小鼠下颌下腺细胞培养及其生物学特性研究

Research on mouse submandibular gland cell culture and the biocharacteristics

目的建立和完善小鼠下颌下腺细胞培养方法,并进行生物学特性研究,为涎腺疾病治疗奠定理论基础,并为组织工程涎腺再生提供技术支持。方法无菌环境下切取小鼠下颌下腺,胶原酶消化,接种于含有胎牛血清、表皮生长因子、胰岛素、氢化可的松及转铁蛋白的低糖DMEM培养基中培养,倒置相差显微镜及HE染色进行形态学观察,透射电镜观察特异性酶原颗粒,生长曲线评估细胞生长特性,特异性α-淀粉酶免疫荧光鉴定细胞来源。结果下颌下腺原代细胞为圆形或多边形,呈铺路石样排列,其生长缓慢,12 d左右汇合至80%。传代细胞与原代形态相同。HE染色显示胞核胞浆对比明显。生长曲线大致呈"S"型,与其他细胞增殖特点相似。特异性α-淀粉酶免疫荧光染色阳性,表明细胞为具有功能的涎腺细胞。结论成功地建立了小鼠下颌下腺细胞原代及传代培养,将为涎腺疾病的治疗和涎腺再生提供实验基础。

Objective To establish and improve the cell culture methods of mouse submandibular glands and to study their biocharacteristics so as to provide theoretical basis for the treatment of salivary gland diseases and technique support for the tissue engineering regeneration of salivary glands. Methods The submandibular glands were removed from mice in a sterile environment;after digested with collagenase,the cell suspension was incubated in DMEM containing fetal bovine serum,epidermal growth factor,insulin, hydrocortisone and transferrin;inverted phase contrast microscope and HP staining were applied to the observation of the morphology of the cultured cells and the special zymogen granule was observed under transmission electron microscope;the growth feature was estimated by growth curve;immunofluorescence staining of special α-amylase was conducted for the determination of the origin of the culture cells. Results The cells of primary culture were round or polygon in the arrangement of typical“flagstone”appearance,the growth was slow and 80% came to a confluence within 12 days;the passage cells were similar to primary cells in morphology;HE staining showed that the contrast between nucleus and cytoplasm was obvious;transmission electron microscope picture showed the special zymogen granule. The growth curve shaped like“S”and the growth characteristics were similar with those of other kinds of cells;the cells were positive with immunofluorescence staining of special α-amylase,which suggested that the cultured cells were functional salivary gland cells. Conclusions The model of primary culture and subculture of mouse salivary gland cells is successfully established;it provides an experimental base for the treatment of salivary gland diseases and the regeneration of salivary glands.