鼓膜上皮干细胞的分布和培养

Epidermal stem cells in the tympanic membrane

目的 确定上皮干细胞在鼓膜上的分布 ,鼓膜穿孔后干细胞的变化 ,鼓膜上皮不同部位的体外培养生长特点 ,建立鼓膜干细胞体外培养和增殖的方法。方法 4只幼年Sprague Dawley (SD)大鼠和 4只成年SD大鼠 ,均为雌性 ,作正常鼓膜观察 ,2 8只成年雌性大鼠作鼓膜紧张部中央 2mm穿孔。于穿孔后不同时间取鼓膜冰冻切片 ,进行 β1整合素和角蛋白 19免疫组化检测。 15只成年雌性SD大鼠 30个鼓膜 ,体外用丝裂霉素C损伤黏膜面 ,分成鼓环上皮和紧张部中央上皮两部分 ,比较鼓环和紧张部细胞倍增时间。以 1h为标准 ,把鼓环细胞按贴壁时间分类。结果 角蛋白 19和β1整合素阳性细胞在紧张部分布于鼓环和锤骨柄区域 ,在松弛部散在分布 ,在紧张部中央无阳性细胞。幼年鼠和成年鼠无差别。紧张部急性穿孔后鼓环和锤骨柄阳性细胞数量增加 ,穿孔边缘无阳性染色。体外培养鼓环上皮细胞倍增时间 (5 1 1± 0 73)h明显短于紧张部中央上皮 (92 87± 5 6 5 )h。 1h内贴壁的细胞 ,生长活跃 ,产生较大的集落 ,富含上皮干细胞。结论 上皮干细胞在鼓膜紧张部分布于鼓环和锤骨柄区域 ,在紧张部的中央没有干细胞。在适当的培养液中 ,干细胞增殖良好 ,并可以通过贴壁时间进行初步的纯化。

Objective To investigate the distributions of epithelial stem cells in the tympanic membrane and the growth characteristics of cultured epithelial cells from different region of tympanic membrane, and to establish culture techniques of stem cells in tympanic membrane. Methods Four young rats and four adult SD rats were used to observe normal tympanic membrane. The other 28 rats were performed 2 mm size perforations in pars tensa. These animals were sacrificed at different periods after perforation. The tympanic membranes were cut in cryostat sections for immunohistochemistry of cytokeratin 19 and integrin β1 Thirty tympanic membranes of rats were treated with Mitomycin C to damage the mucosal surface, then divided into two parts: the annulus region and center region of pars tensa, cultured in medium with high amount of epidermal growth factor and low amount of calcium. Results The immunostaining cells of cytokeratin 19 and integrin β1 were displayed in both the handle of malleus and annular regions, but there were no staining positive cells in the intermediate region of pars tensa. The positive cells distribution had no significant difference between adult and infancy rats. In the pars flaccida, the positive cells scattered in the basal layer. The positive cells increased after perforation in the annulus and handle of malleus region,but no immunostaining cells were found at the edge of perforation. The redouble time of the culture cells from the annulus region was shorter than the center of pars tensa. The cells adherent within 1 hour formed larger and more colonies, and contained more positive cells. Conclusion The epithelial stem cells in tympanic membrane were located in both the handle of malleus and annular regions, but no stem cells could be found in the intermediate region of pars tensa. The stem cells of tympanic membrane can be simply purified according to the adherent time.