腺体组织损伤后体外分离下颌下腺干/祖细胞后的克隆化培养

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10-14 d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.

Cloning culture of submandibular gland stem/progenitor cells in vitro isolated from damaged gland tissue

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 Gu.izhou 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.