WO2021212592A1 - 一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基 - Google Patents

一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基 Download PDF

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WO2021212592A1
WO2021212592A1 PCT/CN2020/092071 CN2020092071W WO2021212592A1 WO 2021212592 A1 WO2021212592 A1 WO 2021212592A1 CN 2020092071 W CN2020092071 W CN 2020092071W WO 2021212592 A1 WO2021212592 A1 WO 2021212592A1
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serum
corneal epithelial
cells
stem cells
mesenchymal stem
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French (fr)
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张炳强
陈梦梦
邹伟
付学奇
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青岛瑞思德生物科技有限公司
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Priority to EP20873344.4A priority Critical patent/EP4141109A1/en
Priority to US17/283,491 priority patent/US20220041982A1/en
Priority to KR1020217008536A priority patent/KR102552191B1/ko
Priority to JP2021516585A priority patent/JP7239686B2/ja
Publication of WO2021212592A1 publication Critical patent/WO2021212592A1/zh

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Definitions

  • the invention relates to the field of induced differentiation of stem cells, in particular to a serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells.
  • the corneal epithelial cell layer is located on the outer surface of the cornea. There are 5 to 6 layers of cells in the upper cornea, each layer is arranged very neatly and tightly, the deepest layer is a single layer of short columnar basal cells; the deep layer has 2 to 3 layers of polygonal wing cells, and the uppermost layer is 2 to 3 layers of polygonal surface. cell.
  • the shallowest layer of surface cells are flat cells that are not keratinized. The gaps between the epidermal cells are not clear.
  • the surface cell membrane is very smooth, the cytoplasm is clear and transparent, and the cytoplasmic protrusions are combined with each other to form a bridge, and the nucleus still exists. This cell has a very strong light transmittance. Corneal epithelial cells have strong resistance and regeneration ability, and deep cells can still undergo mitosis. After the corneal epithelium suffers trauma and infection, it usually recovers quickly.
  • Corneal transplantation is currently the only effective way to cure corneal blindness. According to statistics, there are about 4 million to 5 million blind corneal diseases nationwide, and most of them can be restored through corneal transplantation. However, China can only carry out about 10,000 corneal transplants each year. The main reason is that the number of donated corneas is severely insufficient, which limits the popularization and application of the surgery. As a result, the vast majority of corneal blind patients cannot see through corneal transplantation. Therefore, in vitro Reconstruction of tissue engineered cornea is an important breakthrough to solve the shortage of donor corneal materials.
  • corneal tissue engineering such as the source of corneal epithelial seed cells.
  • the study of corneal epithelium has enabled people to have a deeper understanding of the physiological and pathological characteristics of the cornea and corneal diseases.
  • the life cycle of differentiated corneal epithelial cells in vitro is very short and can only pass 2 to 3 generations.
  • the small quantity and high cost limit the research of corneal tissue and the construction of tissue engineered cornea. Therefore, how to obtain corneal epithelial cells with strong proliferation ability and continuous division and growth to supplement the continuous renewal of cells has become the primary task of obtaining corneal epithelial seed cells.
  • the establishment of corneal epithelial cell lines can provide the required cells for different research purposes stably for a long time, such as epithelial proliferation and differentiation, testing of ophthalmic drugs, development of new treatment methods for corneal diseases, etc.
  • Mesenchymal stem cells have a wide range of sources and are easy to obtain materials. They are convenient for autologous transplantation and have strong proliferation ability. They can always maintain their multi-directional differentiation potential during long-term culture in vitro. They can differentiate into osteoblasts, cartilage, tendons, etc. under specific conditions. Muscle cells, fat cells, nerve cells and liver cells are an ideal seed cell for tissue engineering. Mesenchymal stem cells have become a hotspot in stem cell research. After induction, they differentiate into corneal epithelial cells, which can be used to repair corneal injuries.
  • the differentiation of stem cells is the selective activation or differential expression of some genes, thereby controlling the cell phenotype and the specific distribution of proteins.
  • the differentiation of mesenchymal stem cells into specific cell types mainly depends on the activation of genes, and the types and concentrations of various factors in the extracellular microenvironment are important factors for gene activation.
  • the serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells of the present invention adopts traditional Chinese medicine components resveratrol, icariin, combined with aspirin, parathyroid hormone, hydrocortisone, and rapamycin Inducing mesenchymal stem cells to differentiate into corneal epithelial cells.
  • the selected induction components are non-toxic, with high induction efficiency, short induction time, good activity of induced corneal epithelial cells, and no rejection after cell transplantation. , No ethical issues, high safety.
  • the purpose of the present invention is to provide a high induction efficiency, few induction steps, short induction time, good activity of induced corneal epithelial cells, no rejection after cell transplantation, and no ethics.
  • the problem is a high-safety serum-free complete medium that induces differentiation of mesenchymal stem cells into corneal epithelial cells.
  • the technical solution adopted by the present invention is: a serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells, prepared by the following method: each 1L of said differentiation-induced serum-free complete medium contains Resveratrol 5-10 ⁇ mol, Icariin 2-4 ⁇ mol, Aspirin 1-3nmol, Parathyroid hormone 1-3nmol, Hydrocortisone 5-10nmol, Rapamycin 1-3mg, Testosterone 2-10 ⁇ g, EPO 2-10 ⁇ g, LIF 2-10 ⁇ g, the remainder is the corneal epithelial cell serum-free medium, which can be filtered and sterilized after mixing.
  • a serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells is prepared by the following method: each 1 L of the serum-free complete medium for inducing differentiation contains 8 ⁇ mol of resveratrol and icariin 3 ⁇ mol, aspirin 2nmol, parathyroid hormone 2nmol, hydrocortisone 7nmol, rapamycin 2mg, testosterone 7 ⁇ g, EPO 7 ⁇ g, LIF 7 ⁇ g, the remainder is the corneal epithelial cell serum-free medium. After mixing, filter and sterilize. Can.
  • the serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells of the present invention has the following advantages: 1. No gene transfection is required, so there is no genetic change and cancer risk; 2. Few induction steps; 3. Induction Short time; 4, 5. High efficiency of inducing differentiation; 5.
  • the inducer of the present invention each component is safe and non-toxic; 6. After the mesenchymal stem cells are induced to differentiate into corneal epithelial cells, there is no rejection after transplantation, and no ethical problems. It is highly safe and has broad clinical application prospects.
  • Figure 1 The appearance of corneal epithelial cells ( ⁇ 400) after differentiation of mesenchymal stem cells induced by the culture medium of the present invention
  • Example 1 A serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells of the present invention.
  • Each component is a commercially available product: resveratrol, brand Sigma, article number R5010; icariin, brand Shanghai Microcrystalline Bio, Item No. 489-32-7; Aspirin, Brand Sigma, Item No. A2093-100G; Parathyroid Hormone, Brand Sigma, Item No. P7036; Hydrocortisone, Brand Sigma, Item No.
  • a serum-free complete medium for inducing the differentiation of mesenchymal stem cells into corneal epithelial cells which is composed of the following components and proportioned at the following concentrations:
  • Each 1L of the serum-free complete medium for inducing differentiation contains resveratrol 8 ⁇ mol, icariin 3 ⁇ mol, aspirin 2nmol, parathyroid hormone 2nmol, hydrocortisone 7nmol, rapamycin 2mg, testosterone 7 ⁇ g, EPO 7 ⁇ g, LIF 7 ⁇ g, the balance is corneal epithelial cell serum-free medium, mixed Filter and sterilize after homogenization.
  • Example 2 A serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells is composed of the following components and proportioned at the following concentrations: each 1L of the serum-free complete medium for inducing differentiation contains white Veratrol 5 ⁇ mol, Icariin 2 ⁇ mol, Aspirin 1nmol, Parathyroid hormone 1nmol, Hydrocortisone 5nmol, Rapamycin 1mg, Testosterone 2 ⁇ g, EPO 2 ⁇ g, LIF 2 ⁇ g, the balance is corneal epithelial cell serum-free culture Base, filter and sterilize after mixing.
  • Example 3 A serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells is composed of the following components and proportioned at the following concentrations: each 1L of the serum-free complete medium for inducing differentiation contains white Veratrol 10 ⁇ mol, Icariin 4 ⁇ mol, Aspirin 3nmol, Parathyroid hormone 3nmol, Hydrocortisone 10nmol, Rapamycin 3mg, Testosterone 10 ⁇ g, EPO 10 ⁇ g, LIF 10 ⁇ g, the balance is corneal epithelial cell serum-free culture Base, filter and sterilize after mixing.
  • Example 4 takes human adipose-derived mesenchymal stem cells as an example, using the induction medium prepared in Example 1, to perform corneal epithelial cell differentiation experiments on mesenchymal stem cells, and the steps are as follows:
  • MSCs of passage 3 are seeded on a 6-well plate pre-placed with sterile coverslips treated with polylysine to prepare cell slides. When the cells are close to 80% confluence and grow vigorously, then induce differentiation.
  • the experiment grouping is shown in Table 1:
  • CK3, CK12, and CK19 are selected as corneal epithelial cell markers, and the expression of CK3, CK12, and CK19 before and after induction is detected by immunocytochemistry. 4 slides are randomly selected, and 5 are randomly selected under a 200-fold field of view Visual field, respectively calculate the proportion of positive cells. The proportion of positive cells to the total cells is the mean ⁇ standard deviation Express. Using SPSS11.0 software for statistical analysis, the experimental data is based on Indicates that the analysis of variance was used for the comparison of multiple groups, and the comparison of the rates between the two groups was performed by the ⁇ 2 test. P ⁇ 0.05 was considered statistically significant. The three groups of induction results are shown in Table 2:
  • the positive cell rate of CK3, CK12, and CK19 in the induction group of the present invention is higher than that of the conditioned medium group and the co-culture group (P ⁇ 0.05), indicating that the induction efficiency of the medium of the present invention is significantly higher than that of the conditioned medium Group and co-cultivation group, reached more than 90%.
  • the serum-free complete medium for inducing differentiation of mesenchymal stem cells into corneal epithelial cells of the present invention has the highest induction efficiency and the shortest induction time, and is worthy of promotion.

Abstract

涉及干细胞诱导分化领域，尤其涉及一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，其通过以下方法制备：每1L所述诱导分化无血清完全培养基中含有白藜芦醇5-10μmol、淫羊藿苷2-4μmol、阿司匹林1-3nmol、甲状旁腺激素1-3nmol、氢化可的松5-10nmol、雷帕霉素1-3mg、睾酮2-10μg、EPO2-10μg、LIF2-10μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。该诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，采用中药成分白藜芦醇、淫羊藿苷联合阿司匹林、甲状旁腺激素、氢化可的松、雷帕霉素、睾酮和生长因子，协同诱导间充质干细胞定向分化为角膜上皮细胞，所选用的诱导成分均无毒，诱导效率高，诱导时间短，诱导获得角膜上皮细胞活性好，细胞移植后无排斥，无伦理问题，安全性高。

Description

一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基 技术领域
本发明涉及干细胞诱导分化领域，尤其涉及一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基。
背景技术
角膜上皮细胞层位于角膜外表面。角膜上层细胞有5～6层，每层排列极为整齐而且紧密，最深层为单层矮柱状的基底细胞；深层上面有2～3层多边形的翼状细胞，最上面是2～3层多边形的表层细胞。表层细胞的最浅层为不角化的扁平细胞，表皮细胞之间的间隙不清晰，表层细胞膜很光滑，胞浆清晰透明，胞浆突起互相结合成桥，核仍存在。此细胞透光度极强。角膜上皮细胞抵抗力、再生能力都很强，深层细胞尚可进行有丝***。角膜上皮遭受外伤和感染后，一般均能很快恢复。
多种致病因素如眼外伤、手术创伤、炎症、药物毒性均可造成角膜缘损伤，造成角膜缘干细胞功能障碍，导致上皮细胞缺失，从而导致增加角膜感染、穿孔、新生血管化的危险。角膜移植是目前角膜盲治愈的惟一有效手段。据统计全国角膜病盲患者约有400万～500万，其中大部分通过角膜移植手术可以复明。但我国每年仅能开展约1万例角膜移植手术，主要原因是捐献的供体角膜数量严重不足，限制了手术的推广应用，致使绝大多数角膜盲患者无法通过角膜移植重见光明，因此体外重建组织工程角膜是目前解决供体角膜材料不足的重要突破口。
但是目前角膜组织工程中遇到一些瓶颈问题,如角膜上皮种子细胞的来源问题。角膜上皮的研究使人们对角膜的生理、病理特点和角膜疾病有了更深入的了解，但由于分化后的角膜上皮细胞体外生长的生命周期很短，只能传2～3代，获得的细胞数量少，花费较高，限制了角膜组织的研究和组织工程化角膜的构建。因此如何获得增殖能力强、能不断***生长的角膜上皮细胞，以补充细胞的不断更新，成为获得角膜上皮种子细胞的首要任务。通过建立角膜上皮细胞系能够长期稳定地为不同研究目的提供所需的细胞，如上皮的增殖、分化、眼科用药的测试、角膜疾病新的治疗方法的开发等。
间充质干细胞来源广泛、取材容易，便于自体移植，具有强大的增殖能力，在体外长期培养过程中可以始终保持其多向分化潜能，在特定条件诱导下可以分化为成骨、软骨、肌腱、肌细胞、脂肪细胞、神经细胞和肝细胞等，是一种理想的组织工程种子细胞。间充质干细胞已经成为干细胞研究的热点，经诱导后分化为角膜上皮细胞，可用于角膜损伤的修复。
干细胞的分化是部分基因选择性地被激活或差异性表达，从而控制细胞表型和蛋白质的特异性分布。间充质干细胞分化为特定细胞类型主要取决于基因的激活，而细胞外微环境中各种因子类型和浓度则是基因激活的重要因素。本发明的一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，采用中药成分白藜芦醇、淫羊藿苷联合阿司匹林、甲状旁腺激素、氢化可的松、雷帕霉素、睾酮和生长因子，协同诱导间充质干细胞定向分化为角膜上皮细胞，所选用的诱导成分均无毒，诱导效率高，诱导时间短，诱导获得角膜上皮细胞活性好，细胞移植后无排斥，无伦理问题，安全性高。
发明内容
本发明的目的是针对现有的诱导培养基及诱导方法存在的缺陷，提供一种诱导效率高，诱导步骤少，诱导时间短，诱导获得角膜上皮细胞活性好，细胞移植后无排斥，无伦理问题，安全性高的诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基。
为实现上述目的，本发明采用的技术方案是：一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，通过以下方法制备：每1L所述诱导分化无血清完全培养基中含有白藜芦醇5-10μmol、淫羊藿苷2-4μmol、阿司匹林1-3nmol、甲状旁腺激素1-3nmol、氢化可的松5-10nmol、雷帕霉素1-3mg、睾酮2-10μg、EPO 2-10μg、LIF 2-10μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
优选的，一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，通过以下方法制备：每1L所述诱导分化无血清完全培养基中含有白藜芦醇8μmol、淫羊藿苷3μmol、阿司匹林2nmol、甲状旁腺激素2nmol、氢化可的松7nmol、雷帕霉素2mg、睾酮7μg、EPO 7μg、LIF 7μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
本发明的一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，具有以下优势：1、无需基因转染，故无基因改变和癌症风险；2、诱导步骤少；3、诱导时间短；4、5、诱导分化效率高；5、本发明的诱导剂，各成分均安全无毒性；6、间充质干细胞诱导分化为角膜上皮细胞后，移植后无排斥，无伦理问题，安全性高，具有广阔的临床应用前景。
附图说明
图1本发明培养基诱导间充质干细胞分化后出现角膜上皮细胞的形态(×400)；
具体实施方式
下述实施例中的实验方法，如无特别说明，均为常规方法。实验所用器具仪器试剂皆可通过商业途径获得。
实施例1本发明的一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基各成分均为市售产品：白藜芦醇，品牌Sigma，货号R5010；淫羊藿苷，品牌上海微晶生物,货号489-32-7；阿司匹林，品牌Sigma，货号A2093-100G；甲状旁腺激素，品牌Sigma,货号P7036；氢化可的松，品牌Sigma,货号H3160；雷帕霉素，品牌TargetMol，货号T1537，睾酮，品牌Sigma,货号T1500；EPO(***)，品牌PeproTech，货号CYT-201；LIF(白血病抑制因子)，品牌PeproTech，货号96-300-05-5；角膜上皮细胞无血清培养基(CEpiCM)，品牌ScienCell，货号6511。
一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，由以下组分，分别按以下浓度配比而成：每1L所述诱导分化无血清完全培养基中含有白藜芦醇8μmol、淫羊藿苷3μmol、阿司匹林2nmol、甲状旁腺激素2nmol、氢化可的松7nmol、雷帕霉素2mg、睾酮7μg、EPO 7μg、LIF 7μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
实施例2一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，由以下组分，分别按以下浓度配比而成：每1L所述诱导分化无血清完全培养基中含有白藜芦醇5μmol、淫羊藿苷2μmol、阿司匹林1nmol、甲状旁腺激素1nmol、氢化可的松5nmol、雷 帕霉素1mg、睾酮2μg、EPO 2μg、LIF 2μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
实施例3一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，由以下组分，分别按以下浓度配比而成：每1L所述诱导分化无血清完全培养基中含有白藜芦醇10μmol、淫羊藿苷4μmol、阿司匹林3nmol、甲状旁腺激素3nmol、氢化可的松10nmol、雷帕霉素3mg、睾酮10μg、EPO 10μg、LIF 10μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
实施例4以人脂肪间充质干细胞为例，采用实施例1制备的诱导培养基，对间充质干细胞进行角膜上皮细胞诱导分化实验，步骤如下：
诱导脂肪间充质干细胞分化为角膜上皮细胞：传3代的MSC，接种于事先放置有经多聚赖氨酸处理的消毒盖玻片的6孔板，制备细胞爬片。待细胞接近80％融合，生长旺盛时再进行诱导分化。实验分组如表1：
表1实验分组
Figure PCTCN2020092071-appb-000001
诱导后细胞进行鉴定：选择CK3、CK12、CK19作为角膜上皮细胞标记物，免疫细胞化学法检测诱导前后CK3、CK12、CK19的表达情况，随机抽取4张玻片，200倍视野下随机选取5个视野，分别计算阳性细胞的比例。阳性细胞占总细胞比例以均数±标准差
Figure PCTCN2020092071-appb-000002
表示。采用SPSS11.0软件进行统计分析，实验数据以
Figure PCTCN2020092071-appb-000003
表示，多组比较采用方差分析，两组间率的比较采用χ 2检验，P<0.05认为有统计学意义。三组诱导结果如表2所示：
表2三组诱导结果(n＝20，
Figure PCTCN2020092071-appb-000004
)
Figure PCTCN2020092071-appb-000005
如表2所示，本发明诱导组诱导分化后CK3、CK12、CK19阳性细胞率高于条件培养基组和共培养组(P<0.05)，说明本发明培养基诱导效率明显高于条件培养基组和共培养组，达到了90％以上。
综上，本发明的一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基诱导效率最高，诱导时间最短，值得推广。

Claims (2)

  1. 一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，通过以下方法制备：每1L所述诱导分化无血清完全培养基中含有白藜芦醇5-10μmol、淫羊藿苷2-4μmol、阿司匹林1-3nmol、甲状旁腺激素1-3nmol、氢化可的松5-10nmol、雷帕霉素1-3mg、睾酮2-10μg、EPO 2-10μg、LIF 2-10μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
  2. 一种诱导间充质干细胞向角膜上皮细胞分化的无血清完全培养基，通过以下方法制备：每1L所述诱导分化无血清完全培养基中含有白藜芦醇8μmol、淫羊藿苷3μmol、阿司匹林2nmol、甲状旁腺激素2nmol、氢化可的松7nmol、雷帕霉素2mg、睾酮7μg、EPO 7μg、LIF 7μg，余量为角膜上皮细胞无血清培养基，混匀后过滤除菌即可。
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