein, DAPI labeled nuclei. The results indicate that Apln expression levels are elevated 3d after injury and subsequently decreased by 7d after spinal cord injury (scale bar: 250 μm).
Example 3: tissue immunostaining Tuj1 and CD31 after intraperitoneal administration of Apelin/PBS after spinal cord injury
1. Construction of rat spinal cord hemisection injury model
The control group and the experimental group are 5 respectively, the experimental group constructs a rat spinal cord hemisection injury model according to the example 1, and the control group exposes the spinal cord without injury.
2. Administration to the abdominal cavity in vivo
After spinal cord injury, the experimental group was intraperitoneally injected with Apelin TFA13 (concentration 1 μm) at 200 μ g/kg/day, and the control group was intraperitoneally injected with PBS for one week.
3. Tissue immunostaining Tuj1 and CD31
Two weeks after administration, SD rats of the experimental group and the control group were perfused and sampled, and the rats were anesthetized by intraperitoneal injection of a mixed anesthetic of 85 mg/kg chloral monohydrate, 42 mg/kg magnesium sulfate, and 17 mg/kg sodium pentobarbital), and perfused with 4% paraformaldehyde via the heart. A 10 mm long spinal cord sample was taken from the proximal end of the injury site to the distal end of the injury site. 4 ℃ overnight in 4% paraformaldehyde, transferred to 30% sucrose, frozen and sectioned after the tissue has settled in 30% sucrose solution. 20 μm rows were frozen longitudinally and attached directly to the slide. Then, immunofluorescent staining was performed. Slides were rewarmed at room temperature and washed three times with PBS for 10 min each. The tissue was circled with a pen, added with immunostaining blocking solution, and incubated at 37 ℃ for 1 h. The blocking solution was discarded, and primary anti-CD 31 and Tuj1 diluted in a certain ratio were added thereto, and incubated overnight at 4 ℃. Then reacted with secondary antibody at room temperature for a further 2 h. Finally, the sections were observed and photographed under a fluorescent microscope (axioiimager M2, Zeiss).
After Apelin/PBS treatment of SD rats after spinal cord hemisection injury, tissue immunofluorescence staining was performed on the spinal cord injury area, and the results are shown in FIG. 3, wherein CD31 marks blood vessels, Tuj1 marks axons, and DAPI marks cell nuclei (scale bar: 500 μm). The results indicate that the vascular and axonal area of the spinal cord injury area of rats treated with Apelin TFA13 was significantly greater than that of the PBS-treated control group.
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 (10)
1. An application of a gene Apln in preparing a medicine for treating diseases related to cell angiogenesis.
2. The use according to claim 1, wherein the medicament is targeted to the molecular intervention of the gene Apln.
3. The use of claim 1, wherein the cellular angiogenesis-related disease is nerve damage.
4. The use of claim 3, wherein the nerve injury is a spinal cord injury.
5. The use according to claim 4, wherein the medicament is for the treatment of secondary injury after spinal cord injury due to loss of blood vessels and loss of blood vessels, the medicament being for promoting angiogenesis and axonal growth.
6. The use of claim 4, wherein said medicament reduces the protein level of the gene Apln following spinal cord injury.
7. The drug for treating the diseases related to the cell angiogenesis is characterized by comprising an Apln drug, wherein the Apln drug is Apelin TFA 13.
8. The medicament of claim 1, further comprising a medically acceptable auxiliary agent.
9. A reagent for detecting the condition of a cell after repairing a hemangioblast-related disease, which comprises a tissue immunofluorescent staining reagent for detecting the level of an Apln protein.
10. The detection reagent of claim 7, wherein the detection reagent further comprises CD31 labeling blood vessels and Tuj1 labeling axons to detect blood vessel and axon status.
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