CN113106054B - 一种将间充质干细胞诱导分化为胰岛细胞的诱导剂 - Google Patents
一种将间充质干细胞诱导分化为胰岛细胞的诱导剂 Download PDFInfo
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Abstract
本发明属于生物医学领域，涉及一种将间充质干细胞诱导分化为胰岛细胞的诱导剂。一种将间充质干细胞诱导分化为胰岛细胞的诱导剂，由以下组分组成：GLP‑1、甲状旁腺激素、对乙酰氨基酚、雷帕霉素、淫羊藿苷、曲美替尼、EPO和VEGF。本发明的一种将间充质干细胞诱导分化成胰岛细胞的诱导剂，各成分均安全无毒性，诱导分化所需步骤少、时间短，诱导效率高。
Description
技术领域
本发明属于生物医学领域，涉及一种将间充质干细胞诱导分化为胰岛细胞的诱导剂。
背景技术
糖尿病(diabetes mellitus，DM)是一种以糖脂代谢紊乱为主要特征的内分泌代谢性疾病。根据世界卫生组织和国际糖尿病联盟公布的预测，到2030年，全球糖尿病患者将达3.7亿。目前我国DM患者已超过4500万，仅次于印度居第二位。尽管其病因不同，但都表现为胰岛细胞数量及功能的缺陷，最终都需要使用胰岛素进行治疗。尽管由于胰岛素的应用，尤其是近年来在胰岛素剂型以及给药途径等方面均取得了一些进展，但实践证明外源性胰岛素并不能像人体自身分泌胰岛素那样完美地控制血糖并恒定维持血糖正常。细胞替代疗法是治疗糖尿病的重要方法，是一种更接近生理方式的有效方法，可实现对血糖进行持续监测和精细调节。近些年，经肝门静脉植入胰岛细胞治疗糖尿病也获得了一些疗效，但胰岛细胞移植面临着两个难题：供体来源不足及严重的免疫排斥反应。
干细胞作为一种具有自我更新及多向分化的细胞，在一定条件下可分化为具有功能性的胰岛细胞，因此可作为胰岛细胞的全新来源。目前由干细胞分化获得胰岛细胞有三种途径：1、胚胎干细胞分化；2、胰岛干细胞或胰岛前体细胞分化；3、成体干细胞分化。由于胚胎干细胞具有伦理争议，且很难获得胰岛干细胞，而成体干细胞来源广泛，无伦理争议，可作为胰岛细胞的来源。
目前的干细胞诱导方式主要有体外诱导、基因修饰、蛋白转导与组织微环境诱导，其中体外诱导是采用不同刺激因子组合，将干细胞诱导分化为目的细胞。各实验室诱导分化条件各异，诱导分化机制尚不明确，诱导分化效率低，胰岛分泌能力仅为正常胰岛1%左右，且诱导过程复杂，诱导时间长，所得细胞数量少，功能低。
发明内容
本发明的目的是为了解决现有技术中存在的上述问题，提供一种将间充质干细胞诱导分化成胰岛细胞的诱导剂，各成分均安全无毒性，诱导分化所需步骤少、时间短，诱导效率高。
为实现上述目的，本发明采用的技术方案是：一种将间充质干细胞诱导分化为胰岛细胞的诱导剂，由以下组分组成：GLP-1、甲状旁腺激素、对乙酰氨基酚、雷帕霉素、淫羊藿苷、曲美替尼、EPO和VEGF。
所述诱导剂各组分的质量浓度配比为：GLP-1 20-40mg/L、甲状旁腺激素6-12mg/L、对乙酰氨基酚2-8mg/L、雷帕霉素2-8mg/L、淫羊藿苷2-8mg/L、曲美替尼0.3-0.6mg/L、EPO2-4ug/L、VEGF 2-4ug/L。
优选的，所述诱导剂各组分的质量浓度配比为：GLP-1 30mg/L、甲状旁腺激素9mg/L、对乙酰氨基酚5mg/L、雷帕霉素5mg/L、淫羊藿苷5mg/L、曲美替尼0.4mg/L、EPO 3ug/L、VEGF 3ug/L。
本发明的一种将间充质干细胞诱导分化为胰岛细胞的诱导剂，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：当前文献报道的方法以两步法或者三步法为主，采用本发明的诱导剂仅需要一步；
3、诱导时间短：当前文献一般诱导需要10天以上，采用本发明的诱导剂仅需要5天；
4、采用本发明的诱导剂将间充质干细胞诱导成胰岛细胞诱导分化效率高；
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胰岛细胞后，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是双硫腙染色反应，示本发明诱导组双硫腙染色后呈红色。
具体实施方式
实施例1 本实施例的将间充质干细胞诱导成胰岛细胞的诱导剂，由以下质量浓度配比的组分组成：GLP-1 30mg/L、甲状旁腺激素9mg/L、对乙酰氨基酚5mg/L、雷帕霉素5mg/L、淫羊藿苷5mg/L、曲美替尼0.4mg/L、EPO 3ug/L、VEGF 3ug/L。将上述组分按质量浓度配比依次加入人间充质干细胞无血清培养基（或者DMEM+10%FBS或者市售的其他类型间充质干细胞培养基），混匀，过滤除菌即可。
本发明的诱导剂各成分均为市售产品：人间充质干细胞无血清培养基，品牌LONZA，货号00190632；GLP-1(胰高血糖素样肽1)，品牌Sigma, 货号scp0153；甲状旁腺激素，品牌Sigma，货号 P7036；对乙酰氨基酚，上海一基实业有限公司，货号 Y903952；雷帕霉素，品牌TargetMol ，货号T1537；淫羊藿苷，上海微晶生物, 货号489-32-7；曲美替尼，品牌美仑生物，货号MB5401；EPO ，品牌PeproTech ，货号CYT-201；VEGF，品牌PeproTech，货号96-100-20-2。
实施例2
本实施例的将间充质干细胞诱导成胰岛细胞的诱导剂，由以下质量浓度配比的组分组成：GLP-1 20mg/L、甲状旁腺激素6mg/L、对乙酰氨基酚2mg/L、雷帕霉素2mg/L、淫羊藿苷2mg/L、曲美替尼0.3mg/L、EPO 2ug/L、VEGF 2ug/L。将上述组分按质量浓度配比依次加入人间充质干细胞无血清培养基（或者DMEM+10%FBS），混匀，过滤除菌即可。
实施例3
本实施例的将间充质干细胞诱导成胰岛细胞的诱导剂，由以下质量浓度配比的组分组成：GLP-1 40mg/L、甲状旁腺激素12mg/L、对乙酰氨基酚8mg/L、雷帕霉素8mg/L、淫羊藿苷8mg/L、曲美替尼0.6mg/L、EPO 4ug/L、VEGF 4ug/L。将上述组分按质量浓度配比依次加入人间充质干细胞无血清培养基（或者DMEM+10%FBS），混匀，过滤除菌即可。
实施例4
以人脐带间充质干细胞为例说明本发明诱导剂效果
一、诱导人脐带间充质干细胞分化为胰岛细胞
传3代的人脐带间充质干细胞，0.125%～0.01％Trypsin-EDTA溶液消化收集细胞，制备细胞悬液，细胞计数板计数活细胞密度，并调整密度1×104/cm2，接种于事先放置有经多聚赖氨酸处理的消毒盖玻片的24孔板内，制备细胞爬片。待细胞达接近80%融合，生长旺盛时再进行诱导分化，分组见表1。
表1诱导分组
| 组别 | 诱导条件 |
| 对照组（空白对照） | 人间充质干细胞无血清培养基。 |
| 诱导组1（常规方法） | 常规诱导分化培养基：烟酰胺1.2g/L、牛磺酸0.50 g/L，GLP-1 30.5mg/L；人间充质干细胞无血清培养基。 |
| 诱导组2 （本发明） | 实施例1中本发明的诱导分化培养基 |
二、诱导后胰岛细胞的鉴定
（一）、双硫腙染色反应：分别取以上三组诱导5天后获得的胰岛细胞（其中诱导组1需要诱导分化10天），移出原培养基，PBS洗2次，各加入2ml PBS和50ul双硫腙工作液，37℃孵育10min, 移出染色液，PBS洗涤两次，观察细胞着色情况并拍照。结果如图所示，两个诱导组双硫腙染色后呈红色（如图1所示），为阳性反应，对照组为阴性。
（二）化学发光免疫分析法检测胰岛素水平：分别取以上三组诱导5天后（其中诱导组1需要诱导分化10天）细胞培养上清，检测胰岛素含量，诱导组2的细胞分泌胰岛素浓度为518.7mU/L，远高于诱导组1的细胞分泌胰岛素浓度212 .1mU/L，而空白对照组检测胰岛素浓度为零，说明本发明的诱导剂可以显著提高诱导效率。
（三）ELISA试剂盒检测用本发明诱导剂诱导获得的胰岛样细胞是否具有胰岛细胞功能：这一检测过程直接按照Mercodia公司所提供的“C-peptide ELISA assays”标准过程操作。最终结果检测出C-肽，说明经本发明的诱导剂诱导分化获得的胰岛样细胞能够分泌胰岛素。
（四）葡萄糖刺激实验：挑取100个本发明诱导剂诱导分化5天的胰岛细胞团（50~150um）至1.5ml离心管中，用PBS清洗2遍，加入1ml无糖DMEM预培养3~6h，然后以300ul含有5.6mmol/L葡萄糖、16.7mmol/L葡萄糖的DMEM依次培养2h，收集上清液，用ELISA法检测上清中不同浓度葡萄糖刺激下胰岛素的分泌量，对照组细胞上清中几乎检测不到胰岛素，而经诱导的胰岛细胞团在5.6mmol/L葡萄糖刺激下有少量分泌，经16.7mmol/L葡萄糖孵育2h后，胰岛素分泌量明显升高（P<0.001），约为低糖条件下的2倍，由此结果可知，诱导后胰岛细胞团对葡萄糖刺激敏感，其胰岛素的分泌受外环境的调控。
（五）体内移植实验：首先制作糖尿病大鼠模型。取成年Wistar大鼠，雌雄不限，体重约180~200g。按照70mg/kg剂量给每只大鼠腹腔注射链脲霉素。链脲霉素粉剂用0.1M柠檬酸缓冲液（PH=4.5）配成液体使用，现配现用。当大鼠血糖升高（≥16.7mmol/L）且稳定一周，表明糖尿病模型已经建成。无菌条件下，向糖尿病大鼠肾包囊下或者肝门脉小分支注入300个本发明的诱导剂诱导得到的胰岛样细胞团（50~150um）。术后，定期观测血糖情况。结果：糖尿病大鼠在植入细胞3天后血糖平均下降7.3mmol/L。表明采用本发明诱导剂诱导分化获得的胰岛细胞团具有显著的降血糖作用。
Claims (2)
1.一种将间充质干细胞诱导分化为胰岛细胞的诱导剂，其特征为，由以下组分组成：GLP-1、甲状旁腺激素、对乙酰氨基酚、雷帕霉素、淫羊藿苷、曲美替尼、EPO和VEGF；各组分的质量浓度配比为：GLP-1 20-40mg/L、甲状旁腺激素6-12mg/L、对乙酰氨基酚2-8mg/L、雷帕霉素2-8mg/L、淫羊藿苷2-8mg/L、曲美替尼0.3-0.6mg/L、EPO 2-4ug/L、VEGF 2-4ug/L；将上述组分按质量浓度配比依次加入人间充质干细胞无血清培养基或者 DMEM+10%FBS 或者其他类型间充质干细胞培养基，混匀，过滤除菌即可。
2.根据权利要求1所述的一种将间充质干细胞诱导分化为胰岛细胞的诱导剂，其特征为，各组分的质量浓度配比为：GLP-1 30mg/L、甲状旁腺激素9mg/L、对乙酰氨基酚5mg/L、雷帕霉素5mg/L、淫羊藿苷5mg/L、曲美替尼0.4mg/L、EPO 3ug/L、VEGF 3ug/L。
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| Publication number | Publication date |
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| KR20210093750A (ko) | 2021-07-28 |
| EP4092106A1 (en) | 2022-11-23 |
| EP4092106A4 (en) | 2024-03-06 |
| JP2022519794A (ja) | 2022-03-25 |
| CN113106054A (zh) | 2021-07-13 |
| JP7087119B2 (ja) | 2022-06-20 |
| WO2021143001A1 (zh) | 2021-07-22 |
| US20210363489A1 (en) | 2021-11-25 |
| KR102459759B1 (ko) | 2022-10-26 |
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