Method for verifying influence of SLC5A2 gene on intervertebral disc degenerative disease

Abstract

The invention discloses a method for verifying the influence of SLC5A2 gene on intervertebral disc degeneration disease, the method comprises animal experiment verification and clinical tissue verification, and the animal experiment verification step comprises an intervertebral disc degeneration model establishment step, an X-ray examination step, an MRI examination step and a histological detection step; the clinical tissue verification step comprises a sample collection and classification step, a histological detection step and a western blot experiment step. The invention solves the problem of lack of a verification method for verifying that the SLC5A2 gene can be used for early screening and preventing the intervertebral disc degenerative disease in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of verification methods for intervertebral disc degeneration, and in particular to a verification method for the influence of SLC5A2 gene on intervertebral disc degeneration. Background Art

[0002] Degenerative disc disease is caused by aging, long-term strain, trauma and other reasons. The intervertebral disc is mainly composed of the nucleus pulposus, the annulus fibrosus that wraps the forehead, and the cartilage endplate that supports these two parts. The nucleus pulposus is isolated from the body's immune microenvironment under physiological conditions. Trauma and other reasons cause the annulus fibrosus of the intervertebral disc to rupture, capillaries invade the nucleus pulposus, inflammatory factors and immune cells infiltrate the nucleus pulposus through blood vessels and induce inflammation, resulting in damage to the nucleus pulposus tissue and thus inducing degenerative disc disease. The intervertebral disc supports and stabilizes the spine. The elasticity and tension function of the degenerated intervertebral disc is reduced, resulting in limited body movement function of the patient. When it is aggravated, it may even compress the spinal nerves and affect the function of the patient's lower limbs, or cause symptoms such as incontinence and lower limb paralysis. Degenerative disc disease brings huge economic and social burdens. In the traditional diagnosis and screening of intervertebral disc degeneration, X-rays, CT or MRI examinations are usually used to determine whether the patient has intervertebral disc degeneration. This screening method can only be used for diagnosis after the onset of the disease and cannot play a preventive role. Therefore, a diagnostic method that can screen and prevent disc degeneration at an early stage is urgently needed. SLC5A2 is a gene that encodes sodium-glucose cotransporter 2 (SGLT2). SGLT2 inhibitors have anti-inflammatory effects and are widely used in the treatment of diabetes and chronic kidney disease. We found that the expression of the SLC5A2 gene was significantly upregulated in the nucleus pulposus tissue of patients with disc degeneration, and the disc degeneration was significantly improved after the application of SGLT2 inhibitors. Therefore, we speculate that SLC5A2 is a key gene for early screening of disc degeneration. However, the existing technology lacks a method for verifying whether a patient has disc degeneration through SLC5A2 gene screening, and the existing technology lacks a method for verifying whether the SLC5A2 gene will affect disc degeneration. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a method for verifying the effect of the SLC5A2 gene on intervertebral disc degeneration, in order to solve the problem that there is a lack of a verification method in the prior art for verifying that the SLC5A2 gene can perform early screening and prevention of intervertebral disc degeneration.

[0004] To achieve the above object, the present invention provides a method for verifying the effect of SLC5A2 gene on intervertebral disc degeneration, including animal experiment verification and clinical tissue verification, wherein the animal experiment verification comprises the following steps: S1. Several rats were randomly divided into a sham operation group, an intervertebral disc degeneration group, and an SGLT2 inhibitor group according to the number table method, and the three groups of rats were anesthetized; S2. Based on the S1 step, the rats in the sham operation group were only punctured in the skin without damaging the intervertebral disc tissue. The rats in the intervertebral disc degeneration group and the SGLT2 inhibitor group were perforated in the 6 / 7 intervertebral disc of the coccyx with a puncture needle, and the puncture needle pillow was rotated 360 degrees and kept in the intervertebral disc for 1 minute. S3. Based on step S2, establish an intervertebral disc degeneration model and culture it for four weeks; S4. Based on the steps in S3, in the SGLT2 inhibitor group, the SGLT2 inhibitor was injected into the intervertebral disc of the rats and an equal volume of dimethyl sulfoxide was injected once a week for four consecutive weeks; S5. Based on step S4, performing X-ray examination on a number of rats after four weeks of culture, and calculating the intervertebral disc height index according to the X-ray film to generate intervertebral disc height data; S6. Based on step S5, several rats cultured for eight weeks were examined by MRI, and the Pfirrmann grade was determined according to the MRI images; S7. Based on step S6, several rats that have undergone X-ray and MRI examinations are killed and corresponding intervertebral discs are taken, several intervertebral discs are fixed with formaldehyde for 48 hours, decalcified with EDTA decalcification solution for three weeks and embedded in paraffin, the paraffin-embedded slices are cut into first slices to be evaluated, the first slices to be evaluated are stained and the histological degree of intervertebral disc degeneration is evaluated on the stained slices; The clinical tissue validation includes the following steps: S8. Collect intervertebral disc tissues from several patients with intervertebral disc degeneration and divide them into a control group and a sample intervertebral disc degeneration group according to the degree of degeneration; S9. Based on step S8, extract the intervertebral disc tissue of the sample intervertebral disc degeneration group, divide the intervertebral disc tissue into a first tissue to be processed and a second tissue to be processed, fix the first tissue to be processed with formaldehyde for 48 hours, decalcify with EDTA decalcification solution for three weeks and embed in paraffin, and cut the paraffin-embedded slices into second slices to be evaluated; S10. Based on step S9, the second slices to be evaluated are divided into a first group of slices to be stained and a second group of slices to be stained, the first group of slices to be stained are stained to obtain a first group of stained slices, the second group of slices to be stained are immunohistochemically stained with a primary anti-SLC5A2 antibody to obtain a second group of stained slices, and the first group of stained slices and the second group of stained slices are compared with negative control slices to evaluate the histological degree of intervertebral disc degeneration; S11. Based on step S9, the second tissue to be processed is subjected to RIPA lysis buffer to collect the nucleus pulposus tissue protein sample. The protein sample is separated on SDS-PAGE gel and detected using SLC5A2 antibody. The chemiluminescent signal is visualized using a detection kit.

[0005] The present invention further provides that: in the step S11, an ECL Western Blot detection kit is used to visualize the chemiluminescent signal.

[0006] The present invention further provides that: the negative control slice in step S10 is a slice incubated with non-specific IgG.

[0007] The present invention further provides that: the first slice to be evaluated and the first group of slices to be stained are both stained with hematoxylin, eosin and safranin o-fast green.

[0008] The advantages of adopting the above technical solution are as follows: animal experiments have shown that SGLT2 inhibitors significantly improve the degeneration of rat intervertebral discs, that is, intervertebral disc degeneration can be clearly observed through X-ray examination images and magnetic resonance imaging images, see the appendix of the manual. Figure 1 and 2 The results of histological tests showed that SGLT2 inhibitors significantly improved the level of intervertebral disc degeneration in rats, which means that SGLT2 inhibitors can effectively improve the level of intervertebral disc degeneration in intervertebral disc degeneration, that is, SGLT2 inhibitors can affect the SLC5A2 gene, see the appendix of the manual Figure 3 ; Western blot experiments in clinical tissue validation showed that the SLC5A2 gene was significantly upregulated in degenerative disc tissue, see the appendix of the manual Figure 4 Through histological testing in clinical tissue verification, namely immunohistochemical images and SO staining images, it can be seen that the SLC5A2 gene is significantly upregulated in degenerative intervertebral discs. Figure 5 ; In the above technology, animal experiments and clinical tissue verification are used to verify that the SLC5A2 gene is significantly upregulated in intervertebral disc degenerative diseases, that is, in order to verify that the SLC5A2 gene can screen whether a patient has intervertebral disc degenerative diseases, the above technology shows that the SLC5A2 gene is significantly upregulated in the intervertebral disc tissue of patients with intervertebral disc degenerative diseases, that is, in actual clinical application and symptom analysis, the SLC5A2 gene can be used as one of the reference standards for whether a patient has intervertebral disc degenerative diseases. The above test method is used to screen whether a patient has intervertebral disc degenerative diseases at an early stage through the SLC5A2 gene and prevent intervertebral disc degenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1This is a result diagram of the intervertebral disc degeneration detected by X-ray film in the present invention; Figure 2 This is a result diagram of the intervertebral disc degeneration detected by nuclear magnetic resonance imaging in the present invention; Figure 3 This is a diagram of the histological test results verified by animal experiments in the present invention; Figure 4 This is a graph showing the results of a Western blot experiment verified by clinical tissues in the present invention; Figure 5 A comparison diagram of the first group of stained sections, the second group of stained sections and the invisible sections in the histological test verified by clinical tissue in the present invention; Figure 6 A schematic diagram of a sample for clinical tissue verification in the present invention; Figure 7 This is an example of the results of the X-ray film detection of intervertebral disc degeneration in the present invention, which is the intervertebral disc height index of the experimental animal after eight weeks; Figure 8 This is an example diagram of the results of the nuclear magnetic resonance detection of intervertebral disc degeneration in the present invention, which is the Pfirrmann grade determined based on the nuclear magnetic resonance image after eight weeks; Fig. 9 This is an example diagram of the histological test results verified by animal experiments in the present invention. DETAILED DESCRIPTION

[0010] The present invention provides a method for verifying the effect of the SLC5A2 gene on intervertebral disc degeneration, including animal experiment verification and clinical tissue verification, wherein the animal experiment verification comprises the following steps: S1. Several rats were randomly divided into a sham operation group, an intervertebral disc degeneration group, and an SGLT2 inhibitor group according to the number table method, and the three groups of rats were anesthetized; S2. Based on the S1 step, the rats in the sham operation group were only punctured in the skin without damaging the intervertebral disc tissue. The rats in the intervertebral disc degeneration group and the SGLT2 inhibitor group were perforated in the 6 / 7 intervertebral disc of the coccyx with a puncture needle, and the puncture needle pillow was rotated 360 degrees and kept in the intervertebral disc for 1 minute. S3. Based on step S2, establish an intervertebral disc degeneration model and culture it for four weeks; S4. Based on the steps in S3, in the SGLT2 inhibitor group, the SGLT2 inhibitor was injected into the intervertebral disc of the rats and an equal volume of dimethyl sulfoxide was injected once a week for four consecutive weeks; S5. Based on step S4, performing X-ray examination on a number of rats after four weeks of culture, and calculating the intervertebral disc height index according to the X-ray film to generate intervertebral disc height data; S6. Based on step S5, several rats cultured for eight weeks were examined by MRI, and the Pfirrmann grade was determined according to the MRI images; S7. Based on step S6, several rats that have undergone X-ray and MRI examinations are killed and corresponding intervertebral discs are taken, several intervertebral discs are fixed with formaldehyde for 48 hours, decalcified with EDTA decalcification solution for three weeks and embedded in paraffin, the paraffin-embedded slices are cut into first slices to be evaluated, the first slices to be evaluated are stained and the histological degree of intervertebral disc degeneration is evaluated on the stained slices; The clinical tissue validation includes the following steps: S8. Collect intervertebral disc tissues from several patients with intervertebral disc degeneration and divide them into a control group and a sample intervertebral disc degeneration group according to the degree of degeneration; S9. Based on step S8, extract the intervertebral disc tissue of the sample intervertebral disc degeneration group, divide the intervertebral disc tissue into a first tissue to be processed and a second tissue to be processed, fix the first tissue to be processed with formaldehyde for 48 hours, decalcify with EDTA decalcification solution for three weeks and embed in paraffin, and cut the paraffin-embedded slices into second slices to be evaluated; S10. Based on step S9, the second slices to be evaluated are divided into a first group of slices to be stained and a second group of slices to be stained, the first group of slices to be stained are stained to obtain a first group of stained slices, the second group of slices to be stained are immunohistochemically stained with a primary anti-SLC5A2 antibody to obtain a second group of stained slices, and the first group of stained slices and the second group of stained slices are compared with negative control slices to evaluate the histological degree of intervertebral disc degeneration; S11. Based on step S9, the second tissue to be processed is subjected to RIPA lysis buffer to collect the nucleus pulposus tissue protein sample. The protein sample is separated on SDS-PAGE gel and detected using SLC5A2 antibody. The chemiluminescent signal is visualized using a detection kit.

[0011] Furthermore: in the step S11, an ECL Western Blot detection kit is used to visualize the chemiluminescent signal.

[0012] Furthermore: the negative control slice in step S10 is a slice incubated with non-specific IgG.

[0013] Furthermore: the first slice to be evaluated and the first group of slices to be stained are both stained with hematoxylin, eosin and safranin o-fast green.

[0014] Animal experiment verification: Eighteen 10-week-old male SD rats were randomly divided into three groups according to the digital table method: sham operation group (n = 6), disc degeneration group (n = 6) and SGLT2 inhibitor group (n = 6). The three groups of rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg). Then the experiment was carried out. The rats in the sham operation group only punctured the skin without damaging their disc tissue. The disc degeneration group and the SGLT2 inhibitor group used a 22g puncture needle to perforate the entire layer of the 6 / 7 intervertebral disc of the coccyx of the rats, and then the needle was rotated 360 degrees and kept in the disc for 1 minute, and then the disc degeneration model was established; after four weeks of culture, in the SGLT2 inhibitor group, the SGLT2 inhibitor (2 ul, 25 μM dimethyl sulfoxide) was injected into the disc using a 33-gauge needle, and an equal volume of dimethyl sulfoxide (2 ul) was given at the same time to fuse the SGLT2 inhibitor, and the injection was maintained once a week for four consecutive weeks; After the fourth week of culture, the rats were anesthetized with pentobarbital and underwent X-ray examination, and the disc height index (DHI) was calculated based on the X-ray film, that is, the disc height was divided by the adjacent disc body height to generate the disc height data; after the eighth week of culture, the rats were anesthetized with pentobarbital and underwent MRI examination, the rats were placed in the corresponding machine and their tails were kept at the same height to obtain imaging results, and the Pfirrmann grade was determined based on the magnetic resonance imaging; After completing the X-ray and MRI examinations, the rats were killed with an overdose of pentobarbital and the corresponding intervertebral discs were taken, decalcified with EDTA decalcification solution for three weeks and embedded in paraffin. The paraffin-embedded sections were cut into 4 μm first sections to be evaluated, and the first sections to be evaluated were stained with hematoxylin and eosin (HE) and safranin o-fast green (SO) and the histological degree of intervertebral disc degeneration was evaluated on the stained sections. Subsequently, hematoxylin and eosin (HE) and safranin o-fast green (SO) staining were used to evaluate the histological degree of intervertebral disc degeneration.

[0015] Clinical tissue validation: The intervertebral disc tissues of 12 patients with intervertebral disc degeneration were collected. According to the degree of degeneration, they were divided into a control group (Pfirrmann grade I) and an intervertebral disc degeneration group (Pfirrmann grade>II) with an age of (41.9±21.5) years old; The intervertebral disc tissue of the sample intervertebral disc degeneration group was extracted, and the intervertebral disc tissue was divided into a first tissue to be processed and a second tissue to be processed. The first tissue to be processed was fixed with formaldehyde for 48 hours, decalcified with EDTA decalcification solution for three weeks and embedded in paraffin. The slices embedded in paraffin were cut into the second slices to be evaluated; The second sections to be evaluated are divided into a first group of sections to be stained and a second group of sections to be stained, the first group of sections to be stained are stained with hematoxylin and eosin (HE) and safranin o-fast green (SO) to obtain a first group of stained sections, the second group of sections to be stained are immunohistochemically stained with a primary antibody SLC5A2 to obtain a second group of stained sections, and the first group of stained sections and the second group of stained sections are compared with negative control sections to evaluate the histological degree of intervertebral disc degeneration, wherein the negative control sections are sections incubated with nonspecific IgG; The second tissue to be processed was lysed using RIPA buffer to collect the nucleus pulposus tissue protein samples, the protein samples were separated on SDS-PAGE gel and detected using SLC5A2 antibody, and the chemiluminescent signal was visualized using the ECL Western Blot detection kit.

[0016] According to the instruction manual Figure 7-8 As shown, Figure 7 and Figure 8 These are schematic diagrams of experimental data obtained from the experiment. Each cell represents a mouse. There are three groups of six mice in total, so there are 18 numbers. The Disc Height 2 Index (DHI) is a quantitative measure of disc height to assess disc degeneration. Disc height is usually measured on imaging (such as X-rays or MRI) and a relative height value is calculated.

[0017] The specific calculation method is: DHI = disc height / adjacent vertebral height Disc height: The height of the disc is measured at the front, middle and back, and the average is usually taken.

[0018] Adjacent vertebral height: measure the average height of two adjacent vertebrae.

[0019] The DHI value can reflect the degree of compression or collapse of the intervertebral disc. The lower the value, the more serious the intervertebral disc degeneration.

[0020] Clinical significance of DHI: Normal disc: A high DHI value indicates that the disc height is normal and there is no obvious compression.

[0021] Degenerative disc: As degeneration worsens, the DHI value decreases, indicating that the disc height gradually decreases, indicating disc dehydration and degeneration.

[0022] 2. Pfirrmann classification The Pfirrmann classification is a widely used grading system for disc degeneration in MRI images. It determines the degree of disc degeneration by evaluating the signal intensity, morphology, structure and other parameters of the disc. This system uses a score from 1 to 5 to quantify disc degeneration, with 5 being the most severe degeneration.

[0023] Pfirrmann classification system: Grade 1: The intervertebral disc signal is uniform, the nucleus pulposus signal is high signal (T2-weighted), the intervertebral disc morphology is normal, and the boundary between the nucleus pulposus and the annulus fibrosus is clear.

[0024] Grade 2: The disc signal is still uniform, but the nucleus pulposus signal is slightly reduced, the disc height is normal, and the boundary between the nucleus pulposus and the annulus fibrosus is still clear.

[0025] Grade 3: The signal of the nucleus pulposus is significantly reduced, the height of the intervertebral disc is slightly reduced, and the boundary between the nucleus pulposus and the annulus fibrosus is blurred.

[0026] Grade 4: The disc signal is further reduced, the signals of the nucleus pulposus and annulus fibrosus are indistinguishable, the disc height is significantly reduced, the disc morphology changes, and it begins to become irregular or flat.

[0027] Grade 5: The disc signal is completely lost, the disc height is severely reduced, the disc is obviously deformed, and the disc shows severe degeneration.

[0028] Clinical significance of Pfirrmann classification: Grade 1 to 2: Normal or mildly degenerative, usually without causing obvious symptoms.

[0029] Grade 3 to 4: Moderate degeneration, often accompanied by low back pain or other symptoms.

[0030] Grade 5: Severe degeneration, complete loss of disc function, usually accompanied by severe low back pain and other symptoms.

[0031] The above technology is based on the instructions attached Figure 7-8 Provide experimental data to improve the feasibility and certainty of this verification method.

[0032] In the above-mentioned techniques, when evaluating intervertebral disc degeneration, the use of Safranin O (SO) staining can effectively detect the content of glycosaminoglycans (GAGs) in intervertebral disc tissue (especially nucleus pulposus and annulus fibrosus), because Safranin O will bind to GAGs and appear red. As the intervertebral disc degenerates, the content of GAGs will decrease, resulting in weakened staining, which corresponds to the Fig. 9 shown.

[0033] Histological Score: SO staining can be used to quantitatively assess the extent of disc degeneration. Generally, the scoring system evaluates disc degeneration based on the following factors: 1. Changes in the nucleus pulposus GAGs content: The GAGs content in the nucleus pulposus decreases, and the red color of the staining gradually fades or disappears.

[0034] Changes in the structure of the nucleus pulposus: The normal nucleus pulposus is a uniform gel-like structure, but after degeneration, it may become stratified, fibrotic, or dehydrated.

[0035] · Staining intensity: The reduction of GAGs was assessed based on the intensity of SO staining.

[0036] 2. Changes in the annulus fibrosus Integrity of the annulus fibrosus: The collagen fibers of a normal annulus fibrosus are neatly arranged, but during degeneration, the fibers may be torn, broken, or disordered.

[0037] Fibrosis degree: As degeneration progresses, the annulus fibrosus will undergo fibrosis, leading to changes in SO staining.

[0038] 3. Changes in cartilage endplates Calcification or sclerosis: During the process of endplate degeneration, the cartilage endplate may calcify or sclerosis, and the staining intensity may decrease.

[0039] 4. Cellular changes Changes in cell density: The cell density in the nucleus pulposus or annulus fibrosus may decrease, or the cell morphology may change irregularly.

[0040] Cell death or proliferation: Cell death (decreased staining) or fibroblast proliferation may occur during degeneration.

[0041] Commonly used Histological Score calculation methods The specific scoring criteria for the histological score vary from study to study, but the following is a common scoring framework used to assess the degree of disc degeneration (usually each factor is scored from 0 to 3 or 0 to 4): 0 points: no degeneration, strong staining, normal tissue structure.

[0042] 1 point: Mild degeneration, slightly weakened staining, and slight changes in tissue structure.

[0043] 2 points: moderate degeneration, with significantly weakened staining, disordered structure, and significantly reduced GAGs.

[0044] 3 points: severe degeneration, almost no staining, obvious structural changes, and severe fibrosis or calcification.

[0045] Finally, the scores of each scoring item are added together to obtain a total score to indicate the overall degree of disc degeneration.

[0046] The above-mentioned technical manual is attached Figure 1 and 2 In the table, a single * indicates P < 0.05, two * indicates P < 0.01, and three * indicates P < 0.001.

[0047] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention shall be defined by the attached claims and their equivalents.

Claims

1. A method for verifying the effect of SLC5A2 gene on intervertebral disc degeneration, characterized in that: It includes animal experiment verification and clinical tissue verification, and the animal experiment verification includes the following steps: S1. Several rats were randomly divided into a sham operation group, an intervertebral disc degeneration group, and an SGLT2 inhibitor group according to the number table method, and the three groups of rats were anesthetized; S2. Based on the S1 step, the rats in the sham operation group were only punctured in the skin without damaging the intervertebral disc tissue. The rats in the intervertebral disc degeneration group and the SGLT2 inhibitor group were perforated in the 6 / 7 intervertebral disc of the coccyx with a puncture needle, and the puncture needle pillow was rotated 360 degrees and kept in the intervertebral disc for 1 minute. S3. Based on step S2, establish an intervertebral disc degeneration model and culture it for four weeks; S4. Based on the steps in S3, in the SGLT2 inhibitor group, the SGLT2 inhibitor was injected into the intervertebral disc of the rats and an equal volume of dimethyl sulfoxide was injected once a week for four consecutive weeks; S5. Based on step S4, performing X-ray examination on a number of rats after four weeks of culture, and calculating the intervertebral disc height index according to the X-ray film to generate intervertebral disc height data; S6. Based on step S5, several rats cultured for eight weeks were examined by MRI, and the Pfirrmann grade was determined according to the MRI images; S7. Based on step S6, several rats that have undergone X-ray and MRI examinations are killed and corresponding intervertebral discs are taken, several intervertebral discs are fixed with formaldehyde for 48 hours, decalcified with EDTA decalcification solution for three weeks and embedded in paraffin, the paraffin-embedded slices are cut into first slices to be evaluated, the first slices to be evaluated are stained and the histological degree of intervertebral disc degeneration is evaluated on the stained slices; The clinical tissue validation includes the following steps: S8. Collect intervertebral disc tissues from several patients with intervertebral disc degeneration and divide them into a control group and a sample intervertebral disc degeneration group according to the degree of degeneration; S9. Based on step S8, extract the intervertebral disc tissue of the sample intervertebral disc degeneration group, divide the intervertebral disc tissue into a first tissue to be processed and a second tissue to be processed, fix the first tissue to be processed with formaldehyde for 48 hours, decalcify with EDTA decalcification solution for three weeks and embed in paraffin, and cut the paraffin-embedded slices into second slices to be evaluated; S10. Based on step S9, the second slices to be evaluated are divided into a first group of slices to be stained and a second group of slices to be stained, the first group of slices to be stained are stained to obtain a first group of stained slices, the second group of slices to be stained are immunohistochemically stained with a primary anti-SLC5A2 antibody to obtain a second group of stained slices, and the first group of stained slices and the second group of stained slices are compared with negative control slices to evaluate the histological degree of intervertebral disc degeneration; S11. Based on step S9, the second tissue to be processed is subjected to RIPA lysis buffer to collect the nucleus pulposus tissue protein sample. The protein sample is separated on SDS-PAGE gel and detected using SLC5A2 antibody. The chemiluminescent signal is visualized using a detection kit.

2. The method for verifying the effect of the SLC5A2 gene on intervertebral disc degeneration according to claim 1, characterized in that: In the S11 step, an ECL Western Blot detection kit is used to visualize the chemiluminescent signal.

3. The method for verifying the effect of SLC5A2 gene on intervertebral disc degeneration according to claim 1, characterized in that: The negative control slice in step S10 is a slice incubated with non-specific IgG.

4. The method for verifying the effect of SLC5A2 gene on intervertebral disc degeneration according to claim 1, characterized in that: The first slice to be evaluated and the first group of slices to be stained are both stained with hematoxylin, eosin and safranin o-fast green.

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