WO2022068258A1 - 用于诱导椎间盘纤维化的物质在制备药物中的用途 - Google Patents

用于诱导椎间盘纤维化的物质在制备药物中的用途 Download PDF

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WO2022068258A1
WO2022068258A1 PCT/CN2021/099562 CN2021099562W WO2022068258A1 WO 2022068258 A1 WO2022068258 A1 WO 2022068258A1 CN 2021099562 W CN2021099562 W CN 2021099562W WO 2022068258 A1 WO2022068258 A1 WO 2022068258A1
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intervertebral disc
substance
fibrosis
inducing
bleomycin
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PCT/CN2021/099562
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English (en)
French (fr)
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赵杰
陈辰
杨骁�
周唐峻
秦安
陈智谦
张凯
周益帆
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上海交通大学医学院附属第九人民医院
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Priority to US18/029,112 priority Critical patent/US20240115658A1/en
Publication of WO2022068258A1 publication Critical patent/WO2022068258A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

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  • the invention relates to the field of medicine, in particular to the use of a substance for inducing intervertebral disc fibrosis in the preparation of medicine.
  • Disc degeneration is a complex process involving three main parts: the nucleus pulposus (NP), the proteoglycan-rich, gelatinous center of the disc; the annulus fibrosus (AF), the partially concentric collagen-rich Fibrocartilaginous tissue; two cartilage endplates—each at the junction of the upper and lower vertebral bodies.
  • NP nucleus pulposus
  • AF annulus fibrosus
  • the self-repair and regeneration function of the intervertebral disc is weak, which may be due to the low number of cells in the intervertebral disc and the lack of nutrient supply in the tissue. Therefore, traditional treatment strategies are largely limited to symptom relief, with limited long-term efficacy. Surgical intervention is usually done to restore height and maintain intervertebral space stability and to help relieve severe neurological symptoms. However, spinal fusion does not restore biomechanical properties, but in fact may lead to degeneration of adjacent vertebral bodies due to increased mechanical stress on adjacent vertebral bodies.
  • the purpose of the present invention is to use a substance inducing intervertebral disc fibrosis to prepare a medicament for treating spinal degenerative diseases, so as to solve the problems in the prior art.
  • one aspect of the present invention provides the use of a substance for inducing intervertebral disc fibrosis in the preparation of a medicament for treating spinal degenerative diseases.
  • the substance inducing intervertebral disc fibrosis comprises bleomycin.
  • the substance inducing intervertebral disc fibrosis comprises a combination of bleomycin and fibroblasts.
  • the fibroblasts are dermal fibroblasts.
  • the fibroblasts are autologous fibroblasts.
  • the substance inducing intervertebral disc fibrosis can induce high expression of TGF- ⁇ in annulus fibrosus cells.
  • the substance inducing intervertebral disc fibrosis can up-regulate the expression of p-Smad2 and/or p-Samd3 in annulus fibrosus cells.
  • the substance inducing intervertebral disc fibrosis can up-regulate the expression of FSP1 in annulus fibrosus cells.
  • the substance inducing intervertebral disc fibrosis can up-regulate the expression of type I collagen in annulus fibrosus cells.
  • the substance inducing intervertebral disc fibrosis is capable of up-regulating the expression of type III collagen in annulus fibrosus cells.
  • the degenerative disease of the spine is selected from intervertebral disc degeneration.
  • the substance for inducing intervertebral disc fibrosis is the only active ingredient.
  • Figure 1 is a schematic diagram of the experimental results in Example 1, wherein Figures 1A and 1B are schematic diagrams of the results of bleomycin stimulating annulus fibrosus cells and activating the TGF- ⁇ /Smad pathway, and Figure 1C is a schematic diagram of bleomycin promoting fibroblast-related protein Western blot schematic diagram of high expression of TGF ⁇ , FSP1 and Col1a1.
  • FIG. 2 is a schematic diagram of the results of local injection of bleomycin in the intervertebral disc for treating spinal degeneration in Example 3, wherein FIG. 2A is a schematic diagram of X-ray results, and FIG. 2B is a schematic diagram of MRI results.
  • FIG. 3 is a schematic diagram showing the staining results of safranin fast green and Sirius red paraffin sections after local injection of bleomycin into the intervertebral disc for spinal degeneration in Example 4 for six weeks.
  • Figure 4 is a schematic diagram of the quantitative statistical results of Safranin Fast Green and Sirius Red staining after local injection of bleomycin into the intervertebral disc for spinal degeneration in Example 4 for six weeks.
  • FIG. 5 is a schematic diagram of the immunofluorescence staining results of TGF ⁇ 1, TGF ⁇ R1, Col1a1, and FSP1 after local injection of bleomycin into the intervertebral disc for spinal degeneration in Example 4 for six weeks.
  • FIG. 6 is a schematic diagram showing the quantitative statistical results of immunofluorescence staining of TGF ⁇ 1, TGF ⁇ R1, Col1a1 and FSP1 after local injection of bleomycin into the intervertebral disc for spinal degeneration in Example 4 for six weeks.
  • FIG. 7 is a schematic diagram showing the results of atomic mechanical analysis of spinal degeneration treated by local injection of bleomycin in the intervertebral disc for six weeks in Example 5.
  • FIG. 7 is a schematic diagram showing the results of atomic mechanical analysis of spinal degeneration treated by local injection of bleomycin in the intervertebral disc for six weeks in Example 5.
  • FIG. 8 is a schematic diagram of quantitative statistical results of atomic mechanical analysis in Example 5.
  • FIG. 9 is a schematic diagram of the results of local injection of bleomycin-assisted fibroblasts to treat spinal degeneration in Example 7, wherein FIG. 9A is a schematic diagram of X-ray results, and FIG. 9B is a schematic diagram of MRI results.
  • a first aspect of the present invention provides the use of a substance for inducing intervertebral disc fibrosis in the preparation of a medicament for treating spinal degenerative diseases.
  • substances used to induce intervertebral disc fibrosis eg, bleomycin, etc.
  • the principle is to maintain or even restore the height of the intervertebral space, prevent nerve root compression and Spinal instability, rapid and adequate fibrosis helps to achieve timely stabilization, which can effectively control spinal degenerative diseases.
  • the substance for inducing intervertebral disc fibrosis can generally be administered between intervertebral discs, so as to induce the migration of intervertebral disc annulus fibrosus cells, secrete fibrotic collagen deposition, achieve effective support to maintain the height of the intervertebral space, and retain A certain degree of activity of the vertebral body, maintaining the stability of the intervertebral disc, and preventing further degeneration of the intervertebral disc.
  • Appropriate methods of administering the above-mentioned substances for inducing intervertebral disc fibrosis to an individual should be known to those skilled in the art, for example, the substances for inducing intervertebral disc fibrosis may be administered by injection or the like.
  • bleomycin (a water-soluble crystalline substance) can be formulated as an aqueous solution, and injected locally between the intervertebral discs as an injection.
  • bleomycin can be prepared as an aqueous solution, and fibroblasts can be dispersed in the aqueous solution, which can be locally injected between the intervertebral discs as an injection.
  • the substance for inducing intervertebral disc fibrosis may include Bleomycin.
  • Bleomycin a known clinical anticancer drug, is a cytotoxic chemotherapeutic compound commonly used to treat lymphomas, leukemias, squamous cell carcinomas, and some genital tract tumors.
  • bleomycin can effectively induce sustained release or high expression of TGF- ⁇ in annulus fibrosus cells, and can also upregulate the expression of p-Smad2 and/or p-Samd3 in annulus fibrosus cells, and also Up-regulating the expression of type I collagen and/or type III collagen in annulus fibrosus cells can effectively induce intervertebral disc fibrosis and restore the height and biomechanical properties of the intervertebral disc to a certain extent.
  • the substance for inducing intervertebral disc fibrosis can also include fibroblasts, and specifically can be a combination of bleomycin and fibroblasts.
  • Fibroblasts also known as fibroblasts, are usually the main cellular components of loose connective tissue, belong to terminally differentiated cells, and can secrete a large number of type I collagen fibers.
  • the fibroblasts may be dermal fibroblasts, and the dermal fibroblasts are mainly derived from the dermis layer of the skin.
  • the skin covers the body surface, from the outside to the inside, the epidermis, the dermis, and the subcutaneous layer.
  • the dermis is mainly composed of fibroblasts and the fibers and matrices they produce.
  • the fibroblasts can be allogeneic and/or xenogeneic, or allogeneic, preferably autologous fibroblasts, so as to effectively avoid rejection by the body and provide better safety.
  • the combination of fibroblasts and bleomycin can more effectively induce continuous release or high expression of TGF- ⁇ in annulus fibrosus cells, and further up-regulate p-Smad2 and TGF- ⁇ in annulus fibrosus cells.
  • the expression of/or p-Samd3 can further upregulate the expression of type I collagen and/or type III collagen in annulus fibrosus cells, which can more effectively induce intervertebral disc fibrosis and restore the height and biomechanical properties of the intervertebral disc.
  • the substance for inducing intervertebral disc fibrosis can be used as a promoter of transforming growth factor- ⁇ (TGF- ⁇ ), for example, the promotion of transforming growth factor- ⁇ in annulus fibrosus cells agent.
  • TGF- ⁇ transforming growth factor- ⁇
  • the substance for inducing intervertebral disc fibrosis can induce continuous release or high expression of TGF- ⁇ from annulus fibrosus cells, so that repeated administration can be avoided.
  • bleomycin or the combination of bleomycin and fibroblasts can activate the TGF- ⁇ /Smad pathway of annulus fibrosus cells, induce phosphorylation of Smad2/3, and thereby make TGF ⁇ highly expressed.
  • the substance for inducing intervertebral disc fibrosis can be used as a promoter of FSP1 (Fibroblast-specific protein 1, Fibroblast specific protein 1), for example, a promoter of FSP1 in annulus fibrosus cells.
  • FSP1 Fibroblast-specific protein 1, Fibroblast specific protein 1
  • the substance for inducing intervertebral disc fibrosis can induce an increase in the expression of FSP1 in annulus fibrosus cells.
  • bleomycin or a combination of bleomycin and fibroblasts can activate the TGF- ⁇ /Smad pathway in annulus fibrosus cells and induce phosphorylation of Smad2/3, thereby enabling the final protein product FSP1 expression increased.
  • the substance for inducing intervertebral disc fibrosis can be used as a promoter of type I collagen, for example, a promoter of type I collagen in annulus fibrosus cells.
  • the substance for inducing intervertebral disc fibrosis can up-regulate the expression of type I collagen in annulus fibrosus cells.
  • bleomycin or the combination of bleomycin and fibroblasts can stimulate annulus fibrosus cells, which can increase the expression of type I collagen.
  • the substance for inducing intervertebral disc fibrosis can be used as a promoter of type III collagen, for example, a promoter of type III collagen in annulus fibrosus cells.
  • the substance for inducing intervertebral disc fibrosis can up-regulate the expression of type III collagen in annulus fibrosus cells.
  • bleomycin or the combination of bleomycin and fibroblasts can stimulate annulus fibrosus cells, which can increase the expression of type III collagen.
  • the substance for inducing intervertebral disc fibrosis can be used as a Smad2/3 phosphorylation promoter, for example, a Smad2/3 phosphorylation promoter in annulus fibrosus cells.
  • the substance for inducing intervertebral disc fibrosis can up-regulate the expression of p-Smad2 and/or p-Samd3 in annulus fibrosus cells.
  • bleomycin or the combination of bleomycin and fibroblasts can activate the TGF- ⁇ /Smad pathway in annulus fibrosus cells, induce phosphorylation of Smad2/3, which is highly expressed in TGF- ⁇
  • its downstream factors p-Smad2 and/or p-Samd3 also showed high expression. It can be seen that through the high expression of TGF- ⁇ , the phosphorylation of Smad2 and/or Samd3 in annulus fibrosus cells can be effectively induced, and the expression of p-Smad2 and/or p-Samd3 in annulus fibrosus cells can be up-regulated to induce intervertebral disc fibrosis.
  • spinal degenerative disease can be intervertebral disc degeneration, specifically can be acute or subacute intervertebral disc degeneration, intervertebral space collapse, intervertebral disc herniation, spinal stenosis, spondylolisthesis and other diseases.
  • the patients corresponding to these diseases can be patients with clinically reduced intervertebral disc height, but no curvature and collapse of the intervertebral space, no obvious spinal stenosis, and no neurological symptoms after surgical or non-surgical treatment.
  • the substance for inducing intervertebral disc fibrosis may be the only medicinal component.
  • a second aspect of the present invention provides a composition comprising a substance for inducing intervertebral disc fibrosis, the substance for inducing intervertebral disc fibrosis comprising a combination of bleomycin and fibroblasts.
  • the composition provided by the present invention may also include at least one pharmaceutically acceptable carrier, which generally refers to a carrier for administration, which does not itself induce the production of antibodies that are harmful to the individual receiving the composition, and which is administered to the individual receiving the composition. There is no excessive toxicity after the drug.
  • a pharmaceutically acceptable carrier which generally refers to a carrier for administration, which does not itself induce the production of antibodies that are harmful to the individual receiving the composition, and which is administered to the individual receiving the composition. There is no excessive toxicity after the drug.
  • the carrier can be a combination including, but not limited to, one or more of saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and the like.
  • “individual” generally includes humans and non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle and the like.
  • a third aspect of the present application provides a method of treatment, comprising: administering to an individual an effective amount of the above-mentioned substance for inducing intervertebral disc fibrosis, or the composition provided in the second aspect of the present application.
  • the "effective amount” generally refers to an amount that, after an appropriate period of administration, will achieve the desired effect, eg, imaging, treatment of disease, and the like.
  • the inventors of the present invention utilize the pro-fibrotic effect of the substance that can induce fibrosis of the intervertebral disc itself, combined with the auxiliary effect of fibroblasts, and administer the two at the same time to achieve a further effect of treating intervertebral disc degeneration, and the treatment
  • the process can reasonably control the degeneration, and achieve the purpose of retreating in an orderly and step-by-step manner, which has clinical transformation value.
  • the therapeutic method of drug binding to cells provided by the present application can make the treatment plan more personalized.
  • the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques have been well described in the existing literature. For details, please refer to Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M. Wassarman and A.P. Wolfe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999 et al.
  • Annulus fibrosus cells (cells provided by the cell bank of the Chinese Academy of Sciences) were cultured in six-well plates under the following conditions: high glucose DMEM medium + 10% fetal bovine serum, 37°C 5% CO2 incubator, at a cell confluency of 70%-80% %, add bleomycin 5, 10 ⁇ g/ml and/or add TGF ⁇ R1 inhibitor LY364947 to co-incubate for 30 minutes or 24 hours.
  • Total cellular proteins were extracted from cultured annulus fibrosus cells (Rush University Medical Center) using RIPA lysis buffer (Roche, Basel, Switzerland) supplemented with phosphatase and protease inhibitors.
  • Equal amounts (20-30 [mu]g) of extracted proteins were separated on 10% or 12.5% SDS-PAGE gels and electroblotted with 0.22 [mu]m PVDF membranes (Merck Millipore). Membranes were blocked with 5% BSA-PBS for 1 hour at room temperature and then incubated with primary antibody (diluted 1:1000 in 5% BSA-PBS) overnight (at least 16 hours) at 4°C.
  • Anti-SMAD2 antibody (Ser308, D43B4; rabbit monoclonal antibody), anti-phospho-SMAD2 antibody (Ser465/467, 138D4; rabbit monoclonal antibody), anti-SMAD3 antibody (C67H9; rabbit monoclonal antibody), anti-phospho-SMAD3 antibody ( Ser423/425, C25A9; Rabbit mAb), Anti-SMAD2/3 Antibody (D7G7; Rabbit mAb), Anti-phospho SMAD2/3 Antibody (Ser465/467; Ser423/425; Rabbit mAb), Anti-SMAD4 Antibodies (D3M6U; rabbit monoclonal antibody) and anti-beta-actin antibody (D6A8; rabbit monoclonal antibody) were purchased from Cell Signaling Technology (Danvers, MA, USA).
  • Anti-FSP1 antibody (S100A4; rabbit monoclonal antibody), transforming growth factor beta receptor I (ab31013; rabbit monoclonal antibody), collagen type I (ab6308; rabbit monoclonal antibody) and transforming growth factor beta 1 (ab64715; rabbit monoclonal antibody) Antibodies) were obtained from Abcam (Cambridge, UK). Membranes were then rinsed in Tris-buffered saline-Tween20 (TBST), followed by incubation with anti-rabbit IgG (H+L) (DyLight) TM 8004 ⁇ PEG conjugate; Cell Signaling Technology) secondary antibody (1:5000 dilution) at room temperature 1 hour in the dark.
  • TST Tris-buffered saline-Tween20
  • Membranes were extensively washed again in TBST, and protein immunobands were detected on the LI-COR Odyssey Fluorescence Imaging System (LI-COR Biosciences, Northeast Lincoln, USA), showing that p-smad2 in annulus fibrosus cells was stimulated by bleomycin. , p-smad3 was activated (as shown in Figure 1A and 1B), and the expression of the terminal protein products Col1a1 and FSP1 increased (as shown in Figure 1C).
  • LI-COR Odyssey Fluorescence Imaging System LI-COR Biosciences, Northeast Lincoln, USA
  • the tail was sterilized with iodinated polyvinylpyrrolidone, and then a longitudinal skin incision was made on the ventral side of the tail to expose the intervertebral disc at 6-10 coccyx.
  • the Co6/7 intervertebral disc was used as the sham control, and the Co7/8, Co8/9, and Co9/10 were used as the experimental group.
  • a sterile 20-gauge needle was used to pierce the disc perpendicular to the skin to ensure passage through the annulus fibrosus and to insert the nucleus pulposus in the center of the disc. The incision was then sutured and the rat was allowed to recover for two weeks.
  • Rats (n 6) were re-operated to expose Co8/9 and Co9/10 intervertebral discs, injected 5 ⁇ l of 10 and 5 ⁇ g/ml bleomycin into each intervertebral disc, respectively, sutured the incision, and given the rats a 4-week treatment period. After the experiment, all the remaining rats were sacrificed, tails were removed, soft tissues were removed, and the spine was fixed with 4% PFA. Co6/7 was used as the control group (sham group), Co7/8 was used as the puncture group (Puncture group), Co8/9, Co9 /10 is the puncture + bleomycin administration group (Bleomycin group).
  • the fixed intervertebral disc tissue specimens (sham group, Puncture group and Bleomycin group) were embedded in paraffin blocks, and then tissue sections (5 ⁇ m thick) were performed.
  • tissue sections were stained with saffron O-fast green and Sirius red according to standard laboratory protocols.
  • immunofluorescence detection tissue sections were deparaffinized with graded xylene, rehydrated in graded alcohol solution, and then incubated in antigen retrieval buffer (Roche) at 37°C for 30 minutes. After cooling to room temperature, the slides were immersed in PBS (pH 7.4) and washed 3 times for 5 minutes each. Add autofluorescence quencher to the sections for 5 min and block with blocking buffer for 30 min at room temperature.
  • Sections were then incubated with primary antibodies overnight in a humidified chamber at 4°C.
  • Primary antibodies were used at 1:100 dilution and included anti-Col1a1, anti-FSP1, anti-TGF ⁇ , and anti-TGF ⁇ R1 (all purchased from Cell Signaling Technologies).
  • Alexa-fluor594-conjugated secondary antibody anti-rabbit, 1:500; Cell Signaling Technology
  • Sections were washed with PBS and then incubated with DAPI solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes in the dark to stain nuclei.
  • Sections were subjected to a final PBS wash, air-dried, and then sealed with anti-fluorescence quencher sheets.
  • Digital fluorescence images were taken under a Leica DM4000 B epifluorescence microscope (Leica Microsystems).
  • the indentation was applied with a z-piezoelectric displacement rate of 10 ⁇ m/s under a maximum load of ⁇ 120 nn, a radius of curvature of 5 nm, and a force constant of 0.4 N/m.
  • the tail was sterilized with iodinated polyvinylpyrrolidone, and then a longitudinal skin incision was made on the ventral side of the tail to expose the intervertebral disc at 6-10 coccyx.
  • the Co6/7 intervertebral disc was used as the sham control, and the Co7/8, Co8/9, Co9/10, and Co10/11 were used as the experimental group.
  • a sterile 20-gauge needle was used to pierce the disc perpendicular to the skin to ensure passage through the annulus fibrosus and to insert the nucleus pulposus in the center of the disc. The incision was then sutured and the rat was allowed to recover for two weeks.
  • 10 ⁇ 5 L929 cells were combined with mycin, the incision was sutured, and the rats were treated for 4 weeks. After the experiment, all the remaining rats were sacrificed, the tail was removed, the soft tissue was removed, and the spine was fixed with 4% PFA. Allogeneic fibroblast therapy group (DFb group), Co9/10 bleomycin administration group (Bleomycin group), Co9/10 bleomycin combined with fibroblast therapy group (Bleomycin+DFb group, Rescure group).
  • the present invention effectively overcomes various shortcomings in the prior art, and has a good transformation prospect and value.

Abstract

一种用于诱导椎间盘纤维化的物质在制备药物中的用途，所述药物用于治疗脊柱退行性疾病。发明人利用能够诱导椎间盘纤维化的物质本身的促纤维化作用，并结合成纤维细胞的辅助作用，通过两者结合同时进行给药，达到更进一步的治疗椎间盘退变的效果，且治疗过程可以合理控制退变，达到退而有序、阶梯治疗的目的，具临床转化价值。此外，所提供的药物结合细胞的治疗方法可以使得治疗方案更具个性化。

Description

用于诱导椎间盘纤维化的物质在制备药物中的用途 技术领域
本发明涉及医药领域，特别是涉及用于诱导椎间盘纤维化的物质在制备药物中的用途。
背景技术
当今社会约6亿患者遭受由于椎间盘退变引起的腰痛困扰，这种退变性疾病的诊疗给所有发展中及发达国家的医疗和社会经济结构带来了巨大的负担。椎间盘退变是一个复杂的过程，涉及三个主要部分：髓核(NP)—椎间盘富含蛋白多糖的胶状中心；纤维环(AF)—围绕在髓核周围的部分同心的富含胶原的纤维软骨组织；两个软骨终板—分别与椎体上下交界。它是一种进行性细胞介导的分子级联反应，涉及到结构和生物力学的变化：首先是髓核蛋白多糖含量的降低及其与胶原的比率降低导致髓核静水压性能损失，这种结构磨损和生物力学功能受损会使得髓核持续脱水并塌陷，椎间盘高度丢失，髓核与纤维环边界逐渐丧失，最终导致整个椎间盘完全退变。严重者甚至会导致脊柱不稳和神经根压迫，从而引起慢性背痛。
椎间盘自我修复和再生功能较弱，这可能是由于椎间盘细胞数量少，组织缺乏营养供应。所以传统的治疗策略在很大程度上局限于症状缓解，长期疗效有限。手术干预通常是为了恢复高度和维持椎间隙的稳定性，并帮助缓解严重的神经症状。然而，脊柱融合术并不能恢复生物力学特性，但事实上，由于相邻椎体承受的机械应力增加，可能导致相邻椎体退变。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于使用诱导椎间盘纤维化的物质以制备用于治疗脊柱退行性疾病的药物，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明一方面提供用于诱导椎间盘纤维化的物质在制备药物中的用途，所述药物用于治疗脊柱退行性疾病。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质包括博来霉素。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质包括博来霉素与成纤维细胞的组合。
在本发明一些实施方式中，所述成纤维细胞为真皮成纤维细胞。
在本发明一些实施方式中，所述成纤维细胞为自体成纤维细胞。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质能够诱导纤维环细胞TGF-β高表达。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质能够上调纤维环细胞中p-Smad2和/或p-Samd3的表达。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质能够上调纤维环细胞中FSP1的表达。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质能够上调纤维环细胞中I型胶原蛋白的表达。
在本发明一些实施方式中，所述诱导椎间盘纤维化的物质能够上调纤维环细胞中III型胶原蛋白的表达。
在本发明一些实施方式中，所述脊柱退行性疾病选自椎间盘退变。
在本发明一些实施方式中，所述用于诱导椎间盘纤维化的物质是唯一有效成分。
附图说明
图1为实施例1中实验结果示意图，其中，图1A、图1B为博来霉素刺激纤维环细胞、激活TGF-β/Smad通路结果示意图，图1C为博来霉素促使成纤维相关蛋白TGFβ、FSP1、Col1a1高表达的免疫蛋白印迹示意图。
图2为实施例3中博来霉素椎间盘局部注射治疗脊柱退变的结果示意图，其中，图2A为X线结果示意图，图2B为MRI结果示意图。
图3为实施例4中博来霉素椎间盘局部注射治疗脊柱退变六周后的番红固绿、天狼星红石蜡切片染色结果示意图。
图4为实施例4中博来霉素椎间盘局部注射治疗脊柱退变六周后的番红固绿、天狼星红染色的量化统计结果示意图。
图5为实施例4中博来霉素椎间盘局部注射治疗脊柱退变六周后的TGFβ1、TGFβR1、Col1a1、FSP1免疫荧光染色结果示意图。
图6为实施例4中博来霉素椎间盘局部注射治疗脊柱退变六周后的TGFβ1、TGFβR1、Col1a1、FSP1免疫荧光染色的量化统计结果示意图。
图7为实施例5中博来霉素椎间盘局部注射治疗脊柱退变六周后的原子力学分析结果示意图。
图8为实施例5中原子力学分析的量化统计结果示意图。
图9为实施例7中博来霉素辅助成纤维细胞进行椎间盘局部注射治疗脊柱退变的结果示意图，其中，图9A为X线结果示意图，图9B为MRI结果示意图。
具体实施方式
为了使本发明的发明目的、技术方案和有益技术效果更加清晰，以下结合实施例对本发明进行进一步详细说明，熟悉此技术的人士可由本说明书所揭露的内容容易地了解本申请发明的其他优点及功效。
本发明第一方面提供用于诱导椎间盘纤维化的物质在制备药物中的用途，所述药物用于治疗脊柱退行性疾病。本发明发明人发现，用于诱导椎间盘纤维化的物质(例如，博来霉素等)可以直接被用于治疗脊柱退行性疾病，其原理为维持甚至恢复椎间隙高度，防止神经根卡压及脊柱不稳定，快速充分的纤维化有助于达到及时稳定，从而可以有效控制脊柱退行性疾病。
本发明中，所述用于诱导椎间盘纤维化的物质通常可以被施用于椎间盘之间，从而可以诱导椎间盘纤维环细胞迁移，分泌纤维化胶原沉积，达到有效的支撑以维持椎间隙高度、并且保留一定的椎体的活动度、维持椎间稳定、防止椎间盘的进一步退行性病变的效果。合适的向个体施用上述用于诱导椎间盘纤维化的物质的方法对于本领域技术人员来说应该是已知的，例如，用于诱导椎间盘纤维化的物质可以通过注射剂等形式被施用。在本发明一具体实施例中，可以将博来霉素(水溶性晶体物质)配置为水溶液，作为注射剂在椎间盘之间进行局部注射。在本发明另一具体实施例中，可以将博来霉素配置为水溶液、并将成纤维细胞分散于水溶液中，作为注射剂在椎间盘之间进行局部注射。
本发明中，所述用于诱导椎间盘纤维化的物质可以包括成博来霉素(Bleomycin)。博来霉素是一种已知的临床用抗癌药，是一种细胞毒性化学治疗化合物，通常用于治疗淋巴瘤、白血病、鳞状细胞癌和一些生殖道肿瘤。在本发明一具体实施例中，通过博来霉素可以有效诱导纤维环细胞持续释放或高表达TGF-β，还可以上调纤维环细胞中p-Smad2和/或p-Samd3的表达，还可以上调纤维环细胞中I型胶原蛋白和/或III型胶原蛋白的表达，从而可以有效诱导椎间盘纤维化，并在一定程度上恢复椎间盘的高度和生物力学特性用。
本发明中，所述用于诱导椎间盘纤维化的物质还可以包括成纤维细胞，具体可以为博来霉素和成纤维细胞的组合。成纤维细胞也称为纤维母细胞，通常是疏松结缔组织的主要细胞成分，属于终末分化细胞，可以分泌大量I型胶原纤维。
本发明中，所述成纤维细胞可以是真皮成纤维细胞，所述真皮成纤维细胞主要源自于皮肤的真皮层。具体来说，皮肤覆盖于体表，从外至内依次为表皮、真皮、皮下层，真皮主要由成纤维细胞及其产生的纤维、基质构成。所述成纤维细胞可以是异体和/或异种的，也可以是同种的，优选为自体成纤维细胞，从而可以有效避免机体的排斥反应，以提供更佳的安全 性。在本发明一具体实施例中，通过成纤维细胞与博来霉素的组合，可以更加有效地诱导纤维环细胞持续释放或高表达TGF-β，还可以进一步上调纤维环细胞中p-Smad2和/或p-Samd3的表达，还可以进一步上调纤维环细胞中I型胶原蛋白和/或III型胶原蛋白的表达，从而可以更加有效诱导椎间盘纤维化，并恢复椎间盘的高度和生物力学特性用。
本发明中，所述用于诱导椎间盘纤维化的物质可以作为转化生长因子-β(transforming growth factor-β，TGF-β)的促进剂，例如，纤维环细胞中的转化生长因子-β的促进剂。所述用于诱导椎间盘纤维化的物质能够诱导纤维环细胞持续释放或高表达TGF-β，从而可以避免反复给药。在本发明一具体实施例中，博来霉素或博来霉素与成纤维细胞的组合可以激活纤维环细胞的TGF-β/Smad通路，诱导Smad2/3磷酸化，从而使得TGFβ高表达。
本发明中，所述用于诱导椎间盘纤维化的物质可以作为FSP1(成纤维细胞特异性蛋白1，Fibroblast specific protein 1)的促进剂，例如，纤维环细胞中的FSP1的促进剂。所述用于诱导椎间盘纤维化的物质能够诱导纤维环细胞中FSP1表达上升。在本发明一具体实施例中，博来霉素或博来霉素与成纤维细胞的组合可以激活纤维环细胞的TGF-β/Smad通路，诱导Smad2/3磷酸化，从而使得终末蛋白产物FSP1表达上升。
本发明中，所述用于诱导椎间盘纤维化的物质可以作为I型胶原蛋白的促进剂，例如，纤维环细胞中的I型胶原蛋白的促进剂。所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中I型胶原蛋白的表达。在本发明一具体实施例中，博来霉素或博来霉素与成纤维细胞的组合可以刺激纤维环细胞，可以使得其I型胶原蛋白表达升高。
本发明中，所述用于诱导椎间盘纤维化的物质可以作为III型胶原蛋白的促进剂，例如，纤维环细胞中的III型胶原蛋白的促进剂。所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中III型胶原蛋白的表达。在本发明一具体实施例中，博来霉素或博来霉素与成纤维细胞的组合可以刺激纤维环细胞，可以使得其III型胶原蛋白表达升高。
本发明中，所述用于诱导椎间盘纤维化的物质可以作为Smad2/3磷酸化促进剂，例如，纤维环细胞中的Smad2/3磷酸化促进剂。所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中p-Smad2和/或p-Samd3的表达。在本发明一具体实施例中，博来霉素或博来霉素与成纤维细胞的组合可以激活纤维环细胞的TGF-β/Smad通路，诱导Smad2/3磷酸化，在TGF-β高表达的同时，其下游因子p-Smad2和/或p-Samd3同样出现高表达。可见，通过TGF-β的高表达，从而可以有效诱导纤维环细胞中Smad2和/或Samd3的磷酸化，上调纤维环细胞中p-Smad2和/或p-Samd3的表达，以诱导椎间盘纤维化。
本发明中，所述脊柱退行性疾病可以是椎间盘退变，具体可以是急性或亚急性椎间盘退 变、椎间隙塌陷、椎间盘突出症、椎管狭窄症、椎滑脱症等疾病。具体来说，这些疾病所对应的患者可以是临床上出现椎间盘高度降低，但是椎间隙未弯曲塌陷，无明显椎管狭窄，手术或非手术治疗后无神经症状的患者。
本发明中，所述用于诱导椎间盘纤维化的物质可以是唯一药效成分。
本发明第二方面提供一种组合物，所述组合物包括用于诱导椎间盘纤维化的物质，所述用于诱导椎间盘纤维化的物质包括博来霉素与成纤维细胞的组合。
本发明所提供的组合物中，还可以包括至少一种药学上可接受的载体，其通常指用于给药的载体，它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域技术人员所熟知的，例如，在Remington’s Pharmaceutical Sciences(Mack Pub.Co.,N.J.1991)中公开了关于药学上可接受的载体的相关内容。具体来说，所述载体可以是包括但不限于盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂等中的一种或多种的组合。本发明中，“个体”通常包括人类、非人类的灵长类，如哺乳动物、狗、猫、马、羊、猪、牛等。
本申请第三方面提供一种治疗方法，包括：向个体施用有效量的上述用于诱导椎间盘纤维化的物质、或本申请第二方面提供的组合物。所述“有效量”通常指一用量在经过适当的给药期间后，能够达到欲求的效果，例如，造影、治疗疾病等。
本发明发明人利用能够诱导椎间盘纤维化的物质本身的促纤维化作用，并结合成纤维细胞的辅助作用，通过两者结合同时进行给药，达到更进一步的治疗椎间盘退变的效果，且治疗过程可以合理控制退变，达到退而有序、阶梯治疗的目的，具临床转化价值。此外，本申请所提供的药物结合细胞的治疗方法可以使得治疗方案更具个性化。
下面通过实施例对本申请的发明予以进一步说明，但并不因此而限制本申请的范围。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press， San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1
博来霉素激活纤维环细胞的TGF-β/Smad通路：
纤维环细胞免疫蛋白印迹的方法：
纤维环细胞(中科院细胞库提供的细胞)于六孔板中培养，培养条件为：高糖DMEM培养基+10％胎牛血清，37℃5％CO2培养箱，于细胞汇合度70％-80％时加入博来霉素5，10μg/ml和/或加入TGFβR1抑制剂LY364947共同培养30分钟或者24小时。使用添加磷酸酶和蛋白酶抑制剂的RIPA裂解缓冲液(瑞士巴塞尔罗氏公司)从培养的纤维环细胞(拉什大学医学中心)中提取细胞总蛋白。用10％或12.5％的SDS-PAGE凝胶对提取的蛋白质进行等量(20-30μg)分离，并用0.22μm PVDF膜(Merck Millipore)电印迹分离蛋白质。在室温下用5％BSA-PBS封闭膜1小时，然后在4℃下与一级抗体(在5％BSA-PBS中稀释1:1000)孵育过夜(至少16小时)。抗SMAD2抗体(Ser308、D43B4；兔单克隆抗体)、抗磷酸化SMAD2抗体(Ser465/467、138D4；兔单克隆抗体)、抗SMAD3抗体(C67H9；兔单克隆抗体)、抗磷酸化SMAD3抗体(Ser423/425，C25A9；兔单克隆抗体)、抗SMAD2/3抗体(D7G7；兔单克隆抗体)、抗磷光体SMAD2/3抗体(Ser465/467；Ser423/425；兔单克隆抗体)、抗SMAD4抗体(D3M6U；兔单克隆抗体)和抗β-肌动蛋白抗体(D6A8；兔单克隆抗体)从Cell Signaling Technology(美国马萨诸塞州丹佛斯)购买。抗FSP1抗体(S100A4；兔单克隆抗体)、转化生长因子β受体Ⅰ(ab31013；兔单克隆抗体)、Ⅰ型胶原(ab6308；兔单克隆抗体)和转化生长因子β1(ab64715；兔单克隆抗体)的一级抗体从Abcam(英国剑桥)获得。然后在Tris缓冲盐水-Tween20(TBST)中冲洗膜，随后用抗兔IgG(H+L)(DyLight)培养 TM 8004×PEG结合物；细胞信号技术)二级抗体(1:5000稀释度)室温下黑暗1小时。再次在TBST中广泛冲洗膜，并在LI-COR Odyssey荧光成像系统(美国东北林肯市LI-COR Biosciences)上检测到蛋白质免疫条带，可见在博来霉素刺激下，纤维环细胞p-smad2、p-smad3激活(如图1A和图1B所示)，终末蛋白产物Col1a1、FSP1表达上升(如图1C所示)。
实施例2
所有动物实验均经上海交通大学医学院第九人民医院动物护理与伦理委员会批准，并按 照美国国立卫生研究院(NIH)实验动物护理和使用指南的原则和程序进行。6只8周龄雄性Sprague-Dawley大鼠(中国上海动物研究中心有限公司上海实验室)在26-28℃和50-65％湿度条件下养育，昼夜12小时。给动物喂食标准的啮齿动物食物，所以动物均可以随意获得淡水。手术前，通过腹腔注射戊巴比妥钠(5mg/100g体重)麻醉大鼠。尾部用碘化聚乙烯吡咯烷酮消毒，然后在尾部腹侧纵行皮肤切口，露出尾骨6-10处的椎间盘。以Co6/7椎间盘为假对照，以Co7/8、Co8/9、Co9/10为实验组。用一个20口径的无菌针垂直于皮肤刺穿椎间盘，以确保通过纤维环并在椎间盘中心插入髓核。然后缝合切口，让大鼠恢复两周。大鼠(n＝6)再次手术暴露Co8/9和Co9/10椎间盘，分别向每个椎间盘注射10和5μg/ml博来霉素5μl，缝合切口，给予大鼠4周的治疗时间。实验结束后，处死所有剩余大鼠，取尾，清除软组织，用4％PFA固定脊柱，Co6/7作为对照组(sham组)，Co7/8作为穿刺组(Puncture组)，Co8/9、Co9/10为穿刺+博来霉素施用组(Bleomycin组)。
实施例3
对实施例2制备的穿刺椎间盘样品(Puncture组和Bleomycin组)的数字X射线成像在前后轴上进行，该探测器提供高达5倍的几何放大倍数(Faxitron VersaVision；Faxitron Bioptics LLC，Tucson，AZ。美国)。在西门子MagnetomPrisma E11(Siemens Healthineers，Erlangen，Germany)上对同一穿刺椎间盘样品进行MRI成像，参数如下：TR 3000ms，TE 80ms，厚度1.1mm，间隔0.22mm，视野160×65mm，体素尺寸0.25×0.1mm。
由数字X线成像结果可见，穿刺组(Puncture组)椎间盘塌陷严重，骨赘生成(如图2A左所示)，由MRI成像结果可知，穿刺对照组椎间盘信号变黑(如图2B左所示)。而对应的博来霉素施用组(Bleomycin组)的数字X线成像和MRI成像结果可知，使用博来霉素治疗后可以有效维持椎间隙高度，虽然椎间盘信号难以保留，但其稳定性无明显影响(如图2A右和图2B右所示)。
实施例4
将固定的椎间盘组织标本(sham组、Puncture组和Bleomycin组)包埋于石蜡块中，然后进行组织切片(5μm厚)。为了进行组织学评估，石蜡切片按照标准的实验室规程进行藏红O-固绿和天狼星红染色。免疫荧光检测时，将组织切片用分级二甲苯脱石蜡，在分级酒精溶液中复水，然后在37℃的抗原回收缓冲液(Roche)中孵育30分钟。冷却至室温后，将载玻片浸入PBS(pH7.4)中，洗涤3次，每次5分钟。在切片中加入自荧光猝灭剂5min，室 温下用阻断缓冲液阻断30min。随后在4℃的湿箱中用一级抗体孵育切片过夜。一级抗体以1:100稀释使用，包括抗Col1a1、抗FSP1、抗TGFβ和抗TGFβR1(均从细胞信号技术购买)。第二天，用PBS冲洗切片，然后用Alexa-fluor594结合二级抗体(抗兔，1:500；细胞信号技术)在室温下黑暗中孵育50分钟。切片用PBS洗涤，然后用DAPI溶液(美国密苏里州圣路易斯市Sigma-Aldrich)在黑暗中孵育10分钟以染色细胞核。切片进行最终PBS洗涤，风干，然后用抗荧光猝灭片密封。数字荧光图像是在徕卡DM4000 B表观荧光显微镜(徕卡微系统公司)下拍摄。
番红固绿染色及天狼星红染色如图3所示，可见穿刺组(Puncture)中髓核脱落，纤维环被破坏，锥间隙塌陷，软骨终版退变严重，但博来霉素施用组(Puncture+Bleomycin)中椎间盘明显椎间隙高度更高，并且纤维环修复完全并向内迁移，分泌大量I型胶原填充椎间隙范围。统计学分析可见，注射10和5μg/ml的博来霉素施用组(Puncture+、Bleomycin 10或5)相对于穿刺组(Puncture+、Bleomycin 0)和对照组(Puncture-、Bleomycin 0)，纤维环染色(绿色)、I型胶原(黄色)、III型胶原(绿色)比例明显上升，如图4所示。免疫荧光结果可见博来霉素施用组(Puncture+Bleomycin)(Puncture+、Bleomycin 10或5)中大量分泌了I型及III型胶原(如图5、图6所示)，显示博来霉素可以快速稳定的使椎间盘纤维化，分泌大量I型及III型胶原填充椎间隙。
实施例5
原子力学分析将提取的穿刺椎骨解剖制成石蜡切片，并在配备有微球胶体尖端的Park NX20(韩国Park Systems)上进行纳米压痕(R<10nm，标称k≈0.2N/m，提示：Si/Tipless/Top，悬臂Si/AI/Top；公园系统)。对于大范围的起伏表面，采用13hz的扫描速率。大扫描速率可以减小漂移，但一般只用于扫描小平面。使用扫描Asyst空气探针，以10μm/s的z压电位移速率，在～120nn的最大载荷下施加压痕，曲率半径为5nm，力常数为0.4N/m。
由原子力学分析结果图7可见，穿刺组(Puncture)中纤维环应力有所上升，但考虑这应该是椎间隙塌陷，挤压椎间盘中成分，使其更致密导致，使用博来霉素刺激后(Puncture+Bleomycin)可以更快速的纤维化，使椎间盘抗形变能力上升，同时维持椎间隙高度。统计学分析可见类似特征，详见图8。
实施例6
所有动物实验均经上海交通大学医学院第九人民医院动物护理与伦理委员会批准，并按 照美国国立卫生研究院(NIH)实验动物护理和使用指南的原则和程序进行。6只8周龄雄性Sprague-Dawley大鼠(中国上海动物研究中心有限公司上海实验室)在26-28℃和50-65％湿度条件下养育，昼夜12小时。给动物喂食标准的啮齿动物食物，所以动物均可以随意获得淡水。手术前，通过腹腔注射戊巴比妥钠(5mg/100g体重)麻醉大鼠。尾部用碘化聚乙烯吡咯烷酮消毒，然后在尾部腹侧纵行皮肤切口，露出尾骨6-10处的椎间盘。以Co6/7椎间盘为假对照，以Co7/8、Co8/9、Co9/10、Co10/11为实验组。用一个20口径的无菌针垂直于皮肤刺穿椎间盘，以确保通过纤维环并在椎间盘中心插入髓核。然后缝合切口，让大鼠恢复两周。大鼠(n＝6)再次手术暴露Co8/9、Co9/10和Co10/11椎间盘，分别向每个椎间盘注射10^5个L929细胞，5ug/ml博来霉素5μl及5ug/ml博来霉素结合10^5个L929细胞，缝合切口，给予大鼠4周的治疗时间。实验结束后，处死所有剩余大鼠，取尾，清除软组织，用4％PFA固定脊柱，Co6/7作为对照组(sham组)，Co7/8作为穿刺组(Puncture组)，Co8/9作为同种异体成纤维细胞治疗组(DFb组)、Co9/10博来霉素施用组(Bleomycin组)、Co9/10博来霉素联合成纤维细胞治疗组(Bleomycin+DFb组，Rescure组)。
实施例7
对实施例6制备的穿刺椎间盘样品(Puncture组和Rescure组)的数字X射线成像在前后轴上进行，该探测器提供高达5倍的几何放大倍数(Faxitron VersaVision；Faxitron Bioptics LLC，Tucson，AZ。美国)。在西门子MagnetomPrisma E11(Siemens Healthineers，Erlangen，Germany)上对同一穿刺椎间盘样品进行MRI成像，参数如下：TR 3000ms，TE 80ms，厚度1.1mm，间隔0.22mm，视野160×65mm，体素尺寸0.25×0.1mm。
由数字X线成像结果可见，穿刺组(Puncture组)椎间盘塌陷严重，骨赘生成(如图9A左所示)，由MRI成像结果可知，穿刺对照组椎间盘信号变黑(如图9B左所示)。而对应的组合施用组(Rescure组)的数字X线成像和MRI成像结果可知，使用博来霉素治疗和成纤维细胞治疗后均可以有效维持椎间隙高度，同时发现两者互相结合治疗效果更佳(如图9A右和图9B右所示)。
综上所述，本发明有效克服了现有技术中的种种缺点，具有良好的转化前景及价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等 效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (10)

  1. 用于诱导椎间盘纤维化的物质在制备药物中的用途，所述药物用于治疗脊柱退行性疾病。
  2. 如权利要求1所述的用途，其特征在于，所述用于诱导椎间盘纤维化的物质包括博来霉素。
  3. 如权利要求2所述的用途，其特征在于，所述用于诱导椎间盘纤维化的物质包括博来霉素与成纤维细胞的组合。
  4. 如权利要求3所述的用途，其特征在于，所述成纤维细胞为真皮成纤维细胞；
    和/或，所述成纤维细胞为自体成纤维细胞。
  5. 如权利要求1所述的用途，其特征在于，所述用于诱导椎间盘纤维化的物质能够诱导纤维环细胞TGF-β高表达。
  6. 如权利要求1所述的用途，其特征在于，所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中p-Smad2和/或p-Samd3的表达。
  7. 如权利要求1所述的用途，其特征在于，所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中FSP1的表达；
    和/或，所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中I型胶原蛋白的表达；
    和/或，所述用于诱导椎间盘纤维化的物质能够上调纤维环细胞中III型胶原蛋白的表达。
  8. 如权利要求1所述的用途，其特征在于，所述脊柱退行性疾病选自椎间盘退变。
  9. 如权利要求1所述的用途，其特征在于，所述用于诱导椎间盘纤维化的物质是唯一有效成分。
  10. 一种组合物，所述组合物包括用于诱导椎间盘纤维化的物质，所述用于诱导椎间盘纤维化的物质包括博来霉素与成纤维细胞的组合。
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