摘要
背景:体外构建组织工程化人工涎腺需获得分化增殖良好、数量充足的种子细胞,但正常下颌下腺中很难分离出成体干细胞。目的:利用腺体组织损伤动物模型体外分离下颌下腺干/祖细胞,并进行克隆化培养。设计、时间和地点:细胞学体外观察,于2006-03/2007-01在遵义医学院贵州省细胞工程实验室完成。材料:8周龄雄性SD大鼠10只,由解放军第三军医大学动物中心提供。方法:10只大鼠采用结扎下颌下腺主导管、抑制腺体分泌来建立组织损伤模型,1周后切取腺体组织,酶消化法体外分离下颌下腺干/祖细胞,原代培养10～14d后,挑取培养皿中形成的小的类圆形、类上皮细胞集落予以纯化,传代后进行单克隆培养。主要观察指标:下颌下腺干/祖细胞免疫细胞化学染色及免疫荧光染色结果,通过绘制生长曲线分析下颌下腺干/祖细胞体外增殖能力。结果:实验获得表达层粘蛋白的细胞具有干细胞特征,CD29呈阳性表达表明其具有高黏附、高增殖等组织干细胞特性,角蛋白19的阳性表达提示下颌下腺干/祖细胞呈上皮源性。其生长曲线近似"S"形,体外培养增殖活跃。结论:实验结果显示,下颌下腺干/祖细胞具有组织干细胞的特征,有望成为组织工程化人工涎腺构建的一类种子细胞来源。
BACKGROUND：Seed cells with good proliferation and enough amounts are need in reconstructing artificial salivary gland in vitro.However,adult stem cells are difficult to be isolated from normal submandibular gland.OBJECTIVE：To in vitro isolate submandibular gland stem/progenitor cells for cloning culture using animal models of damaged gland tissue.DESIGN,TIME AND SETTING：Cytological in vitro experiment was performed at the Guizhou Provincial Key Laboratory of Cell Tissue Engineering,Zunyi Medical College from March 2006 to January 2007.MATERIALS：A total of 10 male Sprague Dawley rats aged 8 weeks were supplied by the Animal Center,Third Military Medical University of Chinese PLA.METHODS：The model of tissue damaged submandibular gland in 10 rats was made by deligation.One week later,the gland tissue was obtained to harvest submandibular gland stem/progenitor cells by enzyme digestion in vitro.Following 10-14 days of primary culture,small round cells were collected,purified and subcultured for monoclonal culture.MAIN OUTCOME MEASURES：Immunocytochemical staining and immunofluorescence staining results were measured in submandibular gland stem/progenitor cells.Growth curve was drawn to analyze the proliferation of submandibular gland stem/progenitor cells in vitro.RESULTS：Cells expressing laminin showed stem cell characteristics.Positive expression of CD29 suggested high-adherent and high-proliferative stem cell properties.Positive expression of keratin-19 indicated epithelium-derived submandibular gland stem/progenitor cells.Growth curve was near to ＂S＂ shape,and in vitro culture and proliferation was active.CONCLUSION：Submandibular gland stem/progenitor cells had the characteristics of tissue stem cells.They might be as a kind of seed cells for tissue engineered artificial salivary gland in further research.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第49期9765-9768,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
贵州省优秀科技教育人才省长资金[黔省专合字(2008)113号]
贵州省教育厅基金[(2006)354号]~~
