东方田鼠皮肤成纤维细胞的分离培养及生物学特性

目的探索和建立东方田鼠皮肤成纤维细胞体外分离、培养的技术方法并观察其生物学特性。方法采用含10%小牛血清的Dulbecco改良Eagle培养液(DMEM)和1640两种培养体系,运用组织块贴壁法和胰酶消化法,分别对出生后1、3 d和5 d的东方田鼠乳鼠皮肤成纤维细胞进行原代分离、培养。苏木素-伊红染色及倒置相差显微镜下观察成纤维细胞形态和生长特性。结果 0.25%胰酶消化分离出生后1 d和3 d东方田鼠乳鼠皮肤较出生后5 d组织可获得较多数量细胞,成纤维细胞在体外快速贴壁生长,一般6～7 d长满培养瓶,细胞纯度高,HE染色细胞呈长梭形,胞核明显;DMEM和1640两种培养液均可用于东方田鼠皮肤成纤维细胞的培养,但细胞传代后生长趋缓,只可传代2～3次。本实验运用组织块贴壁法未能培养出皮肤成纤维细胞。结论确定了有效分离东方田鼠皮肤成纤维细胞的日龄和方法,为进一步深入研究提供了技术方法和操作依据。

Isolation,culture and characterization of skin fibroblasts from Microtus fortis

Objective The aim of this study was to isolate,culture,and characterize the skin fibroblasts from Microtus fortis.Methods The primary skin fibroblasts were isolated from Microtus fortis at different ages by trypsin digestion and tissue adherence methods.The growth behavior and the characteristics of Microtus fortis skin fibroblasts（MfSF）cultured in DMEM and 1640 culture medium were studied,respectively.Results The number of cells isolated from 1-3-day old animals was more profit than that from 5-day-old Microtus fortis,isolated by trypsin digestion,and the cell purity was high.The MfSF grew well and attached to the bottle wall with a high purity and good ability of proliferation in the primary passage in DMEM or 1640 culture medium,and the time of confluence was 6-7 days,but the growth became gradually poor in the subsequent passages.HE staining showed that the MfSF had an oval nucleus.It was unsuccessful to isolate and culture MfSF by tissue adherence method in this study.Conclusion A successful and efficient method of isolation and culture of skin fibroblasts from Microtus fortis has been established.It provides a useful method and protocol to obtain MfSF to serve further studies.