CN110923205A - 一种淋巴管内皮细胞培养基及其制备方法和用途 - Google Patents
一种淋巴管内皮细胞培养基及其制备方法和用途 Download PDFInfo
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Abstract
本发明提供一种淋巴管内皮细胞培养基,包括基础培养基和添加剂,所述添加剂包括:胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素。本发明的细胞培养基有效促进淋巴内皮细胞的增殖。本发明中,各个生长因子及组分的配合添加,能够有效地维持原代淋巴内皮细胞的性状,在体外扩充了8代以后,细胞仍能有较大程度维持原有性状,延长了生存时间。培养基中添加的各个组分容易获得,配制方便,降低了培养内皮细胞的成本。
Description
技术领域
本发明属于细胞培养技术领域,具体涉及一种淋巴管内皮细胞培养基及其制备方法和用途。
背景技术
淋巴管内皮细胞(lymphatic endothelial cells,LECs)是衬覆于淋巴管内表面的一种单层扁平上皮,具有选择性屏障、止血、抗凝和运输等作用。LECs不但参与维持体液平衡、调节淋巴细胞再循环和机体免疫反应等生理过程,还与炎症反应、肿瘤转移等病理过程密切相关。
近几年,对LECs的研究逐渐增多,而该细胞的体外扩增培养则成为是研究及应用过程的最基础环节。但是目前研究中,LECs均为原代培养,在分离过程中常有可能混有杂细胞,影响内皮细胞纯度,且细胞仅传了6~8代后就发生分化和凋亡。因此,如何提高LECs生长速度,增加传代次数,维持传代后细胞性状是需要进一步研究及解决的问题。
目前市场上所售内皮培养基配方保密且价格昂贵,均大多在培养血管内皮细胞中具有相对不错的效果。另外,血管内皮细胞与淋巴管内皮细胞有所区别,因此寻找稳定性好,成本低的LECs的培养体系,成为深入研究和应用LECs的必要前提,对于利用LECs体外研究多种疾病显得尤其重要。
发明内容
本发明要解决的技术问题是提供一种淋巴管内皮细胞培养基及其制备方法和用途,能够有效地维持原代淋巴内皮细胞的性状,延长细胞生存时间;培养基中添加的各个组分容易获得,配制方便,降低了培养内皮细胞的成本。
为解决上述技术问题,本发明的实施例提供一种淋巴管内皮细胞培养基,包括基础培养基和添加剂,所述添加剂包括:胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素。
其中,所述的淋巴管内皮细胞培养基采用如下配方:
胎牛血清 50ml;
胰岛素生长因子 20μg;
碱性成纤维细胞生长因子 10μg;
表皮生长因子 5μg;
血管内皮生长因子 0.5μg;
维生素C 1mg;
转铁蛋白 5g;
牛血清白蛋白 10g;
皮质醇 0.2mg;
青霉素 1×105U;
链霉素 100g;
上述组分用基础培养基定容至1L。
优选的,所述基础培养基为1640培养基。
本发明还提供一种淋巴管内皮细胞培养基的制备方法,包括如下步骤:
1)配置1640基础培养基;
2)将所述胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素分别溶解后依次加入步 骤1)中的基础培养基中;
3)在所述步骤2)中配置的培养基中加入所述胎牛血清并混合均匀;
4)将溶液用0.2μm PVDF滤膜过滤除菌,即得。
本发明还提供一种淋巴管内皮细胞培养基的用途,用于淋巴管内皮细胞的培养。
本发明的上述技术方案的有益效果如下:
本发明的细胞培养基有效促进淋巴内皮细胞的增殖。本发明中,各个生长因子及组分的配合添加,能够有效地维持原代淋巴内皮细胞的性状,在体外扩充了8代以后,细胞仍能有较大程度维持原有性状,延长了生存时间。培养基中添加的各个组分容易获得,配制方便,降低了培养内皮细胞的成本。
附图说明
图1为本发明中HLEC细胞形态图;
图2为本发明中HLEC增值率示意图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合具体实施例进行详细描述。
本发明提供了一种淋巴管内皮细胞培养基,包括基础培养基和添加剂,所述添加剂包括:胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素。
所述的淋巴管内皮细胞培养基采用如下配方:
胎牛血清 50ml;
胰岛素生长因子 20μg;
碱性成纤维细胞生长因子 10μg;
表皮生长因子 5μg;
血管内皮生长因子 0.5μg;
维生素C 1mg;
转铁蛋白 5g;
牛血清白蛋白 10g;
皮质醇 0.2mg;
青霉素 1×105U;
链霉素 100g;
上述组分用基础培养基定容至1L。
所述基础培养基为1640培养基。
本发明还提供一种淋巴管内皮细胞培养基的制备方法,包括如下步骤:
1)配置1640基础培养基;
2)将所述胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素分别溶解后依次加入步 骤1)中的基础培养基中;
3)在所述步骤2)中配置的培养基中加入所述胎牛血清并混合均匀;
4)将溶液用0.2μm PVDF滤膜过滤除菌,即得。
本发明提供的淋巴管内皮细胞培养基用于淋巴管内皮细胞的培养。
图1所示为利用本发明制备的淋巴管内皮细胞培养基培养的HLEC细胞形态图;图2所示为利用本发明制备的淋巴管内皮细胞培养基培养的HLEC增值率示意图。
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,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种淋巴管内皮细胞培养基,其特征在于,包括无血清基础培养基和添加剂,所述添加剂包括:胎牛血清、胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素。
2.根据权利要求1所述的淋巴管内皮细胞培养基,其特征在于,采用如下配方:
胎牛血清50~100ml;
胰岛素生长因子 10~30μg;
碱性成纤维细胞生长因子 5~15μg;
表皮生长因子 3~10μg;
血管内皮生长因子 0.2~0.6μg;
维生素C 0.5~1.5mg;
转铁蛋白 3~10g;
牛血清白蛋白 8~25g;
皮质醇 0.1~0.5mg;
青霉素 1×105U;
链霉素 100g;
上述组分用基础培养基定容至1L。
3.根据权利要求1或2所述的淋巴管内皮细胞培养基,其特征在于,所述基础培养基为1640培养基。
4.一种淋巴管内皮细胞培养基的制备方法,其特征在于,包括如下步骤:
1)配置1640基础培养基;
2)将所述胰岛素生长因子、碱性成纤维细胞生长因子、表皮生长因子、血管内皮生长因子、维生素C、转铁蛋白、牛血清白蛋白、皮质醇、青霉素和链霉素分别溶解后依次加入步 骤1)中的基础培养基中;
3)在所述步骤2)中配置的培养基中加入所述胎牛血清并混合均匀;
4)将溶液用0.2μm PVDF滤膜过滤除菌,即得。
5.一种淋巴管内皮细胞培养基的用途,其特征在于,用于淋巴管内皮细胞的培养。
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