WO2020233119A1 - 一种促间充质干细胞增殖的无血清多肽组合物 - Google Patents

一种促间充质干细胞增殖的无血清多肽组合物 Download PDF

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WO2020233119A1
WO2020233119A1 PCT/CN2019/128193 CN2019128193W WO2020233119A1 WO 2020233119 A1 WO2020233119 A1 WO 2020233119A1 CN 2019128193 W CN2019128193 W CN 2019128193W WO 2020233119 A1 WO2020233119 A1 WO 2020233119A1
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content
serum
mscs
polypeptide composition
stem cells
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陈海佳
葛啸虎
王小燕
戚康艺
姜交华
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广州赛莱拉干细胞科技股份有限公司
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Priority to US17/613,941 priority Critical patent/US20220251512A1/en
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Definitions

  • the present invention relates to the technical field of cell proliferation, in particular to a serum-free polypeptide composition for promoting the proliferation of mesenchymal stem cells.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • MSCs have broad clinical application prospects, are the first choice seed cells for cell replacement therapy and tissue engineering, and are the research hotspots in the field of transplantation and the treatment of autoimmune diseases.
  • FBS fetal bovine serum
  • the composition of FBS is complex and contains heterogeneous proteins, and it is easy to carry viruses or be infected by mycoplasma.
  • FBS has large differences between batches and unstable sources, which has a greater impact on the process of large-scale expansion of MSCs in vitro.
  • Current studies have shown that MSCs can phagocytose the proteins in the medium during the culture process.
  • the medium contains bovine serum albumin, which can cause the recipient’s body to produce anti-bovine albumin antibodies to cause an immune response, which may lead to or especially repeated infusions. After injection of MSCs treatment, it fails. Therefore, researchers began to develop alternatives to FBS.
  • Serum-Free Medium does not require the addition of animal or human serum in cell culture.
  • raw materials that replace serum functions are usually added to the culture medium, mainly including binding proteins, growth factors, adhesion factors, hormones, and trace elements.
  • the research and development of SFM applied to mesenchymal stem cells has gone through four major stages:
  • the first generation is a serum-free medium in the general sense, which uses various biological materials that can replace serum functions and contains a large number of animal and plant sources. Proteins and unknown components, such as animal or human platelet lysates, have the advantage of higher experimental accuracy, repeatability, and stability than serum-containing media.
  • the second generation is serum-free and animal-derived protein-free medium, and its additive components do not use animal-derived proteins at all, instead of various recombinant proteins or
  • the advantage of animal and plant protein hydrolysates is that the stability is improved compared with the first generation, the cost is reduced, and the effect is correspondingly improved.
  • the third generation is a limited-edition medium, also known as double-free medium.
  • the added components contain no serum, no protein or very low protein content, and the protein contained in it is clear.
  • the third generation SFM is currently The main products on the market have obvious advantages. The cell culture and production are easy to be constant, the separation and purification of the target protein is easier, and the cost is greatly reduced. However, at this stage, these products all have the disadvantage of insufficient cell passage capacity.
  • SFM R&D companies urgently need to break through the bottleneck, and have high specificity for cultured cells, so there are fewer cell lines suitable for culture, and product development is extremely difficult;
  • the fourth generation is a chemical composition limited medium with added ingredients It does not contain serum and does not contain any protein. It is mainly composed of compounds instead of unstable protein substances. It can be sterilized at high temperature. It is a versatile medium suitable for the growth of a variety of different cells. There is no such product on the market yet, and it is still in the research and development stage.
  • Polypeptide is a compound formed by linking ⁇ -amino acids together by peptide bonds, and is an intermediate product of protein hydrolysis.
  • Compounds formed by the dehydration and condensation of two amino acid molecules are called dipeptides.
  • dipeptides By analogy, there are tripeptides, tetrapeptides, pentapeptides, etc.
  • compounds formed by dehydration and condensation of three or more amino acid molecules can be called polypeptides.
  • Bioactive peptides have a variety of physiological functions, such as hormone action, immune regulation, anti-thrombosis, anti-hypertension, cholesterol-lowering, antibacterial, antiviral, and anticancer effects.
  • Peptides are currently widely used in skin care products, with anti-aging, whitening and skin repair functions.
  • the technical problem solved by the present invention is to provide a serum-free polypeptide composition, which can effectively promote the rapid growth of mesenchymal stem cells.
  • a serum-free polypeptide composition for promoting the proliferation of mesenchymal stem cells including:
  • the content of the tripeptide-1 is 3-18 ⁇ g/L.
  • the content of the tripeptide-2 is 3-18 ⁇ g/L.
  • the content of the hexapeptide-9 is 4-18 ⁇ g/L.
  • the content of palmitoyl hexapeptide-12 is 3-17 ⁇ g/L.
  • the content of the laminin-derived peptide is 30-65 ⁇ g/L.
  • the content of the tripeptide-1 is 10 ⁇ g/L
  • the content of the tripeptide-2 is 10 ⁇ g/L
  • the content of the hexapeptide-9 is 10 ⁇ g/L
  • the content of the palmitoyl hexapeptide It is 10 ⁇ g/L
  • the content of the laminin-derived peptide is 60 ⁇ g/L.
  • the content of the non-essential amino acid is 1 vol%
  • the content of the glutamine is 1 to 4 mmol/L
  • the content of the lipid mixture is 1 vol%
  • the content of the ITS is 1 vol%.
  • the content of recombinant human albumin is 2.5 g/L
  • the content of recombinant human epidermal growth factor is 20 ⁇ g/L
  • the content of recombinant human fibronectin is 10 ⁇ g/L
  • the content of L-glutathione is The content is 2 mg/L
  • the content of ⁇ -mercaptoethanol is 2 mg/L.
  • the application provides a polypeptide composition, which comprises a mixture of non-essential amino acids, glutamine, lipid mixture, ITS, recombinant human albumin, recombinant human epidermal growth factor, recombinant human fibronectin, L-glutathione
  • a basic serum-free medium composed of peptides, ⁇ -mercaptoethanol and ⁇ -MEM basic medium, including tripeptide-1, tripeptide-2, hexapeptide-9, palmitoyl hexapeptide-12 and laminin A polypeptide composition composed of derived peptides.
  • the above-mentioned composition is supplemented with a combination of polypeptides in the basic medium, the above-mentioned polypeptide has the function of promoting adhesion instead of collagen and fibronectin, and can effectively promote cell elastin, collagen, The synthesis of macromolecules such as laminin and integrin promotes cell proliferation and migration. Therefore, the peptide composition as a culture medium has a clear chemical composition, no animal origin, and no serum, which can realize the rapid proliferation of mesenchymal stem cells and solve the problem of insufficient cell numbers. At the same time, it maintains the biological characteristics and immunophenotypic stability of mesenchymal stem cells.
  • Figure 1 is a morphological diagram (40 ⁇ ) of UC-MSCs provided by the experimental group and the control group of the present invention
  • Figure 2 is a graph of the growth curve of UC-MSCs provided by the experimental group and the control group of the present invention
  • Figure 3 is a diagram showing the adipogenic differentiation effect of UC-MSCs provided by the experimental group and the control group of the present invention (400 ⁇ );
  • Figure 4 is a diagram (40 ⁇ ) of the osteogenic differentiation effect of UC-MSCs provided by the experimental group and the control group of the present invention.
  • this application provides a polypeptide composition, which can promote the rapid proliferation of mesenchymal stem cells as a serum-free medium.
  • a serum-free polypeptide composition for promoting the proliferation of mesenchymal stem cells including:
  • tripeptide-1 in the above composition, tripeptide-1, tripeptide-2, hexapeptide-9, palmitoyl hexapeptide-12 and laminin-derived peptide are used as the polypeptide combination of the polypeptide composition.
  • tripeptide-1 can promote the production of elastin and collagen;
  • the ⁇ -MEM basal medium is used as a constant volume substrate, and its content is 1-20 ⁇ g/L, in specific embodiments, its content is 3-18 ⁇ g/L .
  • the tripeptide-2 is an active tripeptide derived from an elastase inhibitor, which can reduce the synthesis of progeria, and its content is 1-20 ⁇ g/L. In a specific embodiment, the content of the tripeptide-2 is 3. ⁇ 18 ⁇ g/L.
  • the hexapeptide-9 is synthesized from six amino acids and is a very stable collagen peptide that can promote the production of cellular collagen, laminin, and integrin; its content is 1-20 ⁇ g/L, which is used in specific implementation In an example, the content of the hexapeptide-9 is 4-18 ⁇ g/L.
  • the palmitoyl hexapeptide-12 has a chemotactic effect and can promote the migration and proliferation of dermal fibroblasts and the synthesis of matrix macromolecules; its content is 1-20 ⁇ g/L. In a specific embodiment, the palmitoyl hexapeptide The content of -12 is 3-17 ⁇ g/L.
  • the laminin-derived peptide has functions similar to laminin and can promote cell adhesion and proliferation; its content is 10-100 ⁇ g/L. In a specific embodiment, the content of the laminin-derived peptide is 30 ⁇ 65 ⁇ g/L.
  • tripeptide-1, tripeptide-2, hexapeptide-9, palmitoyl hexapeptide-12 and laminin-derived peptides gradually increase in performance as the content increases, but performance beyond the upper limit of the application is not greatly improved.
  • the above-mentioned polypeptide synergistically cooperates with the basic medium, so that the polypeptide composition can promote the rapid proliferation of mesenchymal stem cells.
  • a serum-free medium polypeptide composition for mesenchymal stem cells also includes a basic serum-free medium, which specifically includes: non-essential amino acids 1-5 vol%; glutamine 1-4 mmol/L; lipids Class mixture 1 ⁇ 5vol%; ITS1 ⁇ 5vol%; recombinant human albumin 1 ⁇ 5g/L; recombinant human epidermal growth factor 5 ⁇ 25 ⁇ g/L; recombinant human fibronectin 5 ⁇ 15 ⁇ g/L; L-glutathione Peptide 1-5mg/L; ⁇ -mercaptoethanol 1-5mg/L; ⁇ -MEM basal medium 10.2g/L.
  • the above-mentioned basic serum-free medium is used for the cultivation of mesenchymal stem cells.
  • the polypeptide composition includes: the content of the tripeptide-1 is 10 ⁇ g/L, the content of the tripeptide-2 is 10 ⁇ g/L, and the content of the hexapeptide-9 is 10 ⁇ g/L.
  • the content of palmitoyl hexapeptide is 10 ⁇ g/L
  • the content of laminin-derived peptide is 60 ⁇ g/L
  • the content of non-essential amino acids is 1 vol%
  • the content of glutamine is 1 ⁇ 4mmol/L
  • the content of the lipid mixture is 1 vol%
  • the content of the ITS is 1 vol%
  • the content of the recombinant human albumin is 2.5 g/L
  • the content of the recombinant human epidermal growth factor is 20 ⁇ g/L
  • the content of the recombinant human fibronectin is 10 ⁇ g/L
  • the content of the L-glutathione is 2 mg/L
  • the content of the ⁇ -mercaptoethanol is 2 mg/L.
  • the polypeptide composition described in this application can be prepared according to a method well known to those skilled in the art. After the components are mixed for a specific time, they are filtered and sterilized in the filter membrane, and then added to the ⁇ -MEM basal medium and mixed, namely Obtain a serum-free medium for promoting the proliferation of mesenchymal stem cells (UC-MSCs).
  • UC-MSCs mesenchymal stem cells
  • the mesenchymal stem cell-promoting polypeptide composition provided by the present invention has clear chemical components, no animal origin, and no serum, can realize the rapid proliferation of UC-MSCs, solve the problem of insufficient cell numbers, and maintain the biological characteristics and immunity of mesenchymal stem cells Phenotypic stability.
  • polypeptide composition for promoting the proliferation of mesenchymal stem cells provided by the present invention will be described in detail in conjunction with examples below.
  • the protection scope of the present invention is not limited by the following examples.
  • ⁇ -MEM basal medium (Cat. No. 41061037) and non-essential amino acid solution (Cat. No. 11140-050) were purchased from Gibco; other components can be purchased from sigma, MP, Gibco and other companies.
  • Non-essential amino acids 1% by volume
  • Lipid mixture 1vol%
  • Recombinant human albumin 2.5g/L;
  • Recombinant human epidermal growth factor 20 ⁇ g/L;
  • Recombinant human fibronectin 10 ⁇ g/L;
  • Tripeptide-1 10 ⁇ g/L;
  • Tripeptide-2 10 ⁇ g/L
  • Hexapeptide-9 10 ⁇ g/L;
  • Palmitoyl hexapeptide-12 10 ⁇ g/L
  • Laminin-derived peptide 50 ⁇ g/L
  • ⁇ -MEM basal medium 10.2g/L
  • the above-mentioned components are dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present application, which is used as experimental group 2. It is filtered and sterilized through a 0.22m filter membrane to promote the proliferation of mesenchymal stem cells.
  • Non-essential amino acid 0.01vol%
  • Glutamine 1 ⁇ 4mmol/L
  • Lipid mixture 1vol%
  • Recombinant human albumin 2.5g/L;
  • Recombinant human epidermal growth factor 20 ⁇ g/L;
  • Recombinant human fibronectin 10 ⁇ g/L;
  • Tripeptide-1 20 ⁇ g/L;
  • Tripeptide-2 20 ⁇ g/L
  • Hexapeptide-9 20 ⁇ g/L;
  • Palmitoyl hexapeptide-12 20 ⁇ g/L
  • Laminin-derived peptide 100 ⁇ g/L
  • ⁇ -MEM basal medium 10.2g/L
  • Non-essential amino acid 1vol%
  • Glutamine 1 ⁇ 4mmol/L
  • Lipid mixture 0.01L/L;
  • Recombinant human albumin 2.5g/L;
  • Recombinant human epidermal growth factor 20 ⁇ g/L;
  • Recombinant human fibronectin 10 ⁇ g/L;
  • ⁇ -MEM basal medium 10.2g/L
  • the above-mentioned components are dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present application, which is used as the experimental group 1, and then filtered through a 0.22m filter membrane to sterilize the mesenchymal stem cells to promote the proliferation of mesenchymal stem cells.
  • Non-essential amino acid 1vol%
  • Glutamine 1 ⁇ 4mmol/L
  • Lipid mixture 1vol%
  • Recombinant human albumin 2.5g/L;
  • Recombinant human epidermal growth factor 20 ⁇ g/L;
  • Recombinant human fibronectin 10 ⁇ g/L;
  • Tripeptide-1 10 ⁇ g/L;
  • Tripeptide-2 10 ⁇ g/L
  • ⁇ -MEM basal medium 10.2g/L
  • the above-mentioned components are dissolved in water and fully dissolved at room temperature to obtain the polypeptide composition of the present application as the control group 3; and then filter and sterilize through a 0.22m filter membrane to promote the proliferation of mesenchymal stem cells.
  • Non-essential amino acid 1vol%
  • Glutamine 1 ⁇ 4mmol/L
  • Lipid mixture 1vol%
  • Recombinant human albumin 2.5g/L;
  • Recombinant human epidermal growth factor 20 ⁇ g/L;
  • Recombinant human fibronectin 10 ⁇ g/L;
  • Hexapeptide-9 10 ⁇ g/L;
  • Palmitoyl hexapeptide-12 10 ⁇ g/L
  • Laminin-derived peptide 50 ⁇ g/L
  • ⁇ -MEM basal medium 10.2g/L
  • the above-mentioned components are dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present application, which serves as the control group 4; then it is filtered through a 0.22m filter and sterilized to promote the proliferation of mesenchymal stem cells.
  • UC-MSCs were inoculated into T25 culture flasks at a density of 1 ⁇ 10 4/ cm 2 with 3 replicates in each group; cultured in a 5% CO 2 incubator at 37°C; cultured for 48 hours
  • the UC-MSCs images were collected and the results are shown in Figure 1.
  • Figure A in Figure 1 is the morphology of UC-MSCs in control group 1
  • Figure B is the UC-MSCs in control group 2
  • Figure C is the UC-MSCs in experimental group 1.
  • Figure D is the morphology of UC-MSCs in experimental group 2
  • Figure E is the morphology of UC-MSCs in experimental group 3.
  • Control group 1 91.75 ⁇ 0.11 Control group 2 93.20 ⁇ 0.25 Control group 3 90.05 ⁇ 0.34 Control group 4 91.86 ⁇ 0.12 Experimental group 1 91.25 ⁇ 0.35 Experimental group 2 98.66 ⁇ 0.21 Experimental group 3 97.44 ⁇ 0.14
  • the experimental group 2 and the experimental group 3 were compared with the control group 1, the control group 3, and the control group 4 respectively, and there were significant differences (p ⁇ 0.05).
  • UC-MSCs The P3 generation of UC-MSCs was selected to carry out the experiment.
  • UC-MSCs were seeded in a 24-well plate at a density of 1 ⁇ 10 4 cells/mL and cultured in a 5% CO 2 incubator at 37°C; cells were collected every day for cell count, each time randomly Collect and calculate 3 wells for 7 consecutive days and draw the cell growth curve. The results are shown in Table 2 and Figure 2.
  • the polypeptide combination culture UC-MSCs of the present invention has higher proliferation activity.
  • the experimental group 2 and the experimental group 3 were compared with the control group 1, the control group 3, and the control group 4 respectively, and there were significant differences (p ⁇ 0.05).
  • UC-MSCs were inoculated in T25 culture flasks at a density of 1 ⁇ 10 4 /cm 2 and cultured in a 5% CO 2 incubator at 37°C. Three days later, UC-MSCs of each group were digested with 0.25% trypsin solution, and the expression of surface markers such as CD105, CD73, CD90, CD34, CD45, HLA-DR, etc. were detected by flow cytometry. The results are shown in Table 4.
  • Control group 2 99.96% 100.0% 95.24% 0.01% 0.00% 0.03%
  • Control group 3 100.0% 98.35% 96.76% 0.00% 0.00% 0.02%
  • Control group 4 97.95% 99.25% 96.33% 0.04% 0.00% 0.02%
  • Experimental group 1 99.45% 100.0% 98.10% 0.00% 0.01% 0.02%
  • Experimental group 2 100.0% 97.46% 99.80% 0.01% 0.00% 0.01%
  • Experimental group 3 100.0% 99.25% 100.0% 0.02% 0.00% 0.01%
  • the P3 generation UC-MSCs were selected for the experiment.
  • the UC-MSCs of the experimental group 1 to 3, the control group 1 and the control group 2 were routinely cultured and passaged to the P5 generation, and inoculated in a 6-well plate at a density of 1 ⁇ 10 5 /mL. Put it into a 5% CO 2 incubator at 37°C.
  • control holes and induction holes are set respectively to induce osteogenic and adipogenic differentiation of UC-MSCs.
  • the cells in the adipogenic differentiation experimental group were stained with Oil Red O, and 21 days later, the cells in the osteogenic differentiation experimental group were stained with Alizarin Red.
  • Figure A in Figure 3 is the adipogenic differentiation effect of UC-MSCs in control group 1
  • Figure B is the adipogenic differentiation effect of UC-MSCs in control group 2
  • Figure C is The effect of adipogenic differentiation of UC-MSCs in experimental group 1
  • Figure D is the effect of adipogenic differentiation of UC-MSCs in experimental group 2
  • Figure E is the effect of adipogenic differentiation of UC-MSCs in experimental group 3
  • Figure 4 In, Figure A is the osteogenic differentiation effect of UC-MSCs in control group 1
  • Figure B is the osteogenic differentiation effect of UC-MSCs in control group 2
  • Figure C is the osteogenic differentiation of UC-MSCs in experimental group 1.
  • Figure D is the osteogenic differentiation effect diagram of UC-MSCs in experimental group 2
  • Figure E is the osteogenic differentiation effect diagram of UC-MSCs in experimental group 3; the experimental results show that the combination of polypeptide culture of the present invention is supplemented UC-MSCs will not affect their adipogenic and osteogenic differentiation potential and maintain their dryness.

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Abstract

本发明提供了一种促间充质干细胞增殖的无血清多肽组合物，主要包括：10～100μg/L的三肽-1；1～20μg/L的三肽-2；1～20μg/L的六肽-9；1～20μg/L的棕榈酰六肽-12；10～100μg/L的层黏连蛋白衍生肽。本申请提供了的无血清多肽组合物，化学成分明确、无动物源、无血清，能够实现间充质干细胞的快速增殖，解决细胞数量不够问题的同时，维持间充质干细胞生物学特性和免疫表型稳定性。

Description

一种促间充质干细胞增殖的无血清多肽组合物
本申请要求于2019年05月23日提交中国专利局、申请号为201910432549.0、发明名称为“一种促间充质干细胞增殖的无血清多肽组合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及细胞增殖技术领域，尤其涉及一种促间充质干细胞增殖的无血清多肽组合物。
背景技术
间充质干细胞(Mesenchymal stem cells，MSCs)来源于发育早期的中胚层，是一类非造血干细胞，其广泛存在于骨髓、皮下脂肪、骨外膜、肌肉、滑膜、滑液、肝脏、外周组织、脐带、脐带血及胎盘等组织。MSCs具有高度自我更新能力和多向分化潜能，可在体外培养扩增，不仅能够支持造血干细胞的生长，还具有免疫调控的作用；不同的诱导条件下，在体外可分化为骨、软骨、肌肉、神经、心肌、内皮和脂肪等，连续传代培养和冷冻保存后仍具有多向分化潜能，可作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。因此，MSCs具有广阔的临床应用前景，是细胞替代治疗和组织工程的首选种子细胞，是移植领域和自身性免疫疾病治疗的研究热点。
现有的间充质干细胞培养方法使用的培养基大都含有动物血清，如最为常见的胎牛血清(fetal bovine serum，FBS)。FBS成分复杂且含有异种蛋白质，容易携带病毒或被支原体感染等；另一方面，FBS批间差异较大，来源不稳定，对体外大规模扩增MSCs工艺影响较大。目前有研究表明，MSCs在培养过程中会吞噬培养基中的蛋白质，培养基中含有牛血清白蛋白，可以使受者体内产生抗牛白蛋白抗体引起免疫反应，从而导致或者尤其是在重复输注MSCs治疗后失效。因此，研究者开始研发FBS的替代物。
目前市面上血清替代物种类较多，但大都仍然含有一些动物源成分，如Ultroser G(Pall BioSepra)。部分血清替代物会使用人血清活血清衍生物，包括人源血清、血小板衍生物、脐血清等。这些产品虽来源于人，但成分仍然不明确，而且资源少，难于实现量产，无法保证MSCs体外大规模培养。
无血清培养基(Serum-Free Medium，SFM)，顾名思义是在细胞培养中不需要添加动物或人源血清。但为了满足细胞生长的要求，通常会向培养基中添加替代血清功能的原料，主要包括结合蛋白、生长因子、粘附因子、激素以及微量元素等加大类别。应用于间充质干细胞的SFM的研发至今，主要经历了四大阶段：第一代是一般意义上的无血清培养基，其采用各种可替代血清功能的生物材料，含大量动、植物来源蛋白和不明成分，如动物或人源血小板裂解物等，其优点是相较于含血清培养基，实验准确性、可重复性、稳定性高一些，但由于添加材料的化学成分不明确，大部分含大量动物源蛋白，不利于目标蛋白的分离纯化，而且成本较高；第二代是无血清无动物源蛋白培养基，其添加成分完全不用动物来源蛋白，取而代之的是各种重组蛋白或动植物蛋白水解物，其优势是稳定性较第一代有所提高，降低了成本，效果也相应提高，但由于成本极高，仅适合需求量少的科研用户，难于被开展大规模生产的企业所使用；第三代是成分限定培养基，又称双无培养基，添加成分完全不含血清、无蛋白或蛋白含量极低，且所含蛋白是成分明确的，第三代SFM是目前市场上主推产品，其优势十分明显，细胞培养与生产易做到恒定，目标蛋白的分离纯化更为容易，成本大为下降，但目前阶段此类产品均具有细胞传代能力不足的缺点，这也是目前SFM研发企业急需突破的瓶颈，而且对培养的细胞具有很高的特异性，所以适于培养的细胞系较少，产品开发难度极大；第四代是化学组分限定培养基，添加成分不含血清、不含任何蛋白，主要由化合物代替不稳定的蛋白类物质，可高温消毒，适合多种不同细胞生长的全能型培养基，目前暂无此类产品上市，仍处于研发阶段。
随着行业的快速发展，无血清培养基产品越来越多，口碑较好及使用较多的基本为国外公司所生产，如GIBCO公司的
Figure PCTCN2019128193-appb-000001
hMSC SFM，Stemcell公司的MesenCult-XF Medium等。这些培养基不仅价格昂贵，不能满足量产的要求，且在培养MSCs时需对培养容器进行明胶包被，有很高的动物源蛋白引入风险。
多肽是α-氨基酸以肽键连接在一起而形成的化合物，是蛋白质水解的中间产物。由两个氨基酸分子脱水缩合而成的化合物叫做二肽，同理类推还有三肽、四肽、五肽等。通常由三个或三个以上氨基酸分子脱水缩合而成的化合物都可 以成为叫多肽。生物活性肽有多种多样的生理功能，如激素作用、免疫调节、抗血栓、抗高血压、降胆固醇、抑菌、抗病毒、抗癌作用等。多肽目前广泛应用于护肤品中，具有抗衰老、美白、修复皮肤等功能。
发明内容
本发明解决的技术问题在于提供一种无血清多肽组合物，该无血清多肽组合物可有效促使间充质干细胞快速增值。
有鉴于此，本申请提供了一种促间充质干细胞增殖的无血清多肽组合物，包括：
Figure PCTCN2019128193-appb-000002
优选的，所述三肽-1的含量为3～18μg/L。
优选的，所述三肽-2的含量为3～18μg/L。
优选的，所述六肽-9的含量为4～18μg/L。
优选的，所述棕榈酰六肽-12的含量为3～17μg/L。
优选的，所述层黏连蛋白衍生肽的含量为30～65μg/L。
优选的，所述三肽-1的含量为10μg/L，所述三肽-2的含量为10μg/L，所 述六肽-9的含量为10μg/L，所述棕榈酰六肽的含量为10μg/L，所述层黏连蛋白衍生肽的含量为60μg/L。
优选的，所述非必需氨基酸的含量为1vol％，所述谷氨酰胺的含量为1～4mmol/L，所述脂类混合物的含量为1vol％，所述ITS的含量为1vol％，所述重组人血白蛋白的含量为2.5g/L，所述重组人表皮生长因子的含量为20μg/L，所述重组人纤连蛋白的含量为10μg/L，所述L-谷胱甘肽的含量为2mg/L，所述β-巯基乙醇的含量为2mg/L。
本申请提供了一种多肽组合物，其包括由非必需氨基酸、谷氨酰胺、脂类混合物、ITS、重组人血白蛋白、重组人表皮生长因子、重组人纤连蛋白、L-谷胱甘肽、β-巯基乙醇和α-MEM基础培养基组成的基础无血清培养基，还包括由三肽-1、三肽-2、六肽-9、棕榈酰六肽-12和层黏连蛋白衍生肽组成的多肽组合物，上述组合物由于在基础培养基中补充了多肽组合，上述多肽具有替代胶原蛋白、纤连蛋白的促粘附功能，而且能有效促进细胞的弹性蛋白、胶原蛋白、层粘蛋白和整合蛋白等大分子合成，促进细胞增殖与迁移，因此多肽组合物作为培养基，化学成分明确、无动物源、无血清，能够实现间充质干细胞的快速增殖，解决细胞数量不够问题的同时，维持间充质干细胞生物学特性和免疫表型稳定性。
附图说明
图1为本发明实验组与对照组提供的UC-MSCs的形态图(40×)；
图2为本发明实验组和对照组提供的UC-MSCs的生长曲线图；
图3为本发明实验组和对照组提供的UC-MSCs的成脂分化效果图(400×)；
图4为本发明实验组和对照组提供的UC-MSCs的成骨分化效果图(40×)。
具体实施方式
为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但是应当理解，这些描述只是为进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。
针对现有技术中间充质干细胞要求的无血清培养基，本申请提供了一种多肽组合物，该多肽组合物作为无血清培养基可促进间充质干细胞快速增殖。具 体的，本发明实施例公开了一种促间充质干细胞增殖的无血清多肽组合物，包括：
Figure PCTCN2019128193-appb-000003
在上述组合物中，三肽-1、三肽-2、六肽-9、棕榈酰六肽-12和层黏连蛋白衍生肽作为多肽组合物的多肽组合。其中，三肽-1可促进弹性蛋白、胶原蛋白生成；以α-MEM基础培养基为定容基底，其含量为1～20μg/L，在具体实施例中，其含量为3～18μg/L。
所述三肽-2是衍生于弹性蛋白酶抑制因子的活性三肽，能减少早衰蛋白的合成，其含量为1～20μg/L，在具体实施例中，所述三肽-2的含量为3～18μg/L。
所述六肽-9由六个氨基酸合成，是一种非常稳定的胶原蛋白肽，可促进细胞胶原蛋白、层黏连蛋白和整联蛋白生成；其含量为1～20μg/L，在具体实施例中，所述六肽-9的含量为4～18μg/L。
所述棕榈酰六肽-12具有趋化作用，可促进真皮成纤细胞迁移、增殖和基质大分子的合成；其含量为1～20μg/L，在具体实施例中，所述棕榈酰六肽-12的含量为3～17μg/L。
所述层黏连蛋白衍生肽具有层黏蛋白类似的功能，可促进细胞粘附、增殖；其含量为10～100μg/L，在具体实施例中，所述层黏连蛋白衍生肽的含量为30～65μg/L。
上述三肽-1、三肽-2、六肽-9、棕榈酰六肽-12和层黏连蛋白衍生肽随含量的增加性能逐渐增加，但是超过本申请的上限范围性能没有太大提高。
本申请通过在多肽组合物中引入上述多肽，上述多肽协同作用与基础培养基配合，使得多肽组合物可促进间充质干细胞快速增殖。
作为间充质干细胞的无血清培养基多肽组合物，其还包括基础无血清培养基，该基础无血清培养基具体包括：非必需氨基酸1～5vol％；谷氨酰胺1～4mmol/L；脂类混合物1～5vol％；ITS1～5vol％；重组人血白蛋白1～5g/L；重组人表皮生长因子5～25μg/L；重组人纤连蛋白5～15μg/L；L-谷胱甘肽1～5mg/L；β-巯基乙醇1～5mg/L；α-MEM基础培养基10.2g/L。上述基础无血清培养基用于间充质干细胞的培养。
在具体实施例中，所述多肽组合物包括：所述三肽-1的含量为10μg/L，所述三肽-2的含量为10μg/L，所述六肽-9的含量为10μg/L，所述棕榈酰六肽的含量为10μg/L，所述层黏连蛋白衍生肽的含量为60μg/L；所述非必需氨基酸的含量为1vol％，所述谷氨酰胺的含量为1～4mmol/L，所述脂类混合物的含量为1vol％，所述ITS的含量为1vol％，所述重组人血白蛋白的含量为2.5g/L，所述重组人表皮生长因子的含量为20μg/L，所述重组人纤连蛋白的含量为10μg/L，所述L-谷胱甘肽的含量为2mg/L，所述β-巯基乙醇的含量为2mg/L。
本申请所述多肽组合物的制备按照本领域技术人员熟知的方法制备即可，具体时间各组分混合后，于滤膜中过滤除菌，再加入α-MEM基础培养基中混匀，即得到用于促间充质干细胞(UC-MSCs)增殖的无血清培养基。
本发明提供的促间充质干细胞多肽组合物，化学成分明确，无动物源，无血清，能够实现UC-MSCs快速增殖，解决细胞数量不够问题的同时，维持间充质干细胞生物学特性和免疫表型稳定性。
为了进一步理解本发明，下面结合实施例对本发明提供的促间充质干细胞增殖的多肽组合物进行详细说明，本发明的保护范围不受以下实施例的限制。
以下实施例中所涉及的组分、试剂均为常规市售产品。如α-MEM基础培养基(货号41061037)、非必须氨基酸溶液(货号11140-050)购自Gibco公司；其他组分均可从sigma、MP、Gibco等公司购得。
实施例1无血清培养基配制
1)以α-MEM基础培养基为基，按照下述原料配比配制无血清培养基：
非必需氨基酸：体积比1％；
谷氨酰胺：1～4mM；
脂类混合物：1vol％；
ITS：1vol％；
重组人血白蛋白：2.5g/L；
重组人表皮生长因子：20μg/L；
重组人纤连蛋白：10μg/L；
L-谷胱甘肽：2mg/L；
β-巯基乙醇：2mg/L；
三肽-1：10μg/L；
三肽-2：10μg/L；
六肽-9：10μg/L；
棕榈酰六肽-12：10μg/L；
层黏连蛋白衍生肽：50μg/L；
α-MEM基础培养基 10.2g/L；
将上述各组分在常温下溶于水中并充分溶解，得到本申请的多肽组合物，作为实验组2；再经0.22m滤膜过滤除菌，用于促间充质干细胞的增殖。
2)以α-MEM基础培养基为基，按照下述原料配比配制无血清培养基：
非必需氨基酸：0.01vol％；
谷氨酰胺：1～4mmol/L；
脂类混合物：1vol％；
ITS：1vol％；
重组人血白蛋白：2.5g/L；
重组人表皮生长因子：20μg/L；
重组人纤连蛋白：10μg/L；
L-谷胱甘肽：2mg/L；
β-巯基乙醇：2mg/L；
三肽-1：20μg/L；
三肽-2：20μg/L；
六肽-9：20μg/L；
棕榈酰六肽-12：20μg/L；
层黏连蛋白衍生肽：100μg/L；
α-MEM基础培养基：10.2g/L；
将上述各组分在常温下溶于水中并充分溶解，得到本申请的多肽组合物，作为实验组3；再经0.22m滤膜过滤除菌，用于促间充质干细胞的增殖。
3)以α-MEM基础培养基为基，按照下述原料配比配制基础无血清培养基：
非必需氨基酸：1vol％；
谷氨酰胺：1～4mmol/L；
脂类混合物：0.01L/L；
ITS：1vol％；
重组人血白蛋白：2.5g/L；
重组人表皮生长因子：20μg/L；
重组人纤连蛋白：10μg/L；
L-谷胱甘肽：2mg/L；
β-巯基乙醇：2mg/L；
α-MEM基础培养基：10.2g/L；
将上述各组分在常温下溶于水中并充分溶解，得到本申请的多肽组合物，作为实验组1；再经0.22m滤膜过滤除菌，用于促间充质干细胞的增殖。
4)以α-MEM基础培养基为基，按照下述原料配比配制无血清培养基：
非必需氨基酸：1vol％；
谷氨酰胺：1～4mmol/L；
脂类混合物：1vol％；
ITS：1vol％；
重组人血白蛋白：2.5g/L；
重组人表皮生长因子：20μg/L；
重组人纤连蛋白：10μg/L；
L-谷胱甘肽：2mg/L；
β-巯基乙醇：2mg/L；
三肽-1：10μg/L；
三肽-2：10μg/L；
α-MEM基础培养基：10.2g/L；
将上述各组分在常温下溶于水中并充分溶解，得到本申请的多肽组合物，作为对照组3；再经0.22m滤膜过滤除菌，用于促间充质干细胞的增殖。
5)以α-MEM基础培养基为基，按照下述原料配比配制无血清培养基：
非必需氨基酸：1vol％；
谷氨酰胺：1～4mmol/L；
脂类混合物：1vol％；
ITS：1vol％；
重组人血白蛋白：2.5g/L；
重组人表皮生长因子：20μg/L；
重组人纤连蛋白：10μg/L；
L-谷胱甘肽：2mg/L；
β-巯基乙醇：2mg/L；
六肽-9：10μg/L；
棕榈酰六肽-12：10μg/L；
层黏连蛋白衍生肽：50μg/L；
α-MEM基础培养基：10.2g/L；
将上述各组分在常温下溶于水中并充分溶解，得到本申请的多肽组合物，作为对照组4；再经0.22m滤膜过滤除菌，用于促间充质干细胞的增殖。
以上述基础无血清培养基为实验组1，分别以上述多肽组合物作为实验组2、实验组3、对照组3和对照组4，以含10％FBS的α-MEM完全培养基作为对照组1，以GIBCO公司的
Figure PCTCN2019128193-appb-000004
hMSC SFM作为对照组2，进行以下实验。
实施例2UC-MSCs形态观察及活性检测
选择P3代UC-MSCs开展实验，UC-MSCs按1×10 4/cm 2密度接种于T25培养瓶中，每组设置3个重复；置于5％CO 2培养箱37℃培养；培养48h后采集UC-MSCs图像，结果如图1所示，图1中图A为对照组1的UC-MSCs形态图，图B为对照组2的UC-MSCs形态图，图C为实验组1的UC-MSCs形态图，图D为实验组2的UC-MSCs形态图，图E为实验组3的UC-MSCs形态图。
由图1可知，各组UC-MSCs均呈单层贴壁生长，大部分细胞呈长梭形，形态不规则，三个实验组的UC-MSCs形态与对照组2更为相似。
培养72h后，0.25％胰蛋白酶溶液消化收集各组UC-MSCs，采用countstar细胞计数仪计算各组细胞数量及细胞活率，结果如表1所示：
表1各组UC-MSCs活性检测结果数据表
实验组别 细胞活率(％)
对照组1 91.75±0.11
对照组2 93.20±0.25
对照组3 90.05±0.34
对照组4 91.86±0.12
实验组1 91.25±0.35
实验组2 98.66±0.21
实验组3 97.44±0.14
实验组2、实验组3分别与对照组1、对照组3、对照组4对比，有显著性差异(p<0.05)。
由表1可知，实验组UC-MSCs活性高于对照组1和对照组2，且实验组2和实验组3的UC-MSCs活性高于实验组1。
实施例3UC-MSCs增殖速率检测
选择P3代UC-MSCs开展实验，UC-MSCs按1×10 4个/mL密度接种于24孔板中，放入5％CO 2培养箱37℃培养；每天收集细胞进行细胞计数，每次随 机收集计算3个孔，连续7天，绘制细胞生长曲线，结果如表2和图2所示。
表2各组UC-MSCs7天细胞计数结果
Figure PCTCN2019128193-appb-000005
对第7天细胞量进行显著性分析，实验组2、实验组3分别与对照组1、对照组3、对照组4对比，均具有显著性差异(p<0.05)。
由表2及图2结果知，与实验组1、对照组相比，本发明所述的多肽组合培养UC-MSCs增殖活性更高。
根据倍增时间计算公式：DT＝t*[lg2/(lgNt-lgNo)]，其中t为培养时间；No为首次记下的细胞数；Nt为t时间后的细胞数，结果如表3所示。
表3各组UC-MSCs倍增时间结果数据表
实验组别 对照组1 对照组2 对照组3 对照组4 实验组1 实验组2 实验组3
倍增时间(h) 32.12±0.15 31.01±0.21 32.22±0.30 32.23±0.32 29.18±0.24 24.77±0.17 26.62±0.33
实验组2、实验组3分别与对照组1、对照组3、对照组4对比，均具有显著性差异(p<0.05)。
由表3可知，实验组2与实验组3的UC-MSCs的倍增时间少于实验组1和四个对照组，说明本发明所述的多肽组合物培养的UC-MSCs增殖速率更高。
实施例4UC-MSCs表面标志物检测
选择P3代UC-MSCs开展实验，UC-MSCs按1×10 4/cm 2密度接种于T25培养瓶中，置于5％CO 2培养箱37℃培养。3天后，0.25％胰蛋白酶溶液消化收集各组UC-MSCs，流式细胞仪检测其表面marker如CD105、CD73、CD90、CD34、CD45、HLA-DR等的表达情况，结果如表4所示。
表4各组UC-MSCs表面marker检测结果数据表
实验组别 CD105 CD90 CD73 CD34 CD45 HLA-DR
对照组1 100.0％ 98.50％ 96.35％ 0.02％ 0.15％ 0.01％
对照组2 99.96％ 100.0％ 95.24％ 0.01％ 0.00％ 0.03％
对照组3 100.0％ 98.35％ 96.76％ 0.00％ 0.00％ 0.02％
对照组4 97.95％ 99.25％ 96.33％ 0.04％ 0.00％ 0.02％
实验组1 99.45％ 100.0％ 98.10％ 0.00％ 0.01％ 0.02％
实验组2 100.0％ 97.46％ 99.80％ 0.01％ 0.00％ 0.01％
实验组3 100.0％ 99.25％ 100.0％ 0.02％ 0.00％ 0.01％
由表4可知，各实验组和对照组的UC-MSCs表面markerCD105、CD73、CD90阳性表达，而CD34、CD45、HLA-DR阴性表达，各组之间无显著性差异；表明本发明所述的多肽组合培养UC-MSCs不影响其表面标记物的表达。
实施例5UC-MSCs多向分化潜能检测
选择P3代UC-MSCs开展实验，实验组1～3、对照组1和对照组2的UC-MSCs分别常规培养传代至P5代，按1×10 5/mL密度接种于6孔板中，放入5％CO 2培养箱37℃培养。待各组UC-MSCs融合度达80％以上，分别设置对照孔和诱导孔，诱导UC-MSCs成骨和成脂分化。14天后对成脂分化实验组细胞进行油红O染色，21天后对成骨分化实验组细胞进行茜素红染色。结果如图3和图4所示，图3中图A为对照组1的UC-MSCs的成脂分化效果图，图B为对照组2的UC-MSCs的成脂分化效果图，图C为实验组1的UC-MSCs的成脂分化效果图，图D为实验组2的UC-MSCs的成脂分化效果图，图E为实验组3的UC-MSCs的成脂分化效果图，图4中，图A为对照组1的UC-MSCs的成骨分化效果图，图B为对照组2的的UC-MSCs的成骨分化效果图，图C为实验组1的UC-MSCs的成骨分化效果图，图D为实验组2的UC-MSCs的成骨分化效果图，图E为实验组3的UC-MSCs的成骨分化效果图；实验结果表明补充本发明所述的多肽组合培养UC-MSCs不会影响其成脂成骨分化潜能，维持其干性。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见 的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (8)

  1. 一种促间充质干细胞增殖的无血清多肽组合物，包括：
    Figure PCTCN2019128193-appb-100001
  2. 根据权利要求1所述的无血清多肽组合物，其特征在于，所述三肽-1的含量为3～18μg/L。
  3. 根据权利要求1所述的无血清多肽组合物，其特征在于，所述三肽-2的含量为3～18μg/L。
  4. 根据权利要求1所述的无血清多肽组合物，其特征在于，所述六肽-9的含量为4～18μg/L。
  5. 根据权利要求1所述的无血清多肽组合物，其特征在于，所述棕榈酰六肽-12的含量为3～17μg/L。
  6. 根据权利要求1所述的无血清多肽组合物，其特征在于，所述层黏连蛋白衍生肽的含量为30～65μg/L。
  7. 根据权利要求1所述的无血清多肽组合物，其特征在于，所述三肽-1 的含量为10μg/L，所述三肽-2的含量为10μg/L，所述六肽-9的含量为10μg/L，所述棕榈酰六肽的含量为10μg/L，所述层黏连蛋白衍生肽的含量为60μg/L。
  8. 根据权利要求7所述的无血清多肽组合物，其特征在于，所述非必需氨基酸的含量为1vol％，所述谷氨酰胺的含量为1～4mmol/L，所述脂类混合物的含量为1vol％，所述ITS的含量为1vol％，所述重组人血白蛋白的含量为2.5g/L，所述重组人表皮生长因子的含量为20μg/L，所述重组人纤连蛋白的含量为10μg/L，所述L-谷胱甘肽的含量为2mg/L，所述β-巯基乙醇的含量为2mg/L。
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