口腔黏膜干细胞治疗口腔白斑的实验研究

Abstract:.Objective: To study the isolation, culture, appraisal and purification of the stem cells in the epithelial and mesenchymal of oral normal and leukoplakia. mucous membrane. Comparing the biological characteristics of Oral mucosa stem cells. To research the stem cells from oral mucosa tissue for the therapy of leukoplakia..Methods: Clinical experimental tissues are from the oral normal and leukoplakia. mucous membrane of the patients, which are after the full inspection suitable for operation and clear diagnosis of the biopsy. A single cell suspension is aquaired by enzymatic digestion and filtration. A limited dilution method is used to culture primary Oral mucosa stem cells isolated,which is expanded by cloning training and detected the expression. MTT assay the proliferation capacity of OMSC. Oral mucosa stem cells will be induced to the differentiation of osteogenesis, fat and neural, respectively. Real-Time PCR and immunofluorescence test are used to identify the expression of related markers at genes and proteins level. Transmission electron microscope(TEM) detect the ultrastructure of the OMSC before and after differentiation...According to reports in the literature, dimethyl benzanthracene (DMBA) induce the golden hamaters to set up the animal model of leukoplakia. Pathology confirme the model to be successful built. Oral mucous membrane stem cells are taken into the animal model of leukoplakia according to certain concentration. Histopathology examine the effectiveness after the treatment.

目的：比较体外分离、培养、.鉴定、纯化的正常口腔黏膜干细胞（Oral mucosa stem cells，OMSC）和白斑上皮及间充质干细胞的生物学特性，研究在口腔白斑动物模型中应用干细胞治疗口腔白斑的可行性。方法：体外实验组织均取自白斑患者和粘液囊肿旁的正常口腔黏膜。制备单细胞悬液，培养分离的原代口腔黏膜上皮和间充质干细胞，并进行克隆化培养，检测干细胞表面标记的表达。MTT法检测所得干细胞的增殖能力。将OMSC分别接种于成骨、成脂、成神经诱导液，检测干细胞的多向分化潜能；Real-Time PCR法和免疫荧光法检测相关标记在基因和蛋白水平的表达。透射电镜观察诱导前后细胞超微结构。同上法制备金黄地鼠OMSC；并建立DMBA诱导金黄地鼠的口腔白斑动物模型。注射一定浓度同种同体（鼠）或异种异体（人）OMSC到动物口腔白斑病损中。从肉眼、镜下和分子水平观察、分析实验白斑的变化及其影响因素。

口腔白斑是公认最典型的癌前病变, 其细胞的生物学行为处于不稳定状态病变能够继续发展最终癌变，目前的研究多关注于上皮细胞的病变，对白斑中间充质干细胞的变化的研究较少，本次研究从需要活检手术的口腔白斑患者的口腔中获得病变组织，正常组织来自于黏液腺囊肿旁的口腔黏膜，阳性对照为骨髓间充质干细胞。采用酶消化及过滤法得到单细胞悬液，有限稀释法培养分离的原代口腔黏膜固有层的干细胞，并进行克隆化培养，其克隆形成和增殖能力增强。采用流式细胞技术鉴定正常口腔黏膜和口腔白斑间充质干细胞，发现其同骨髓间充质干细胞一样，出现间充质干细胞标志物STRO-1, CD105, CD90阳性表达，造血干细胞CD34阴性表达。正常口腔黏膜和白斑的间充质干细胞均具有成骨、成脂和成神经多向分化能力。但是口腔白斑间充质干细胞的多向分化能力降低。本次研究结果为口腔白斑间充质干细胞在疾病中显示出功能性的损伤，这为探究口腔黏膜间充质干细胞在白斑中的作用、以及其对白斑癌变或转归的影响奠定了一定基础。