CN116574670B - 一种诱导间充质干细胞向胰岛样细胞分化的方法及其用途 - Google Patents
一种诱导间充质干细胞向胰岛样细胞分化的方法及其用途 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方法以及其应用。其包含诱导间充质干细胞向胰岛样细胞分化的诱导剂，由以下组分组成：尼克酰胺、碱性成纤维细胞生长因子、GLP‑1、β‑‑巯基乙醇、人血白蛋白和亚硒酸以及基础培养基。本发明的一种将间充质干细胞诱导分化成胰岛细胞的方法，诱导步骤简单，诱导周期短，获得的胰岛样细胞胰岛素分泌功能显著提高，并能显著降低糖尿病模小鼠血糖的浓度。
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方法。
背景技术
糖尿病是严重危害人类健康的常见多发病，目前治疗以注射胰岛素和药物为主，不仅给患者带来很大痛苦，对社会和家庭也造成沉重负担。近几年采用胰岛细胞或胰腺移植治疗糖尿病取得了一些疗效，但遗憾的是仍然面临两大难题：供体匮乏和免疫排斥。干细胞是一群较原始的细胞，具有自我更新的能力。间充质干细胞因来源广泛、易于培养和自体移植的特点，从而更加受到学者的青睐。
干细胞的优势是可自体来源,避免了免疫抑制剂的使用,将为糖尿病的细胞替代治疗提供了新的思路。干细胞是一类具有自我更新和分化潜能的细胞,分为胚胎干细胞(embryon ic stem cel,IESC)和成体干细胞(adu lt stem ce l1,ASC)。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方法及其应用，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包括以下步骤：
1)间充质干细胞的分离，间充质干细胞可以来源于包括但不限于骨髓、脾源性胰腺干细胞、巢蛋白(nestin)阳性干细胞、神经源性胰腺干细胞、肝源性胰腺干细胞、肠源性胰腺干细胞等，通过常规方法均可以分离得到间充质干细胞。
2)配置诱导培养基：以高糖DMEM为基础培养基，添加尼克酰胺15-20mmol/L、细胞生长因子150-500pmol/L、GLP-1 5-15mg/L、β--巯基乙醇0.05-0.3mmol/L，最后加入10-20％(V/V)的人血白蛋白以及0.5-1％亚硒酸(V/V)，混匀，4℃保存备用。
所述的细胞生长因子可以为碱性成纤维细胞生长因子、表皮细胞生长因子、肝细胞生长因子、角化细胞生长因子中的一种或多种。
3)体外诱导分化：将所述的间充质干细胞加入诱导培养基至于4-6％CO2、94-96％饱和湿度的36-38°培养箱中培养，每3-5X105个的细胞中加入3ml诱导培养基，悬浮培养。
4)每24h半量换液，培养4-6天，得胰岛分泌细胞。
本发明的一种将间充质干细胞诱导分化为胰岛细胞的方法，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、显著缩短周期：本发明的诱导培养基仅需要5天即可到达诱导峰值。
3、显著提高诱导细胞胰岛素分泌功能。
4、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。
具体实施方式
实施例1：配置诱导培养基
诱导培养基1：以高糖DMEM为基础培养基，添加尼克酰胺15mmol/L、碱性成纤维细胞生长因子150pmol/L、GLP-1 5mg/L、β--巯基乙醇0.05mmol/L，最后加入10％(V/V)的人血白蛋白以及0.5％亚硒酸(V/V)，混匀，4℃保存备用。
诱导培养基2：以高糖DMEM为基础培养基，添加尼克酰胺20mmol/L、碱性成纤维细胞生长因子500pmol/L、GLP-1 15mg/L、β--巯基乙醇0.3mmol/L，最后加入20％(V/V)的人血白蛋白以及1％亚硒酸(V/V，混匀，4℃保存备用。
对比例1
高糖DMEM基础培养基，通过商购即可获得，
对比例2
常规培养基1：高糖DMEM基础培养基+尼克酰胺15mmol/L+碱性成纤维细胞生长因子150pmol/L+GLP-1 5mg/L+β--巯基乙醇0.05mmol/L+10％(V/V)的人血白蛋白
对比例3
常规培养基2：高糖DMEM基础培养基+尼克酰胺15mmol/L+碱性成纤维细胞生长因子150pmol/L+GLP-1 5mg/L+β--巯基乙醇0.05mmol/L+0.5％亚硒酸(V/V)
对比例4
常规培养基3：高糖DMEM基础培养基+尼克酰胺15mmol/L+碱性成纤维细胞生长因子150pmol/L+GLP-1 5mg/L+10％(V/V)的人血白蛋白+0.5％亚硒酸(V/V)
对比例5
常规培养基4：高糖DMEM基础培养基+尼克酰胺15mmol/L+碱性成纤维细胞生长因子150pmol/L+β--巯基乙醇0.05mmol/L+10％(V/V)的人血白蛋白+0.5％亚硒酸(V/V)
对比例6
常规培养基5：高糖DMEM基础培养基+尼克酰胺15mmol/L+GLP-1 5mg/L+β--巯基乙醇0.05mmol/L+10％(V/V)的人血白蛋白+0.5％亚硒酸(V/V)
对比例7
常规培养基6：高糖DMEM基础培养基+碱性成纤维细胞生长因子150pmol/L+GLP-15mg/L+β--巯基乙醇0.05mmol/L+10％(V/V)的人血白蛋白+0.5％亚硒酸(V/V)
对比例8
常规培养基7：高糖DMEM基础培养基+尼克酰胺15mmol/L+碱性成纤维细胞生长因子150pmol/L+GLP-1 5mg/L
实施例2：诱导分化。
诱导步骤：将人骨髓间充质干细胞加入诱导培养基至于4-6％CO2、94-96％饱和湿度的36-38°培养箱中培养，每3-5X105个的细胞中加入3ml诱导培养基，悬浮培养。
每24h半量换液，培养4-6天，得胰岛分泌细胞。
上述培养基分别选自实施例1中的培养基。
实施例3：诱导细胞的鉴定
(一)、双硫腙染色反应：分别取以上诱导获得的胰岛细胞(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记录。
表1：细胞着色情况
如表1所示，本申请的诱导培养基以及诱导方法可明显缩短从间充质干细胞向胰岛样细胞分化的周期。
(二)葡萄糖刺激实验胰岛素以及C肽量分析：挑取150个本发明诱导剂诱导分化的胰岛细胞团(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、25mmol/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，基础培养基细胞上清中几乎检测不到胰岛素和C-肽，而经诱导的胰岛细胞团在5.6mmol/L葡萄糖刺激下均有一定程度的分泌，但诱导培养基组的分泌胰岛素量和C-肽量与常规组由显著差异(P<0.01)，经25mmol/L葡萄糖孵育2h后，胰岛素和C-肽分泌量明显升高(P<0.01)，由此结果可知，诱导后胰岛细胞团对葡萄糖刺激敏感，其胰岛素和C-肽的分泌受外环境的调控。
表2：葡萄糖刺激实验胰岛素以及C肽量分析
a：与其他组相比，胰岛素和C-肽释放量差异极显著(P＜0.01)C：与低糖组相比，高糖组胰岛素和C-肽释放量差异极显著(P＜0.01)表明本申请的诱导培养基以及诱导方法能显著提高诱导的样细胞胰岛素和C-肽的分泌量
(三)、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200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后血糖浓度即开始下降，10天后下降到11mmol/L以下，平均下降8.1mmol/L。这表明诱导的胰岛样细胞具有确切的生理功能，能够在体内降低糖尿病模型血糖的浓度。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种将骨髓间充质干细胞诱导分化为胰岛细胞的诱导剂，其特征在于，由以下组分组成：尼克酰胺15-20 mmol/L、碱性成纤维细胞生长因子150-500 pmol/L、GLP-1 5-15 mg/L、β-巯基乙醇0.05-0.3 mmol/L、10-20%（V/V）的人血白蛋白以及0.5-1%（V/V） 亚硒酸。
2.根据权利要求1所述的诱导剂，其特征在于，各组分的含量为尼克酰胺20 mmol/L、碱性成纤维细胞生长因子150 pmol/L、GLP-1 5 mg/L、β-巯基乙醇0.05 mmol/L、10%（V/V）的人血白蛋白以及0.5% （V/V）亚硒酸。
3.根据权利要求1所述的的诱导剂，其特征在于，各组分的含量为尼克酰胺15 mmol/L、碱性成纤维细胞生长因子500 pmol/L、GLP-1 15 mg/L、β-巯基乙醇0.3 mmol/L、20 %（V/V）的人血白蛋白以及1 %（V/V） 亚硒酸。
4.一种将骨髓间充质干细胞诱导分化为胰岛细胞的培养基，其特征在于，包含高糖DMEM基础培养基以及权利要求1-3任一项所述的诱导剂。
5.一种体外将骨髓间充质干细胞诱导分化为胰岛细胞的方法，其特征在于使用权利要求1-3任一项所述的诱导剂或权利要求4所述的培养基进行诱导培养。
6.根据权利要求5所述的方法，其具体步骤为：
1）通过人工分离或商购获取骨髓间充质干细胞；
2）配置诱导培养基，在包含高糖DMEM基础培养基里添加权利要求1-3任一项所述的诱导剂；
3）诱导分化，将所述的骨髓间充质干细胞置于所述培养基里培养；培养时间为5-6天；
4）对诱导细胞进行鉴定。
7.权利要求1-3任一项所述的诱导剂或权利要求4所述的培养基在体外诱导骨髓间充质干细胞向胰岛细胞分化中的应用。
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