WO2019057219A1 - Ghrelin活性剂用于诱导干细胞向软骨细胞分化 - Google Patents

Ghrelin活性剂用于诱导干细胞向软骨细胞分化 Download PDF

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WO2019057219A1
WO2019057219A1 PCT/CN2018/114449 CN2018114449W WO2019057219A1 WO 2019057219 A1 WO2019057219 A1 WO 2019057219A1 CN 2018114449 W CN2018114449 W CN 2018114449W WO 2019057219 A1 WO2019057219 A1 WO 2019057219A1
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stem cells
ghrelin
differentiation
tgf
active agent
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French (fr)
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葛子钢
樊丽彤
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北京大学
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Definitions

  • the present invention relates to a composition comprising a ghrelin active agent, and a method of using the same to induce differentiation of stem cells into chondrocytes, and in particular to a method of using the same for treating osteoarthritis, a method for treating cartilage damage, and a corresponding use.
  • cartilage tissue damage has become one of the most common injuries in the clinic.
  • Articular cartilage has no blood vessels and nerves, relying on joint fluid nutrition, once the damage can not repair itself.
  • the chondrocytes of adult animals are terminally differentiated cells, which have limited sources. They are traumatic, difficult to obtain, poor in proliferative ability in vitro, easy to dedifferentiate and lose function, and difficult to meet the conditions of seed cells as cartilage tissue engineering.
  • MSCs Mesenchymal stem cells
  • BMSCs bone marrow MSCs
  • the currently widely used induced stem cell cartilage differentiation medium is based on TGF- ⁇ and cell concentration, and TGF- ⁇ is a multi-functional growth factor by cell accumulation conditions (pellets or micromass)
  • the culture system adds a cartilage-inducing medium to induce chondrogenesis of stem cells. This method does induce stem cells to differentiate into cartilage.
  • Physical means, chemical factors, and biological factors are all used to improve the efficiency of cartilage differentiation. Physical means such as topological structure, force stimulation and hypoxia. Chemical factors such as small molecules and the chemical properties of some materials. Biological factors are mainly proteins and peptides, which are also very useful for the differentiation of MSC into cartilage.
  • TGF- ⁇ is capable of initiating chondrogenic differentiation via the SMAD pathway.
  • PHTrP inhibits the hypertrophic phenotype of primary chondrocytes.
  • BMP-2 is also a member of the TGF- ⁇ superfamily and is capable of inducing chondrogenic differentiation and hypertrophy.
  • the addition of different growth factors in stages can also promote chondrogenic differentiation and subsequent phenotypic maturation.
  • the method has low induction efficiency, accelerates chondrocyte hypertrophy, induces differentiated chondrocytes, and has low expression of cartilage-related genes, chondrocyte subtypes, and mostly growth plate cartilage rather than articular cartilage.
  • Ghrelin also known as ghrelin, is a 28-amino acid intestinal peptide found in the stomach of rats in 1999 by Kojima and is recognized as a growth hormone secretagogue receptor (GHS-R).
  • Source ligand (Kojima et al., 1999). Its biological activity is mainly achieved by interacting with its cell surface specific receptor GHS-R (including GHSR 1 ⁇ and GHSR 1 ⁇ subtypes).
  • Ghrelin and its receptors are widely distributed in peripheral tissues and central nervous system, and play important biological roles, such as promoting the release of growth hormone, increasing appetite, regulating energy metabolism, inhibiting inflammatory reactions and regulating immune function.
  • the present inventors have unexpectedly found that ghrelin can effectively induce differentiation of stem cells into chondrocytes, and has completed the present invention.
  • the invention provides an inducement differentiation composition comprising a ghrelin active agent and a pharmaceutically acceptable carrier.
  • the ghrelin active agent is a mammalian ghrelin, such as human ghrelin, or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67% with the ghrelin amino acid sequence.
  • a peptide having ghrelin activity preferably the ghrelin active agent has the same amino acid as ghrelin at a position corresponding to amino acids 1 to 5 of ghrelin.
  • the amino acid sequence of the human ghrelin is GSSFLSPEHQ RVQQRKESKK PPAKLQPR (SEQ ID NO.
  • the ghrelin active agent is a mammalian ghrelin or human ghrelin, or a peptide in which a mammalian ghrelin or human ghrelin has been replaced, deleted or added with one or more amino acids and has ghrelin activity, preferably The ghrelin active agent has the same amino acid as ghrelin at a position corresponding to amino acids 1 to 5 of ghrelin.
  • the ghrelin active agent is in a non-naturally occurring form, such as chemically or biologically synthesized.
  • the ghrelin active agent can be free or conjugated, for example, to a structure that increases molecular weight (eg, PEG, albumin, or immunoglobulin Fc segments).
  • the ghrelin active agent is octanoylated at a position corresponding to the 3rd serine of human ghrelin.
  • the induced differentiation composition further comprises at least another differentiation inducer, preferably the differentiation inducer is selected from the group consisting of type I collagen, BMP, TGF, FGF, IGF, WNT, GDF, kartogenin, and the like.
  • the BMP is selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, BMP-8, BMP-9, such as BMP- 2 and/or BMP-4
  • the TGF is selected from TGF- ⁇ or TGF- ⁇ , such as TGF- ⁇
  • the FGF is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF- 5.
  • FGF-6, FGF-7, FGF-8, FGF-9, FGF-10 for example FGF-2
  • said IGF being selected from the group consisting of IGF-1 and IGF-2, such as IGF-1
  • said WNT It is selected from WNT-3a, WNT-5a and WNT-7
  • the GDF is selected from GDF-5; more preferably the differentiation inducing agent is TGF- ⁇ , particularly TGF- ⁇ 3.
  • the ghrelin active agent and other differentiation inducing agents can be physically separated in space, located in different compartments. This makes it convenient to apply the ghrelin active agent and other differentiation inducers in different ways, for example by adding different differentiation inducers in time. Specifically, the ghrelin active agent is added first, and the other differentiation inducer is added. Alternatively, the other differentiation inducer may be added first, and the ghrelin active agent may be added.
  • the induced differentiation composition is a medium or medium supplement for inducing differentiation, or a pharmaceutical composition.
  • the present invention provides a method of inhibiting stem cell proliferation comprising exposing the stem cell to the differentiation-inducing composition of the first aspect of the invention.
  • the method can be carried out in vivo and/or in vitro, or it can be achieved by endogenously stimulating the stem cells themselves to produce the ghrelin active agent.
  • the present invention provides a method of inducing differentiation of stem cells into chondrocytes, comprising exposing said stem cells to the differentiation-inducing composition of the first aspect of the invention.
  • the present invention provides a method of enhancing or promoting the ability of TGF- ⁇ to induce differentiation of stem cells into chondrocytes, comprising: rendering said stem cells before, during or after said stem cells are exposed to TGF- ⁇ Exposure to the differentiation-inducing composition of the first aspect of the invention.
  • the present invention provides a method of producing chondrocytes, comprising
  • the present invention provides a method of preparing cartilage tissue, comprising
  • the stem cells of the present invention may be pluripotent stem cells, such as adult stem cells or induced pluripotent stem cells (iPS), and may also be embryonic stem cells.
  • the stem cell is a mesenchymal stem cell.
  • the stem cells are first cultured in the presence of the ghrelin active agent, and then the stem cells are cultured in the absence of the ghrelin active agent.
  • other differentiation inducing agents of the invention such as TGF-[beta] are present during the two-stage culture described above.
  • the present invention provides a method of treating osteoarthritis comprising administering to a subject in need thereof the differentiation-inducing composition of the first aspect of the invention.
  • the present invention provides a method of treating cartilage damage comprising administering to a subject in need thereof the differentiation-inducing composition of the first aspect of the invention.
  • the administering is carried out in at least two stages, wherein the composition applied in the first stage comprises the ghrelin active agent, in the second
  • the composition to be administered in stages does not comprise a ghrelin active agent
  • the composition, optionally administered in the first and second stages comprises at least one of the other differentiation inducing agents, for example, the other differentiation inducer comprises TGF- ⁇ .
  • the nanosecond pulse electric field has a pulse width of 50-200 ns, more preferably 100 ns; a field strength of 5-30 kV/cm, more preferably 20 kV/cm, and an electric shock number of 1-50 times, for example 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 times.
  • the present invention provides a ghrelin active agent for use in a method corresponding to the above second to eighth aspects, for example, for inhibiting stem cell proliferation, for inducing differentiation of stem cells into chondrocytes, for use in Enhances or promotes the ability of TGF-[beta] to induce differentiation of stem cells into chondrocytes, for the production of chondrocytes, for the preparation of cartilage tissue, for the treatment of osteoarthritis, and/or for the treatment of cartilage damage.
  • the ghrelin active agent is preferably provided in the form of a composition as described in the first aspect of the invention.
  • the invention provides the use of a ghrelin active agent for the preparation of a medicament, the use corresponding to the methods of the second to eighth aspects above, in particular the methods of the seventh and eighth aspects.
  • the ghrelin active agent is preferably provided in the form of a composition according to the first aspect of the invention.
  • the present invention can at least provide advantages and benefits of at least one of the following aspects.
  • the invention can significantly improve the efficiency of cartilage formation and avoid the common side effects of accelerating chondrocyte hypertrophy.
  • the ghrelin active agent can be combined with other differentiation inducers to induce differentiation into cartilage, and can have a synergistic effect with other differentiation inducers.
  • Figure 1 Effect of different concentrations of ghrelin on proliferation of rat bone marrow mesenchymal stem cells.
  • Figure 2 Different concentrations of ghrelin and TGF- ⁇ induce the expression of cartilage-related genes in rat bone marrow mesenchymal stem cells after differentiation into chondrocytes.
  • Figure 3 Different expression strategies of ghrelin and TGF- ⁇ induce the expression of cartilage-related genes in rat bone marrow mesenchymal stem cells after differentiation into chondrocytes.
  • FIG. 4 Ghrelin and TGF- ⁇ addition strategies for the expression of glycosaminoglycan (GAG) in cells after induction of differentiation of rat bone marrow mesenchymal stem cells into soft bone cells.
  • GAG glycosaminoglycan
  • Figure 5 Different addition strategies of ghrelin and TGF- ⁇ After induction of differentiation of rat bone marrow mesenchymal stem cells into chondrocytes, histological sections were stained with alexin blue.
  • Figure 6 Expression of cartilage-associated genes in cells after induction of differentiation of rat bone marrow mesenchymal stem cells into chondrocytes by ghrelin and BMP-2.
  • Figure 7 Expression of cartilage-related genes in cells after induction of differentiation of human embryonic stem cells into chondrocytes by ghrelin and TGF- ⁇ .
  • Figure 8 Nanosecond pulsed electric field (nsPEPE) and ghrelin promote cartilage differentiation.
  • A Experimental grouping and corresponding processing methods.
  • the ghrelin active agent refers to a substance capable of providing ghrelin activity in vivo, in vitro, and ex vivo, and does not limit the manner and type of ghrelin activity production.
  • the ghrelin active agent can be a mammalian ghrelin, especially human ghrelin.
  • the ghrelin active agents of the invention may exert ghrelin activity upon in vivo bioprocessing (eg, conformational changes, degradation, structural modifications such as fatty acidation, glycosylation, etc.) upon entry into an animal.
  • the ghrelin active agent itself may undergo ghrelin activity or may not exhibit ghrelin activity prior to in vivo bioprocessing.
  • the term “medium” refers to a mixture of cells for supporting the growth, proliferation and/or differentiation of cells outside the organism, which contains the nutrients and other material conditions necessary to support these cellular processes, for example, may contain sugars, amino acids, various nutrients. , serum, growth factors, inorganic substances, etc.
  • selected from means that one or more elements are selected from a group of elements.
  • treatment refers to a treatment that is expected to provide any improvement in the health of the subject to be treated or to reduce or avoid any health risks that may occur to the subject to be treated.
  • Bone marrow-derived rat bone marrow mesenchymal stem cells were obtained from 6 week old Sprague Dawley (SD) rats. After 6-week-old male SD rats were anesthetized with an excess of chloral hydrate, the tibia and fibula of the two hind legs were removed, and the whole bone marrow adherence method was used (Lin C W, Zhou S H. Journal of Clinical Rehabilitative Tissue Engineering). Research, 2010, 14(14): 2508-2512), the bone marrow inside the bone was rinsed into the culture dish with PBS.
  • SD Sprague Dawley
  • the isolated cells were washed with PBS, and then a medium containing 90% DMEM (Dulbecco's modified Eagle's medium, Gibco), 10% fetal bovine serum (FBS, Gibco), and 0.1% penicillin/streptomycin (PS, Amresco). Monolayer cell culture was carried out in a humidified incubator at 37 ° C, 5% CO 2 , and changed twice a week. When the confluence rate of the cultured cells reached 85%, BMSCs were trypsinized with 0.25% trypsin (Invitrogen). Passage, bone marrow mesenchymal stem cells were collected for subsequent trials when passed to passage 4.
  • DMEM Dynamic fetal bovine serum
  • PS penicillin/streptomycin
  • the rat bone marrow mesenchymal stem cells prepared in Example 1 were cultured for 7 days by adding different concentrations of ghrelin (Kang Peptide Biotechnology (Beijing) Co., Ltd., 031-30), and the control group (Control group) was added with the same amount of medium, respectively. After 3 days and 7 days of culture, each group of stem cells were collected and seeded in a 96-well plate at a density of 3.5 ⁇ 10 3 cells/well, cultured in DMEM medium (37° C., 5% CO 2 ) for 4 hours, according to CCK. The stem cell proliferation test was performed on the instructions of the -8 cell counting kit (CK04, Dojindo). 10 ⁇ l of CCK-8 solution was added to each well of a 96-well plate, incubated at 37 ° C for 2 hours, and then the absorbance at a wavelength of 490 nm was measured with a microplate reader. 4 holes.
  • Fig. 1 After adding ghrelin at different concentrations for 3 days and 7 days, stem cell proliferation was inhibited to a certain extent, and inhibition of stem cell proliferation was necessary for differentiation.
  • the rat bone marrow mesenchymal stem cells prepared in Example 1 were made into a suspension with serum-free cartilage differentiation induction medium, placed in a conical tube at 2.5 ⁇ 10 5 stem cells/tube, centrifuged at 600 g for 10 min, and the pellet was collected at The culture medium was cultured in a serum-free cartilage differentiation induction medium in an incubator (37 ° C, 5% CO 2 ), and changed every 3 days.
  • the serum-free cartilage differentiation induction medium is 10 -7 M dexamethasone, 50 ⁇ g/ml vitamin C, 1 mM sodium pyruvate, 4 mM proline, and 1% v/v ITS+premix (BD Bioscience). Inc., Franklin Lakes, NJ) High glucose DMEM medium.
  • TGF- ⁇ 10 ng/ml human TGF- ⁇ 3, Peprotech, 100-36E
  • ghrelin 10 ng/ml human TGF- ⁇ 3, Peprotech, 100-36E
  • CM group control group only Chrondrogenic differentiation media was added.
  • the specific primer sequences of each gene to be detected and the control gene are as follows:
  • the method for inducing rat bone marrow mesenchymal stem cells was the same as that of Example 3 except that the time limit for adding TGF- ⁇ and ghrelin to the medium was different during the induction process (14 days), and the specific groups were as follows:
  • the concentration of TGF- ⁇ added in each group was 10 ng ml -1 , and the concentration of ghrelin was 10 -9 M.
  • cartilage-related genes ACAN, SOX 9 and COL II were significantly increased in TT group, GG group, TG-TG group and TG-T group compared with CM group without inducer.
  • the expression of COL X and COL I in cartilage hypertrophy was only slightly increased.
  • the expression of cartilage-related genes SOX 9 and COL II in the TG-T group was most significantly increased, and there was a significant difference from the other groups.
  • the GAG content of each group of cells was determined by using the total content of glycosaminoglycan (GAG) dimethylmethylene blue (DMMB) colorimetric assay kit (341088, Sigma); Each group of cells was digested with 0.5 mg/ml proteinase K at 56 ° C for 12 hours, then detected by DMMB, the digestion solution was shaken for 30 min, and then centrifuged at 10000 g for 10 min. The obtained precipitate was dissolved in the decomplexing solution, and the detection solution was at 630 nm. Absorbance, and the GAG content in each group of cell blocks was calculated from a standard curve prepared by chondroitin sulfate.
  • GAG glycosaminoglycan
  • DMMB dimethylmethylene blue
  • the GAG content of the T-T group, the G-G group, the TG-TG group, and the TG-T group was significantly higher than that of the CM group without the inducer.
  • the rat bone marrow mesenchymal stem cells were induced in the same manner as in Example 3 except that during the induction process (14 days), the inducer added to the medium was BMP-2 (100 ng/ml, R&D Systems, 355-BM). As well as BMP-2 plus ghrelin, the control group (CM) only added serum-free cartilage differentiation induction medium. After 14 days of culture, the cells of each group were collected, total RNA was extracted by conventional methods, and the expression of cartilage-related genes (COL X, COL I, SOX 9, ACAN and COL II) in each group was detected by real-time quantitative fluorescent PCR.
  • ghrelin can enhance the cartilage differentiation-inducing effect of BMP-2 on rat mesenchymal stem cells.
  • Example 3 The operation of this example was carried out in accordance with Example 3, in which cells were replaced by rat bone marrow mesenchymal stem cells into a human embryonic stem cell line, WiCell #04-W308 (WA01), by the Chinese Academy of Medical Sciences, Department of Plastic Surgery Dr. Fu Wei from the hospital gave a gift.
  • each group was separately added with TGF- ⁇ (10 ng/ml) or TGF- ⁇ (10 ng/ml) and ghrelin (10 -9 M) to the culture medium.
  • Real-time quantitative fluorescent PCR was used to detect the expression of cartilage-related genes in each group (COL X, COL I, SOX 9 and COL II and ACAN).
  • the specific primer sequences of each gene to be detected and the control gene are as follows:
  • Nanosecond pulsed electric field treatment Rat bone marrow mesenchymal stem cells prepared in Example 1 were made into a suspension with serum-free cartilage differentiation induction medium, and nanosecond electric pulse device was used for nanosecond electric pulse stimulation. The specific parameter was pulse. It is 100ns wide and has a field strength of 20kV/cm, stimulating 5 pulses. Then, 2.5 ⁇ 10 5 stem cells/tubes were placed in a conical tube, centrifuged at 600 g for 10 min, and the pellet was cultured in an incubator (37° C., 5% CO 2 ) with serum-free cartilage differentiation medium every 3 days. Change the fluid once.
  • the serum-free chondrocyte differentiation induction medium is 10 -7 M dexamethasone, 50 ⁇ g/mL vitamin C, 1 mM sodium pyruvate, 4 mM valine, and 1% v/v ITS+premix (BD Bioscience). Inc., Franklin Lakes, NJ) High glucose DMEM medium.
  • TGF- ⁇ 10 ng/ml human TGF- ⁇ 3, Peprotech, 100-36E
  • TG group indicated that TGF- ⁇ and ghrelin were added to the medium
  • TG+nsPEF group indicated Nanosecond pulsed electric field treatment (specific parameters of 100 ns, 20 kV/cm, stimulation of 5 pulses) was also performed before the addition of TGF- ⁇ and ghrelin (see Figure 8A).
  • the GAG content of each group of cells was determined by using the cell glycosaminoglycan (GAG) total dimethylmethylene blue (DMMB) colorimetric quantitative detection kit (341088, Sigma).
  • GAG cell glycosaminoglycan
  • DMMB dimethylmethylene blue
  • the specific determination steps are as follows: each group of cells is digested with 0.5 mg/mL proteinase K at 56 ° C for 12 hours, then detected by DMMB, the digestion solution is shaken for 30 min, and then centrifuged at 10000 g for 10 min, and the obtained precipitate is dissolved in the solution. The absorbance of the solution at 630 nm was measured, and the GAG content (mainly glycosaminoglycan, sGAG) in each group of cell blocks was calculated from a standard curve prepared by chondroitin sulfate.
  • Fig. 8B The results are shown in Fig. 8B.
  • the nanosecond pulsed electric field was used for pretreatment, and the expression of cartilage-related genes ACAN, SOX 9 and COL II was further significantly improved, among which SOX 9 gene expression was compared.
  • the TG group was increased by more than 3 times, and the expression of COL II and ACAN genes was also significantly higher than that of the TG group; at the same time, the COL X and COL I related genes of cartilage hypertrophy were not significantly improved.
  • the TG group and the TG+ nanosecond electric pulse group were compared with the control group, and the sGAG content was increased. Histological sections of each group of cell pellets were stained with alimin blue, and the results showed (see Fig. 8D) that the secretion of extracellular matrix was significantly increased after the addition of nanosecond electric pulse stimulation.
  • nanosecond pulsed electric field treatment can further enhance the ability of ghrelin to induce stem cells to differentiate into cartilage.

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Abstract

本发明提供包含ghrelin活性剂的组合物，和使用其诱导干细胞向软骨细胞分化的方法，具体地还提供使用其治疗骨关节炎的方法以及使用其治疗软骨损伤的方法。

Description

Ghrelin活性剂用于诱导干细胞向软骨细胞分化
交叉引用
本申请要求发明名称为“Ghrelin活性剂用于诱导干细胞向软骨细胞分化”于2017年9月19日提交到中国专利局的中国专利申请201710852799.0的优先权，其内容通过引用以整体并入本文。
技术领域
本发明涉及包含ghrelin活性剂的组合物，和使用其诱导干细胞向软骨细胞分化的方法，具体地还涉及使用其治疗骨关节炎的方法，使用其治疗软骨损伤的方法，以及相应的用途。
背景技术
随着社会人口老龄化和骨关节炎等疾病发病率的增加，软骨组织损伤已成为临床最常见的损伤之一。关节软骨无血管和神经，依靠关节液营养，一旦损伤不能自我修复。无论是通过组织工程还是再生医学的方法，高效获取大量软骨细胞都是软骨损伤治疗的核心问题。成年动物的软骨细胞是终末分化细胞，其来源有限，取材时创伤、难度大，体外增殖能力差，容易去分化而失去功能，难以满足作为软骨组织工程种子细胞的条件。间充质干细胞(mesenchymal stem cell，MSC)是来源于中胚层的具有多向分化潜能的成体干细胞，可以分化成大多数细胞类型，包括骨、软骨、脂肪、肌肉等。MSC尤其是骨髓MSC(BMSC)出色的多向分化能力使其成为软骨组织工程和软骨再生医学中最有潜力的细胞来源。如何更好的理解MSC向软骨定向分化的机理，提高BMSC软骨方向分化的效率是目前科研工作的重要课题之一。
当前广泛使用的诱导干细胞软骨分化培养基基于TGF-β和细胞浓集，TGF-β是一种有多种功能的生长因子，通过在细胞积聚条件下(小球(pellet)或微团(micromass)培养体系)添加软骨诱导培养基，诱导干细胞的软骨形成。这个方法确实能够诱导干细胞成软骨分化。物理手段、化学因子以及生物因子都被应用在提高成软骨分化效率中。物理手段如拓扑结构、力刺激和缺氧等。化学因子如小分子以及一些材料的化学特性等。 生物因子主要是蛋白质和多肽，对于MSC成软骨分化是必须也是十分有用的。TGF-β能够通过SMAD途径起始成软骨分化。PHTrP能够抑制原代软骨细胞的肥大表型。BMP-2也是TGF-β超家族的一员，能够诱导成软骨分化和肥大。当然分阶段的添加不同的生长因子也是能够促进成软骨分化和之后的表型成熟的。然而就目前的技术手段来讲，该方法存在诱导效率低、加速软骨细胞肥大、诱导分化的软骨细胞中成软骨相关基因表达不高、软骨亚型混杂且多为生长盘软骨而非关节软骨，以及对于间充质干细胞向软骨细胞分化的不同阶段没有不同的分化策略等问题。
Ghrelin也称饥饿激素，是1999年由Kojima在大鼠胃中发现的一种具有28个氨基酸的肠肽，被识别为生长激素促分泌素受体(growth hormone secretagogue receptor，GHS-R)的内源性配体(Kojima等人,1999)。它的生物活性主要是通过与其细胞表面特异性受体GHS-R(包括GHSR 1α和GHSR 1β两种亚型)相互作用来完成的。Ghrelin及其受体在外周组织及中枢神经系统中广泛分布,并发挥着重要的生物学作用,如促进生长激素的释放、增加食欲、调节能量代谢、抑制炎性反应及调节免疫功能等。
发明内容
本发明人出乎意料地发现，ghrelin能有效诱导干细胞向软骨细胞分化，并在此基础上完成了本发明。
在第一个方面中，本发明提供一种诱导分化组合物，其包含ghrelin活性剂和药学可接受的载体。
在一个实施方案中，所述ghrelin活性剂是哺乳动物ghrelin如人ghrelin，或者是与ghrelin氨基酸序列具有至少60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％一致性并具有ghrelin活性的肽，优选地所述ghrelin活性剂在对应于ghrelin第1至5位氨基酸的位置具有与ghrelin相同的氨基酸。具体地，所述人ghrelin的氨基酸序列是GSSFLSPEHQ RVQQRKESKK PPAKLQPR(SEQ ID NO.1)。在一个替代实施方案中，所述ghrelin活性剂是哺乳动物ghrelin或人ghrelin，或者是哺乳动物 ghrelin或人ghrelin经过替换、缺失或添加一个或更多个氨基酸并且具有ghrelin活性的肽，优选地所述ghrelin活性剂在对应于ghrelin第1至5位氨基酸的位置具有与ghrelin相同的氨基酸。
在另一个实施方案中，所述ghrelin活性剂是非天然存在的形式，例如是化学或生物学方法合成的。另外，所述ghrelin活性剂可以是游离的或者是缀合物，例如与增加分子量的结构(如PEG、白蛋白或免疫球蛋白Fc段)相缀合。
在另一个实施方案中，所述ghrelin活性剂在对应于人ghrelin第3位丝氨酸的位置上被辛酰化。
在另一个实施方案中，所述诱导分化组合物还至少包含另一种分化诱导剂，优选地所述分化诱导剂选自I型胶原、BMP、TGF、FGF、IGF、WNT、GDF、kartogenin等具有软骨诱导活性的小分子中的一种或更多种，例如所述BMP选自BMP-2、BMP-4、BMP-6、BMP-7、BMP-8、BMP-9，例如是BMP-2和/或BMP-4，所述TGF选自TGF-α或TGF-β，例如是TGF-β，所述FGF选自FGF-1、FGF-2、FGF-3、FGF-4、FGF-5、FGF-6、FGF-7、FGF-8、FGF-9、FGF-10，例如是FGF-2，所述IGF选自IGF-1和IGF-2，例如是IGF-1，所述WNT选自WNT-3a、WNT-5a和WNT-7，和/或所述GDF选自GDF-5；更优选地所述分化诱导剂是TGF-β，特别是TGF-β3。
在一个具体实施方案中，在所述组合物中，ghrelin活性剂和其他分化诱导剂可以在空间上物理分离，位于不同的区隔中。这可以方便地实现以不同方式施加ghrelin活性剂和其他分化诱导剂，例如在时间上先后添加不同的分化诱导剂。具体来说，先添加所述ghrelin活性剂，再添加所述其他分化诱导剂。或者先添加所述其他分化诱导剂，再添加所述ghrelin活性剂。
在再一个实施方案中，所述诱导分化组合物是用于诱导分化的培养基或者培养基补充剂，或者是药物组合物。
在第二个方面中，本发明提供一种抑制干细胞增殖的方法，其包括使所述干细胞暴露于本发明第一方面所述的诱导分化组合物。特别地，所述方法可以在体内和/或体外进行，也可以是通过内源性刺激干细胞自身产生ghrelin活性剂来实现。
在第三个方面中，本发明提供一种诱导干细胞向软骨细胞分化的方 法，其包括使所述干细胞暴露于本发明第一方面所述的诱导分化组合物。
在第四个方面中，本发明提供一种增强或促进TGF-β诱导干细胞向软骨细胞分化之能力的方法，其包括在所述干细胞暴露于TGF-β之前、期间或之后，使所述干细胞暴露于本发明第一方面所述的诱导分化组合物。
在第五个方面中，本发明提供一种产生软骨细胞的方法，其包括
1)提供干细胞，和
2)使所述干细胞暴露于本发明第一方面所述的诱导分化组合物并培养所述干细胞。
在第六个方面中，本发明提供一种制备软骨组织的方法，其包括
1)提供干细胞，和
2)使所述干细胞暴露于本发明第一方面所述的诱导分化组合物并培养所述干细胞。
在一个实施方案中，本发明所述的干细胞可以是多能干细胞，例如是成体干细胞或诱导的多能干细胞(iPS)，还可以是胚胎干细胞。在一个具体实施方案中，所述干细胞是间充质干细胞。
在另一个实施方案中，在实施本发明各种方法时，先使所述干细胞在所述ghrelin活性剂存在下培养，随后使所述干细胞在缺少所述ghrelin活性剂的条件下培养。任选地，在上述两阶段培养过程中均存在本发明所述的其他分化诱导剂，例如TGF-β。
在第七个方面中，本发明提供一种治疗骨关节炎的方法，其包括向有此需要的对象施用本发明第一方面所述的诱导分化组合物。
在第八个方面中，本发明提供一种治疗软骨损伤的方法，其包括向有此需要的对象施用本发明第一方面所述的诱导分化组合物。
在一个实施方案中，在本发明第七和第八方面的方法中，所述施用至少分两个阶段进行，其中在第一阶段施用的所述组合物包含所述ghrelin活性剂，在第二阶段施用的所述组合物不包含ghrelin活性剂，任选地在第一阶段和第二阶段施用的所述组合物均包含至少一种所述其他分化诱导剂，例如所述其他分化诱导剂包含TGF-β。
在一个实施方案中，在本发明第二至第八方面的方法中，在所述将所 述干细胞暴露于第一个方面的组合物之前，还包括利用纳秒脉冲电场对所述干细胞进行电击的步骤；优选的，所述纳秒脉冲电场的脉宽为50-200ns，更优选为100ns；场强为5-30kV/cm，更优选为20kV/cm，电击次数1-50次，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、30、40、50次。
在第九个方面中，本发明提供ghrelin活性剂，其用于与以上第二至第八个方面相对应的方法中，例如用于抑制干细胞增殖，用于诱导干细胞向软骨细胞分化，用于增强或促进TGF-β诱导干细胞向软骨细胞分化之能力，用于产生软骨细胞，用于制备软骨组织，用于治疗骨关节炎，和/或用于治疗软骨损伤。优选地以本发明第一方面所述的组合物的形式提供所述ghrelin活性剂。
在第十个方面中，本发明提供ghrelin活性剂用于制备药物的用途，所述用途对应于以上第二至第八个方面的方法，尤其是第七和第八方面的方法。优选地以本发明第一方面所述组合物的形式提供所述ghrelin活性剂。
本发明内容仅仅举例说明了要求保护的一些具体实施方案，其中一个或更多个技术方案中所记载的技术特征可以与任意的一个或更多个技术方案相组合，这些经组合而得到的技术方案也在本申请保护范围内，就像这些经组合而得到的技术方案已经在本发明公开内容中具体记载一样。
通过以上技术方案，本发明至少可以提供以下至少一个方面的优势和利益。本发明能够在显著提升软骨形成的效率，避免常见的加速软骨细胞肥大的副作用，ghrelin活性剂跟其他分化诱导剂可以联合应用于诱导向软骨分化，与其他分化诱导剂可以具有协同效应。
附图说明
图1：不同浓度的ghrelin对大鼠骨髓间充质干细胞增殖的影响。
图2：不同浓度的ghrelin和TGF-β对大鼠骨髓间充质干细胞向软骨细胞诱导分化后，细胞中成软骨相关基因表达情况。
图3：Ghrelin和TGF-β不同添加策略对大鼠骨髓间充质干细胞向软骨细胞诱导分化后，细胞中成软骨相关基因表达情况。
图4：Ghrelin和TGF-β不同添加策略对大鼠骨髓间充质干细胞向软 骨细胞诱导分化后，细胞中硫酸葡萄糖胺聚糖(GAG)含量情况。
图5：Ghrelin和TGF-β不同添加策略对大鼠骨髓间充质干细胞向软骨细胞诱导分化后，组织学切片进行阿利新蓝染色结果。
图6：Ghrelin和BMP-2对大鼠骨髓间充质干细胞向软骨细胞诱导分化后，细胞中成软骨相关基因表达情况。
图7：Ghrelin和TGF-β对人胚胎干细胞向软骨细胞诱导分化后，细胞中成软骨相关基因表达情况。
图8：纳秒脉冲电场(nsPEPE)和ghrelin促进软骨分化。(A)实验分组以及对应处理方式。(B)SOX9、COL II、COL I、COL X和ACAN基因表达的qRT-PCR分析。每组n＝4。*，P<0.05；**，P<0.01；***，P<0.001。(C)TG+nsPEF组诱导的硫酸葡萄糖胺聚糖(sGAG)定量分析。每组n＝4。*，P<0.05；**，P<0.01；***，P<0.001，表示与对照组相比的显著差异。(D)细胞团块中sGAG的HE染色和阿尔新蓝染色。每组n＝4。
具体实施方式
还可进一步通过实施例来理解本发明，其中所述实施例说明了一些制备或使用方法。然而，要理解的是，这些实施例不限制本发明。现在已知的或进一步开发的本发明的变化被认为落入本文中描述的和以下要求保护的本发明范围之内。
本发明中，ghrelin活性剂是指能够在体内、体外、离体提供ghrelin活性的物质，此处不限制ghrelin活性产生的方式和类型。例如所述ghrelin活性剂可以是哺乳动物ghrelin，尤其是人ghrelin。例如，在一些实施方案中，本发明的ghrelin活性剂可以在进入动物体内以后经过体内生物加工(如构象变化、降解、结构修饰如脂肪酸化、糖基化等)发挥出ghrelin活性。在这种情况下，经过体内生物加工之前的ghrelin活性剂本身可以表现出ghrelin活性，也可以不表现出ghrelin活性。
术语“培养基”是指用于支持细胞在生物体外的生长、增殖和/或分化的混合物，它含有支持这些细胞过程必需的营养和其他物质条件，例如可以包含糖、氨基酸、各种营养物质、血清、生长因子、无机质等。
本文中使用时，术语“选自”表示从一组要素中选择一种或更多种要 素。
本文中使用时，术语“治疗”是指一种处理措施，它预期能够使待治疗对象健康状况有任何改善或者降低或避免待治疗对象可能发生的任何健康风险。
实施例1
大鼠骨髓间充质干细胞的分离和扩增
从6周龄的Sprague Dawley(SD)大鼠获得骨髓来源的大鼠骨髓间充质干细胞(BMSC)。将6周龄的雄性SD大鼠使用过量水合氯醛麻醉致死后，取下两条后腿的胫骨和髌骨，利用全骨髓贴壁法(Lin C W,Zhou S H.Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(14):2508-2512)，将骨里面的骨髓用PBS冲洗到培养皿中。
分离获得的细胞用PBS清洗，然后用含90％的DMEM(Dulbecco’s modified Eagle’s medium,Gibco)、10％胎牛血清(FBS,Gibco)以及0.1％青霉素/链霉素(PS，Amresco)的培养基在37℃，5％CO 2的潮湿培养箱中进行单层细胞培养，每周换液2次，当培养细胞汇合率达到85％时，用0.25％胰蛋白酶(Invitrogen)对BMSC进行胰蛋白酶消化传代，当传至第4代时收集骨髓间充质干细胞用于后续试验。
实施例2
Ghrelin对大鼠骨髓间充质干细胞增殖的影响
将实施例1制备的大鼠骨髓间充质干细胞添加不同浓度的ghrelin(康肽生物科技(北京)有限公司，031-30)培养7天，对照组(Control组)添加等量培养基，分别在培养3天和7天后收集各组干细胞并接种于96孔板中，密度为3.5x10 3个/孔，采用DMEM培养基孵箱培养(37℃，5％CO 2)4小时后，按照CCK-8细胞计数试剂盒(CK04，Dojindo)的说明书进行干细胞增殖测试，96孔板每孔中添加10μl CCK-8溶液，37℃孵育2小时，然后用酶标仪检测490nm波长处吸光度，每组4孔。
结果如图1所示，添加了不同浓度的ghrelin培养3天和7天后，干细胞增殖受到了一定程度的抑制作用，而干细胞增殖受抑制是分化所必须的。
实施例3
Ghrelin对TGF-β诱导的大鼠骨髓间充质干细胞向软骨分化的影响
将实施例1制备的大鼠骨髓间充质干细胞用无血清软骨分化诱导培养基制成悬液，按2.5x 10 5个干细胞/管置于锥形管中，600g离心10min，收集团块在孵箱(37℃，5％CO 2)中用无血清软骨分化诱导培养基进行培养，每3天换液一次。所述无血清软骨分化诱导培养基为包含10 -7M的地塞米松、50μg/ml的维生素C、1mM的丙酮酸钠、4mM的脯氨酸和1％v/v ITS+premix(BD Bioscience Inc.,Franklin Lakes,NJ)的高糖DMEM培养基。
分组：各组分别向培养基中添加不同终浓度的TGF-β(10ng/ml人TGF-β3，Peprotech，100-36E)和/或ghrelin(参见实施例2)，对照组(CM组)仅添加无血清软骨分化诱导培养基(Chondrogenic differentiation Media)。
在培养14天后，收集各组团块细胞，使用Trizol试剂按照厂家说明书指引提取总RNA，总RNA用超微量分光光度计进行定量，取1000ng总RNA在PCR仪中采用M-MLV逆转录酶进行逆转录反应；采用实时定量荧光PCR方法检测各组中成软骨相关基因的表达(COL X、COL I、SOX 9、COL II和ACAN)，在实时定量荧光PCR中，采用GAPDH作为对照，各基因的相对表达通过2-ΔΔCt方法进行计算(由PCR仪直接测量的Ct值(PCR循环数)扣除GAPDH内部对照)。各待检测基因和对照基因的特异性引物序列如下：
Figure PCTCN2018114449-appb-000001
Figure PCTCN2018114449-appb-000002
结果如图2所示，在向培养基中添加终浓度10 -7M和10 -9M的ghrelin后，SOX 9基因表达较单加TGF-β组提高了2-4倍左右；COL II基因表达较单加TGF-β组提高了2-5倍左右，同时不会引起软骨肥大相关基因COL X和COL I的显著提高。
实施例4
Ghrelin对TGF-β诱导的大鼠骨髓间充质干细胞向软骨分化的添加策略研究
大鼠骨髓间充质干细胞诱导方法同实施例3，区别仅在于，在诱导过程(14天)中，培养基添加TGF-β和ghrelin的时限有所不同，具体分组如下：
Figure PCTCN2018114449-appb-000003
各组中TGF-β的添加浓度为10ng ml -1，ghrelin的添加浓度为10 -9M。
(1)按照上述添加策略培养14天后，按照实施例3的方法检测各组中成软骨相关基因的表达(COL X、COL I、SOX 9、ACAN和COL II)， 其他基因特异性引物同实施例3。
结果如图3所示，T-T组、G-G组、TG-TG组和TG-T组较不添加诱导剂的CM组相比，成软骨相关基因ACAN、SOX 9和COL II表达明显提高，而引起软骨肥大相关基因COL X和COL I表达仅微弱提高。并且，在各组中，TG-T组的成软骨相关基因SOX 9和COL II表达提高最为明显，并且与其他各组存在显著性差异。
(2)在各组细胞培养21天后，采用细胞糖胺多糖(GAG)总含量二甲基亚甲基蓝(DMMB)比色法定量检测试剂盒(341088，Sigma)测定各组细胞的GAG含量；具体为：各组团块细胞用0.5mg/ml蛋白酶K在56℃消化12小时，随后采用DMMB进行检测，消化溶液震荡30min，然后10000g离心10min，获得的沉淀溶解于解络溶液中，检测溶液630nm处的吸光度，并以硫酸软骨素制备的标准曲线计算各组细胞图块中GAG含量。
结果如图4所示，T-T组、G-G组、TG-TG组和TG-T组较不添加诱导剂的CM组相比，GAG含量明显提高。
(3)对各组细胞团块进行组织学切片进行阿利新蓝染色，结果显示(图5)，添加了ghrelin后细胞外基质分泌明显增加。
上述结果提示，在诱导间充质干细胞向软骨分化过程中，早期添加ghrelin而后期不添加ghrelin可以获得最佳的诱导效果。
实施例5
Ghrelin对BMP-2诱导的大鼠骨髓间充质干细胞向软骨分化的影响
大鼠骨髓间充质干细胞诱导方法同实施例3，区别仅在于，在诱导过程(14天)中，培养基中添加的诱导剂分别为BMP-2(100ng/ml，R&D Systems，355-BM)以及BMP-2加ghrelin，对照组(CM)仅添加无血清软骨分化诱导培养基。在培养14天后，收集各组团块细胞，按常规方法提取总RNA，采用实时定量荧光PCR方法检测各组中成软骨相关基因的表达(COL X、COL I、SOX 9、ACAN和COL II)。
结果如图6所示，与实施例4类似，ghrelin可以提高BMP-2对大鼠间充质干细胞的软骨分化诱导效果。
实施例6
Ghrelin对TGF-β诱导的胚胎干细胞向软骨分化的影响
本实施例的操作参照实施例3进行，其中将细胞由大鼠骨髓间充质干细胞替换为人胚胎干细胞系，所述人胚胎干细胞系为WiCell#04-W308(WA01)，由中国医学科学院整形外科医院傅歆博士赠与。
参照实施例3，各组分别向培养基中添加TGF-β(10ng/ml)或者TGF-β(10ng/ml)和ghrelin(10 -9M)。用实时定量荧光PCR方法检测各组中成软骨相关基因的表达(COL X、COL I、SOX 9和COL II和ACAN)。各待检测基因和对照基因的特异性引物序列如下：
Figure PCTCN2018114449-appb-000004
结果如图7所示，在向培养基中添加终浓度10 -9M的ghrelin后，成软骨相关基因ACAN、SOX 9和COL II表达明显提高，其中SOX 9基因表达较单加TGF-β组提高了1-2倍左右，COL II基因表达较单加TGF-β组提高了2-3倍左右，ACAN基因表达较单加TGF-β组提高了1-2倍左右；同时不会引起软骨肥大相关基因COL X和COL I的显著提高。
实施例8
Ghrelin与纳秒脉冲电场联用对胚胎干细胞向软骨分化的影响
纳秒脉冲电场处理：将实施例1制备的大鼠骨髓间充质干细胞用无血清软骨分化诱导培养基制成悬液，并利用纳秒电脉冲设备进行纳秒电脉冲刺激，具体参数为脉宽100ns，场强20kV/cm，刺激5个脉冲。再将2.5×10 5个干细胞/管置于锥形管中，600g离心10min，收集团块在孵箱(37℃，5％CO 2)中用无血清软骨分化培养基进行培养，每3天换液一次。所述无血清软骨分化诱导培养基为包含10 -7M的地塞米松、50μg/mL的维生素C、1mM的丙酮酸钠、4mM的脯氨酸和1％v/v ITS+premix(BD Bioscience Inc.,Franklin Lakes,NJ)的高糖DMEM培养基。
分组：对照组表示仅向培养基中添加TGF-β(10ng/ml人TGF-β3，Peprotech，100-36E)；TG组表示向培养基中添加TGF-β和ghrelin；TG+nsPEF组表示在添加TGF-β和ghrelin前还进行纳秒脉冲电场处理(具体参数为100ns，20kV/cm，刺激5个脉冲)(见图8A)。
在培养7天后，收集各组团块细胞，使用Trizol试剂按照厂家说明书指引提取总RNA，总RNA用超微量分光光度计进行定量。取1000ng总RNA在PCR仪中采用M-MLV逆转录酶进行逆转录反应；采用实时定量荧光PCR方法检测各组中成软骨相关基因的表达(COL X、COL I、SOX 9、COL II和ACAN)。在实时定量荧光PCR中，采用GAPDH作为对照，各基因的相对表达通过2-ΔΔCt方法进行计算(由PCR仪直接测量的Ct值(PCR循环数)扣除GAPDH内部对照)。各待检测基因和对照基因的特异性引物序列同实施例6：
在各组细胞培养21天后，采用细胞糖胺多糖(GAG)总含量二甲基亚甲基蓝(DMMB)比色法定量检测试剂盒(341088，Sigma)测定各组细胞的GAG含量。具体测定步骤如下：将各组团块细胞用0.5mg/mL蛋白酶K在56℃消化12小时，随后采用DMMB进行检测，消化溶液震荡30min，然后10000g离心10min，获得的沉淀溶解于解络溶液中，检测溶液在630nm处的吸光度，并以硫酸软骨素制备的标准曲线计算各组细胞图块中GAG含量(主要是硫酸葡萄糖胺聚糖，sGAG)。
结果如图8B所示，在添加ghrelin和TGF-β前，使用纳秒脉冲电场进行预处理，成软骨相关基因ACAN、SOX 9和COL II表达得到了进一 步的显著提升，其中SOX 9基因表达较TG组提高了3倍以上，COL II和ACAN基因的表达也较TG组明显提高；同时不会引起软骨肥大相关基因COL X和COL I的显著提高。
另外如图8C所示，TG组和TG+纳秒电脉冲组较对照组，sGAG含量提高。对各组细胞团块进行组织学切片，进行阿利新蓝染色，结果显示(参见图8D)，添加了纳秒电脉冲刺激后细胞外基质分泌明显增加。
以上结果表明，纳秒脉冲电场处理能够进一步提高ghrelin诱导干细胞向软骨分化的能力。
Figure PCTCN2018114449-appb-000005
Figure PCTCN2018114449-appb-000006
Figure PCTCN2018114449-appb-000007
Figure PCTCN2018114449-appb-000008
Figure PCTCN2018114449-appb-000009
Figure PCTCN2018114449-appb-000010
Figure PCTCN2018114449-appb-000011
Figure PCTCN2018114449-appb-000012
Figure PCTCN2018114449-appb-000013
Figure PCTCN2018114449-appb-000014
Figure PCTCN2018114449-appb-000015
Figure PCTCN2018114449-appb-000016

Claims (17)

  1. 一种诱导分化组合物，其包含ghrelin活性剂和药学可接受的载体。
  2. 根据权利要求1的组合物，其中所述ghrelin活性剂是ghrelin或是与ghrelin氨基酸序列具有至少60％、65％、70％、75％、80％、85％、90％或95％一致性并具有ghrelin活性的肽，优选地所述ghrelin活性剂在对应于ghrelin第1至5位氨基酸的位置具有与ghrelin相同的氨基酸。
  3. 根据权利要求1或2的组合物，其还至少包含另一种分化诱导剂，优选地所述分化诱导剂选自I型胶原、BMP、TGF、FGF、IGF、WNT、GDF和kartogenin中的一种或更多种；例如所述BMP选自BMP-2、BMP-4、BMP-6、BMP-7、BMP-8、BMP-9，例如是BMP-2和/或BMP-4，所述TGF选自TGF-α或TGF-β，例如是TGF-β，所述FGF选自FGF-1、FGF-2、FGF-3、FGF-4、FGF-5、FGF-6、FGF-7、FGF-8、FGF-9、FGF-10，例如是FGF-2，所述IGF选自IGF-1和IGF-2，例如是IGF-1，所述WNT选自WNT-3a、WNT-5a和WNT-7，和/或所述GDF选自GDF-5；更优选地所述分化诱导剂是TGF-β，特别是TGF-β3。
  4. 根据权利要求1至3中任一项的组合物，其中所述组合物是用于诱导分化的培养基或者培养基补充剂，或者是药物组合物。
  5. 一种抑制干细胞增殖的方法，其包括
    使所述干细胞暴露于根据权利要求1至4中任一项的组合物。
  6. 一种诱导干细胞向软骨细胞分化的方法，其包括
    使所述干细胞暴露于根据权利要求1至4中任一项的组合物。
  7. 一种增强或促进TGF-β诱导干细胞向软骨细胞分化之能力的方法，其包括
    在所述干细胞暴露于TGF-β之前、期间或之后，使所述干细胞暴露于根据权利要求1至4中任一项的组合物。
  8. 一种产生软骨细胞的方法，其包括
    1)提供干细胞，和
    2)使所述干细胞暴露于根据权利要求1至4中任一项的组合物并培养所述干细胞。
  9. 一种制备软骨组织的方法，其包括
    1)提供干细胞，和
    2)使所述干细胞暴露于根据权利要求1至4中任一项的组合物并培养所述干细胞。
  10. 根据权利要求5至9中任一项的方法，其中所述干细胞是多能干细胞，例如是间充质干细胞或诱导多能干细胞。
  11. 根据权利要求5至10中任一项的方法，其中至少分两个阶段培养所述干细胞，在第一阶段先在所述ghrelin活性剂存在下培养所述干细胞，随后在第二阶段在不存在所述ghrelin活性剂的条件下培养所述干细胞；任选地在第一和第二阶段中存在至少一种所述其他分化诱导剂，例如所述其他分化诱导剂包含TGF-β。
  12. 一种治疗骨关节炎的方法，其包括向有此需要的对象施用根据权利要求1至4中任一项的组合物。
  13. 一种治疗软骨损伤的方法，其包括向有此需要的对象施用根据权利要求1至4中任一项的组合物。
  14. 根据权利要求12或13的方法，其中所述施用至少分两个阶段进行，其中在第一阶段施用的所述组合物包含所述ghrelin活性剂，在第二阶段施用的所述组合物不包含ghrelin活性剂，任选地在第一阶段和第二阶段施用的所述组合物均包含至少一种所述其他分化诱导剂，例如所述其他分化诱导剂包含TGF-β。
  15. 根据权利要求5至14中任一项所述的方法，其中在所述将所述干细胞暴露于根据权利要求1至4中任一项的组合物之前，还包括利用纳秒脉冲电场对所述干细胞进行电击的步骤；优选的，所述纳秒脉冲电场的脉宽为50-200ns，更优选为100ns；场强为5-30kV/cm，更优选为20kV/cm，电击次数1-50次，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、30、40、50次。
  16. Ghrelin活性剂，其用于诱导干细胞向软骨细胞分化，用于治疗骨关节炎，和/或用于治疗软骨损伤，优选地以根据权利要求1至4中任一项组合物的形式提供所述ghrelin活性剂。
  17. Ghrelin活性剂用于制备用于治疗骨关节炎的药物中的用途，优选地以根据权利要求1至4中任一项组合物的形式提供所述ghrelin活性 剂。
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