鼠颊黏膜干细胞的分离及鉴定

Isolation, culture and identification of rat buccal mucosa stem cells

目的探讨鼠颊黏膜干细胞的体外分离和培养方法，以获得稳定生长的黏膜干细胞。方法DispaseⅡ酶和Trypsin．EDTA两步法分离昆明小鼠颊黏膜上皮细胞，接种于以小鼠成纤维细胞NIH3T3细胞为滋养层的培养板中。达尔伯克氏必需基本培养基（Dulbecco’s minimum essential medium，DMEM）培养24h后换用角质细胞无血清培养基（keratinocyte serum—free medium，K-SFM）继续培养，部分细胞72h后行成骨、成脂诱导和改良型Kaplow碱性磷酸酶（alkaline phosphatase，ALP）染色鉴定；部分细胞继续培养5d后，消化转移至Ⅳ型胶原预包被的培养板中，加入黏膜干细胞培养基培养，观察细胞克隆形成和成骨、成脂诱导、兔抗鼠角蛋白和ALP染色结果。结果初次分离的上皮细胞群83．96％处于G0或G1期，培养72h后可见克隆样生长细胞，成骨、成脂诱导和ALP染色均呈阳性；细胞在Ⅳ型胶原包被的培养板上快速贴壁，呈圆形或卵圆形，胞体小，折光性强，克隆样生长，兔抗鼠角蛋白、ALP染色和成骨、成脂诱导均呈阳性。结论运用滋养层细胞培养法获得大量干细胞样上皮细胞后，Ⅳ型胶原黏附结合K-SFM与NIH3T3培养上清液混合培养法可实现鼠颊黏膜干细胞的初步纯化和快速培养。

Objective To explore a method for isolation and cultui＇e Dispase Ⅱ and Trypsin-EDTA. Cells were seeded onto mitomycin C-treated of rat buccal mucosa stem cells and to identify the stem cells. Methods Epithelial cell mass were obtained by digesting rat buccal mucosa with 3T3 Swiss albino layer and cultured in DMEM for 24 hours, followed by K-SFM culturing. Some cells were induced to osteocytes and adipocytes and underwent ALP testing after 72 hours. Five days later, the primary cells were digested with trypsin and inoculated onto collagen Ⅳ-coated flasks and cultured at room temperature for 20 minutes. The adherent cells continued to be cultured with epithelial stem cell medium, then examined for identifying the clones, osteocytes, adipocytes, cytokeratin and ALP staining. Results 83. 96 percent of the primary epithelial cell mass were in G0/G1 phase by flow cytometry test. The clones were seen after 72 hours on 3T3 Swiss albino layer, and the osteocytes and adipocytes were positive. Cells were adhered quickly to collagen IV, in a shape of round or orbicular-ovate with strong refraction. The induced-osteocyte and adipocyte, cytokeratin and ALP were all positive. Conclusions The stem cell-like epithelial cells could be obtain using the 3T3 Swiss albino layer method. Sieved by collagen IV and cultured in epithelial stem cell medium could make the epithelial stem cells depurate and proliferate quickly.