Method for scale-up culture of nucleus pulposus cells derived from intervertebral disc and use thereof

Abstract

The invention mainly discloses a method for obtaining nucleus pulposus cells from an intervertebral disc and carrying out in-vitro amplification culture on the nucleus pulposus cells. The invention aims to provide application of the nucleus pulposus cell obtained by the method to manufacture a medical composition for treating lower back pain, and the application of the medical composition containing the nucleus pulposus cell to treat chronic lower back pain.

Description

Technical Field

[0001] The present invention relates to the technical field of cell amplification and culture methods, and in particular to the technical field of an amplification and culture method of nucleus pulposus cells derived from an intervertebral disc and a use thereof. Background Art

[0002] Intervertebral disc tissue is located between the vertebrae, and is composed of the annulus fibrosus and the nucleus pulpous. It can buffer the friction between the vertebrae caused by movement. Degenerated disc disease often causes low back pain, which is more common in adults aged 30 to 50. In severe cases, it can cause the disc to collapse. According to statistics, 80% of adults have experienced low back pain, and about a quarter of them have been affected by back pain at work.

[0003] Generally, when the initial symptoms of disc degeneration occur, clinicians will first give patients painkillers to temporarily relieve the patient's physical pain. Commonly used drugs include non-steroidal anti-inflammatory analgesics (NSAIDs), acetaminophen, steroid drugs, etc., but these drugs cause imbalance in cell secretion and may cause gastrointestinal side effects. Severe disc degeneration requires disc replacement treatment, but the material of artificial disc implants is a mixture of high molecular polymers and metal substances, and there is no biocompatibility, which can easily cause more serious side effects. None of the above methods can directly treat the degenerated disc, and the patient's quality of life has not been significantly improved.

[0004] When the disc structure is damaged or degenerated, the nucleus pulposus may become unstable and may protrude outward or even pass through the annulus fibrosus and escape due to increased weight or pressure. This protruding nucleus pulposus thus compresses the surrounding nerve structures, such as the spinal cord or nerve roots, causing symptoms such as pain, radiating pain, numbness or muscle weakness. Therefore, there is a close relationship between disc herniation and the location and pressure of the nucleus pulposus.

[0005] In view of the limitations and shortcomings of current treatments for intervertebral disc degeneration, and based on the relationship between intervertebral disc degeneration and nucleus pulposus tissue, the inventor of this case hopes to obtain nucleus pulposus cells from the nucleus pulposus tissue of the patient's intervertebral disc in the early stage of intervertebral disc degeneration, amplify and culture them in vitro, and freeze them. When the patient needs them, they are thawed, re-cultured, and implanted into the patient's affected part, so that they can grow in the patient's affected part without immune rejection, thereby achieving the purpose of nucleus pulposus tissue regeneration and treatment of intervertebral disc degeneration. Therefore, the inventor of this case has made great efforts to study and invent, and finally developed a method of amplifying and culturing nucleus pulposus cells derived from intervertebral discs in vitro and a use of the nucleus pulposus cells obtained by the aforementioned method to manufacture a pharmaceutical composition for treating low back pain. Summary of the invention

[0006] The main purpose of the present invention is to provide a method for amplifying and culturing nucleus pulposus cells derived from intervertebral discs in vitro, comprising:

[0007] a) providing a nucleus pulposus;

[0008] b) hydrolyzing the nucleus pulposus tissue with an enzyme, and separating the unhydrolyzed nucleus pulposus tissue from the primary nucleus pulposus cells detached from the tissue by sieving and centrifuging;

[0009] c) primary culturing the primary nucleus pulposus cells obtained in b);

[0010] d) when the primary nucleus pulposus cells in primary culture in c) reach a certain cell density, taking out the primary nucleus pulposus cells for subculture;

[0011] e) screening nucleus pulposus cells from the primary nucleus pulposus cells subcultured in d) based on the expression level of one or more genes, wherein the one or more genes are selected from the following gene modules: nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus specific genes, nucleus pulposus degeneration genes and annulus fibrosus specific genes; and

[0012] f) amplifying and culturing the nucleus pulposus cells obtained in e).

[0013] In the above method, the nucleus pulposus development gene is KDM4E, the cartilage differentiation genes include: SOX9, COL2A1, Agc1, the nucleus pulposus specific gene is PAX1, the nucleus pulposus degeneration gene is SAA1, and the annulus fibrosus specific gene is CD90.

[0014] In addition, in the above method, the enzyme used to hydrolyze the nucleus pulposus tissue is collagenase, trypsin or a combination thereof, and when the primary nucleus pulposus cells subjected to primary culture in c) reach a cell density of 50-100% after 0-14 days, preferably after reaching a cell density of 80-90% after 5-7 days, the primary nucleus pulposus cells are taken out for subculture.

[0015] Furthermore, in the above method, the expression level of the gene is detected by quantitative polymerase chain reaction (qPCR), Northern blotting, Western blotting or DNA microarray.

[0016] Meanwhile, another object of the present invention is to provide a use of the nucleus pulposus cells obtained by the above method for preparing a pharmaceutical composition for treating low back pain, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0017] The pharmaceutical composition can be used to obtain the nucleus pulposus cells from the nucleus pulposus tissue of a patient, after amplification and culture, and then re-transplanted into the patient's affected area, i.e., the site of intervertebral disc degeneration, so that they can grow in the affected area, achieve the purpose of treatment through the regeneration of the nucleus pulposus tissue, and avoid the possibility of immune rejection in the patient receiving the transplant. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression level of the nucleus pulposus development gene KDM4E in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups;

[0019] Figure 2A The expression level of the cartilage differentiation gene SOX9 in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups;

[0020] Figure 2B The expression level of the cartilage differentiation gene COL2A1 in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups;

[0021] Figure 2C The expression level of the cartilage differentiation gene: Agc1 in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups;

[0022] Figure 3 The expression level of the nucleus pulposus specific gene: PAX1 in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups;

[0023] Figure 4 The expression level of the nucleus pulposus degeneration gene: SAA1 in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups;

[0024] Figure 5 The expression level of the annulus fiber specific gene: CD90 in the nucleus pulposus cells obtained by the amplification culture method of the intervertebral disc-derived nucleus pulposus cells of the present invention in different age groups; and

[0025] Figure 6 Shown is a flow chart of the method for amplifying and culturing nucleus pulposus cells derived from intervertebral discs of the present invention and its use. DETAILED DESCRIPTION

[0026] All technical and scientific terms used in this specification, unless otherwise defined, have the meanings commonly understood by those with common knowledge in the art. The present invention will be illustrated by the following examples, which are only illustrative and not limiting. The present invention is not limited by the following examples. Unless otherwise specified, the materials used in the present invention are all commercially available and easily available. The following are only examples of available channels.

[0027] Example 1 Processing of Nucleus Pulposus Specimens

[0028] 1. First, in the operating room of the hospital, the nucleus pulposus tissue of the patient with intervertebral disc degeneration or intervertebral disc herniation is removed in a sterile manner, placed in a sterile closed carrier containing an antibiotic composition and physiological saline, and then sent to the sterile operating table of the Good Tissue Practice (GTP) laboratory for processing;

[0029] 2. Record the weight of the nucleus pulposus tissue specimen and the basic information of the patient;

[0030] 3. Place the specimen in a 10 cm dish containing 2 mL of P0 medium (medium of passage number 0), and cut the specimen into small pieces with an average size of less than 1 mm. 3 ; one of the 10cm dishes contains about 5g of the specimen. If the specimen is large, the nucleus pulposus tissue is divided into several culture dishes according to this ratio, wherein the P0 medium includes the following components: DMEM (Dubeka's modified Eagle's medium), Human Platelet Lysate (human platelet lysate) and Antibiotic (antibiotic);

[0031] 4. Prepare collagenase solution: 7mL P0 medium + 1mL type I collagenase (type I collagen), that is, the ratio of P0 medium and type I collagenase in the collagenase solution is 7:1 (volume ratio);

[0032] 5. Add the prepared collagenase solution to the culture dishes containing the chopped nucleus pulposus tissue. Add 8 mL of collagenase solution to each culture dish and culture in an incubator at 37°C and 5% CO2 overnight.

[0033] 6. After overnight culture, the nucleus pulposus tissue specimens in each culture dish were sieved through a 100 μm cell strainer. During the sieving process, the tissues were gently ground with the piston of a 10 mL syringe. After grinding, the cell strainer was washed with 10 mL of DPBS (Dulbecco's phosphate-buffered saline).

[0034] 7. Centrifuge the cell suspension collected after sieving at 200 x g at room temperature for 5 minutes and remove the supernatant;

[0035] 8. Wash the cell pellet twice with 20 mL DPBS;

[0036] 9. Then resuspend the cell pellet in 2 mL of P0 medium and count the cells using trypan blue to calculate the number of cells and their survival rate (80-99%).

[0037] 10. Take 5-10×10 5 The cells were seeded into a 10 cm dish containing 10 mL of P0 medium. The cells at this time were P0 (cells of passage number 0), i.e., primary nucleus pulposus cells, and cultured in an incubator at 37°C and 5% CO2 for primary culture.

[0038] 11. Continue to observe the growth of the primary nucleus pulposus cells, and on the third day, remove the old P0 medium, wash once with 6 mL DPBS, and then add 10 mL of new P0 medium to continue culturing; and

[0039] 12. After the primary nucleus pulposus cells have reached a cell density of 50-100% after 0-14 days of culture, or preferably after 5-7 days of culture when the cell density reaches 80-90%, the primary nucleus pulposus cells can be taken out for the first subculture.

[0040] Example 2 Subculture of primary nucleus pulposus cells

[0041] 1. When the cell density of the primary nucleus pulposus cells in Example 1 reaches 50-100% or preferably 80-90%, subculture is performed;

[0042] 2. Remove the old P0 medium and wash the primary nucleus pulposus cells attached to the bottom of the 10 cm dish twice with 6 mL DPBS;

[0043] 3. Add 1 mL of 0.05% trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid) to a 10 cm dish and incubate it at 37°C in a 5% CO2 incubator for 3 minutes;

[0044] 4. After the reaction is completed, tap the 10 cm dish with your hand to make the primary nucleus pulposus cells detach from the bottom of the 10 cm dish;

[0045] 5. Add 4 mL of P0 medium to a 10 cm dish to neutralize trypsin, then collect the primary nucleus pulposus cells suspended in P0 medium, place in a centrifuge tube and centrifuge at 200 x g for 5 minutes at room temperature;

[0046] 6. After removing the supernatant from the centrifuge tube, resuspend the primary nucleus pulposus cells in an appropriate volume of culture medium. Adjust the volume of culture medium required for resuspending the primary nucleus pulposus cells in proportion, for example, resuspend the primary nucleus pulposus cells in 1 mL culture medium per 1-2 10 cm dishes, wherein the culture medium includes the following components: DMEM and Human Platelet Lysate;

[0047] 7. Count the cells using trypan blue to confirm the number of primary nucleus pulposus cells and their survival rate (80-99%);

[0048] 8. Place the resuspended primary nucleus pulposus cells in a 10 cm dish containing 1x10 6The primary nucleus pulposus cells are cultured in 10 mL of culture medium at a ratio of the number of cells per passage. The primary nucleus pulposus cells at this time are P1 (i.e., cells of passage number 1).

[0049] 9. Perform subculture approximately every 3 to 4 days, i.e. repeat steps 1 to 8 above.

[0050] Example 3 Evaluation of gene expression

[0051] 1. When P2 (second subculture cells), P3 (third subculture cells), and P4 (fourth subculture cells) were subcultured using the subculture method in Example 2, about 5×10 5 The primary nucleus pulposus cells were washed twice with 10 mL DPBS;

[0052] 2. The collected primary nucleus pulposus cells were resuspended in 1 mL of DPBS and transferred to a centrifuge tube, and then centrifuged at 200 x g for 5 minutes at room temperature;

[0053] 3. Remove the supernatant from the centrifuge tube, add 1 mL of GENEzol to the centrifuge tube, and then store the sample in the centrifuge tube at -80°C; and

[0054] 4. Extract RNA from the sample (primary nucleus pulposus cells) in step 3 above, and detect the expression of each gene in the primary nucleus pulposus cells in the sample by qPCR (quantitative polymerase chain reaction), wherein each gene detected is a gene selected from the following five gene modules: I. nucleus pulposus development gene, II. cartilage differentiation gene, III. nucleus pulposus specific gene, IV. nucleus pulposus degeneration gene and V. annulus fibrosus specific gene. Further, the nucleus pulposus development gene is KDM4E, the cartilage differentiation gene includes: SOX9, COL2A1, Agc1, the nucleus pulposus specific gene is PAX1, the nucleus pulposus degeneration gene is SAA1, and the annulus fibrosus specific gene is CD90.

[0055] Example 4 Cell Cryopreservation

[0056] 1. When the primary nucleus pulposus cells are continuously expanded and cultured to P2 or P3, when the cell density reaches 80-90%, the cells can be frozen;

[0057] 2. Remove the old culture medium from the 10 cm dish and wash twice with 6 mL DPBS;

[0058] 3. Add 1 mL of trypsin-EDTA to a 10 cm dish and incubate at 37°C in a 5% CO2 incubator for 3 minutes;

[0059] 4. After the action is completed, tap the 10 cm dish with your hand to make the P2 or P3 primary nucleus pulposus cells detach from the bottom of the 10 cm dish;

[0060] 5. Add 4 mL of culture medium to a 10 cm dish to neutralize trypsin, then collect the P2 or P3 primary nucleus pulposus cells suspended in the culture medium, place in a centrifuge tube and centrifuge at 200 x g for 5 minutes at room temperature;

[0061] 6. After removing the supernatant from the centrifuge tube, resuspend the P2 or P3 primary nucleus pulposus cells in an appropriate volume of culture medium. Adjust the volume of culture medium required for resuspending the P2 or P3 primary nucleus pulposus cells in proportion, for example, resuspend the P2 or P3 primary nucleus pulposus cells in 1 mL culture medium for every 1 to 2 10 cm dishes;

[0062] 7. Count the cells using trypan blue to confirm the number of P2 or P3 primary nucleus pulposus cells and their survival rate (80-99%);

[0063] 8. Centrifuge the P2 or P3 primary nucleus pulposus cells resuspended in culture medium in the centrifuge tube at 200 x g for 5 minutes at room temperature, and then remove the supernatant;

[0064] 9. 2×10 6 / mL cell concentration, resuspend the P2 or P3 primary nucleus pulposus cells in an appropriate volume of cell freezing solution;

[0065] 10. Dispense the P2 or P3 primary nucleus pulposus cell suspension into cryovials at a ratio of 1 mL / vial; and

[0066] 11. Place the cryovials into a cell freezing box, then place the cell freezing box into a -80°C freezer, and transfer the cryovials to liquid nitrogen for storage the next day.

[0067] The following table 1 is the source of the reagents used in the above embodiments:

[0068] Table 1

[0069]

[0070]

[0071]

[0072] Example 5 Expression of KDM4E, a gene of nucleus pulposus development in primary nucleus pulposus cells

[0073] Figure 1 The results are shown for the expression of the nucleus pulposus development gene KDM4E obtained by analyzing the primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3. Figure 1 As can be seen from the histogram in , the expression level of the nucleus pulposus development gene: KDM4E in the primary nucleus pulposus cells obtained from the age groups less than 35 years old and 36-50 years old is higher than that in the age group greater than 51 years old. And from the expression of the specific nucleus pulposus development gene: KDM4E, it can be confirmed that the primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 of the present invention are indeed derived from the development process of nucleus pulposus tissue.

[0074] Example 6 Expression levels of cartilage differentiation genes of primary nucleus pulposus cells: SOX9, COL2A1 and Agc1

[0075] Figures 2A to 2C The results are shown for the expression of cartilage differentiation genes: SOX9, COL2A1 and Agc1 in primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 in different age groups, and analyzed by the gene expression evaluation method of Example 3. Figure 2A The histogram in the figure shows that the expression of the cartilage differentiation gene SOX9 in the primary nucleus pulposus cells obtained from the age group under 35 years old is significantly higher than that in the age group of 36-50 years old and over 51 years old. SOX9 is an important early transcription factor in cartilage differentiation. Figure 2B From the histogram in Figure 2, we can see that the expression of the cartilage differentiation gene COL2A1 in the primary nucleus pulposus cells obtained from the age groups of less than 35 years old and 36-50 years old is higher than that in the age group of more than 51 years old. In addition, the expression of the COL2A1 gene in the age group of less than 35 years old is also significantly higher than that in the age groups of 36-50 years old and more than 51 years old. COL2A1 is an important cartilage matrix gene in cartilage differentiation. Figure 2C The histogram in FIG. 1 shows that the expression level of the cartilage differentiation gene Agc1 in the primary nucleus pulposus cells obtained by the amplified culture method of the present invention is the highest in the age group under 35 years old, the second highest in the age group between 36 and 50 years old, and the lowest in the age group over 51 years old. Agc1 is an important cartilage matrix gene in cartilage differentiation. Figures 2A to 2CIt can also be seen that the primary nucleus pulposus cells obtained by the amplification culture method of the above-mentioned embodiments 1 to 2 of the present invention all express important cartilage differentiation genes related to nucleus pulposus tissue, and the expression level decreases with the increase of age.

[0076] Example 7 Expression of a nucleus pulposus-specific gene: PAX1 in primary nucleus pulposus cells

[0077] Figure 3 The results are shown for the expression of a nucleus pulposus-specific gene: PAX1, obtained by analyzing the primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3. Figure 3 From the histogram in the figure, we can see that the expression of the PAX1 gene in the primary nucleus pulposus cells obtained from the age group under 35 years old is significantly higher than that in the age group of 36-50 years old and over 51 years old. The PAX1 gene is the precursor cell gene of the nucleus pulposus tissue. The younger the nucleus pulposus tissue, the higher the expression of the PAX1 gene. The higher the expression, the higher the repair ability of the nucleus pulposus tissue. Therefore, Figure 3 It can be seen that the primary nucleus pulposus cells obtained by the amplification culture method of the above-mentioned embodiments 1 to 2 of the present invention all have the expression of the PAX1 gene related to the repair ability of the nucleus pulposus tissue, and the expression level is significantly higher in young people.

[0078] Example 8 Expression of the nucleus pulposus degeneration gene: SAA1 in primary nucleus pulposus cells

[0079] Figure 4 The results are shown for the expression of the nucleus pulposus degeneration gene: SAA1 obtained by analyzing the primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3. Figure 4 The histogram in FIG. 1 shows that the expression of the nucleus pulposus degeneration gene SAA1 in the primary nucleus pulposus cells obtained by the amplified culture method of the present invention is the highest in the age group over 51 years old, the second highest in the age group 36-50 years old, and the lowest in the age group under 35 years old. SAA1 is expressed during nucleus pulposus degeneration and nucleus pulposus cell apoptosis, and increases with age. Therefore, Figure 4 It can be seen that the expression level of the nucleus pulposus degeneration gene: SAA1 of the primary nucleus pulposus cells obtained by the amplified culture method of the above embodiments 1 to 2 of the present invention shows that the older the age, the higher the expression. That is, the primary nucleus pulposus cells amplified and cultured by the method of the present invention have the characteristics expressed by the cells in the nucleus pulposus tissue.

[0080] Example 9 Expression of annulus fiber-specific gene: CD90 in primary nucleus pulposus cells

[0081] Figure 5 The results are shown for the expression of the annulus fiber specific gene CD90 obtained by analyzing the primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3. Figure 5 It can be seen from the histogram in that the primary nucleus pulposus cells obtained by the amplified culture method of the present invention have very low expression levels of the annulus fibrosus-specific gene: CD90 in different age groups. That is, the very low expression level of the annulus fibrosus-specific gene: CD90 can reversely verify (explain) that the primary nucleus pulposus cells obtained by the amplified culture method of the present invention are not derived from the annulus fibrosus.

[0082] Table 2 below shows the results of five types of gene modules obtained by analyzing the primary nucleus pulposus cells obtained by the amplification culture method of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3: nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus specific genes, nucleus pulposus degeneration genes and annulus fibrosus specific genes.

[0083] Table 2

[0084]

[0085] As shown in Table 2, Figures 1 to 5 The data represented by the histogram is the average value of the gene expression data obtained in each age group. Figures 1 to 5 As shown in Table 2, we can clearly know that through the method for amplifying and culturing primary nucleus pulposus cells and the gene expression evaluation method in the in vitro amplified culture method of nucleus pulposus cells derived from intervertebral discs provided by the present invention, that is, by using the expression levels of five types of gene modules: nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus-specific genes, nucleus pulposus degeneration genes and annulus fibrosus-specific genes, nucleus pulposus cells with the function of repairing nucleus pulposus tissue can be obtained, and then the obtained nucleus pulposus cells can be used to manufacture a pharmaceutical composition for treating low back pain, and the chronic low back pain caused by intervertebral disc degeneration can be treated through the use of the pharmaceutical composition containing the nucleus pulposus cells.

[0086] Example 10 A method for amplifying and culturing nucleus pulposus cells from intervertebral discs and a process for their use

[0087] Figure 6The flowchart of the method for amplifying and culturing nucleus pulposus cells derived from intervertebral discs of the present invention and its use is shown. First, as described in the technical content of Example 1, a nucleus pulposus tissue specimen is obtained from the spine of a patient with intervertebral disc degeneration or intervertebral disc herniation in a sterile manner through surgery, and then the nucleus pulposus tissue specimen is transported to a sterile operating table of a human cell tissue good operation specification laboratory in a sterile and constant temperature manner for processing, and finally a primary nucleus pulposus cell is obtained; then, the primary nucleus pulposus cell obtained in Example 1 is subcultured using the primary nucleus pulposus cell subculture method described in Example 2, and the primary nucleus pulposus cell obtained is amplified, and then the primary nucleus pulposus cell is evaluated using the gene expression evaluation method described in Example 3, and the primary nucleus pulposus cell is simultaneously evaluated. A sterility safety test is performed to confirm that the primary nucleus pulposus cells are not contaminated, for example, by Mycoplasma. Then, based on the results of the aforementioned gene expression evaluation and sterility safety test, a personalized report is prepared as to whether the primary nucleus pulposus cells obtained from the patient's nucleus pulposus tissue specimen contain nucleus pulposus cells with regenerative ability and other nucleus pulposus cell characteristics. Based on this report, the nucleus pulposus cells with regenerative ability and other nucleus pulposus cell characteristics are used to customize a pharmaceutical composition for treating low back pain (intervertebral disc degeneration or intervertebral disc herniation) for the patient. Finally, the pharmaceutical composition is implanted into the patient's affected part to allow the nucleus pulposus cells with regenerative ability to grow in the patient's affected part, thereby achieving the purpose of nucleus pulposus tissue regeneration and treating intervertebral disc degeneration or intervertebral disc herniation.

[0088] The above description has completely and clearly described the amplification culture method of intervertebral disc-derived nucleus pulposus cells and its use of the present invention. It must be emphasized that the above detailed description is a specific description of a feasible embodiment of the present invention, but the embodiment is not used to limit the patent scope of the present invention. Any equivalent implementation or change that does not deviate from the technical spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for amplifying and culturing nucleus pulposus cells derived from an intervertebral disc in vitro, comprising: a) providing a nucleus pulposus; b) hydrolyzing the nucleus pulposus tissue with an enzyme, and separating the unhydrolyzed nucleus pulposus tissue from the primary nucleus pulposus cells by sieving and centrifuging; c) primary culturing the primary nucleus pulposus cells obtained in b); d) when the primary nucleus pulposus cells in primary culture in c) reach a certain cell density, taking out the primary nucleus pulposus cells for subculture; e) selecting nucleus pulposus cells from the primary nucleus pulposus cells subcultured in d) based on the expression level of one or more genes, wherein: The one or more genes are selected from the following gene modules: nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus specific genes, nucleus pulposus degeneration genes and annulus fibrosus specific genes; and f) amplifying and culturing the nucleus pulposus cells obtained in e).

2. The method according to claim 1, wherein: The nucleus pulposus development gene is KDM4E.

3. The method according to claim 1, wherein: The cartilage differentiation genes include: SOX9, COL2A1, and Agc1.

4. The method according to claim 1, wherein: The nucleus pulposus specific gene is PAX1.

5. The method according to claim 1, wherein: The nucleus pulposus degeneration gene is SAA1.

6. The method according to claim 1, wherein: The annulus fibrosus specific gene is CD90.

7. The method according to claim 1, wherein: The enzyme used to hydrolyze the nucleus pulposus tissue is collagenase, trypsin or a combination thereof.

8. The method according to claim 1, wherein: The cell density is 80-90% cell density.

9. The method according to claim 1, wherein: The expression level of the gene is detected by quantitative polymerase chain reaction (qPCR), Northern blotting, western blotting or DNA microarray.

10. Use of the nucleus pulposus cells obtained by the method according to claim 1 for preparing a pharmaceutical composition for treating low back pain.

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