大鼠肺泡Ⅱ型上皮细胞的原代分离

Primary separation of rat alveolar epithelial cells type Ⅱ

目的建立大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial cells typeⅡ,AECⅡ)分离、纯化、原代培养及鉴定的方法。方法用4.2U/ml的弹性蛋白酶通过气管插管注入肺泡内,消化分离AECⅡ。把细胞悬液接种到包被有大鼠IgG的塑料平皿中纯化细胞。用电镜、碱性磷酸酶显色法、改良巴氏染色法、单宁酸染色法、免疫组化染色法鉴定AECⅡ。结果细胞纯度达到90%以上,倒置显微镜下可见细胞呈岛屿状生长。电镜下可见细胞内有大量板层小体,包膜上有绒毛结构。碱性磷酸酶染色法(BCIP/NBT)可见胞浆内有蓝色颗粒。改良巴氏染色法、单宁酸染色法可见胞浆内有黑色颗粒。抗大鼠肺泡表面活性蛋白A(surfactant protein A,SP-A)免疫组化染色呈阳性反应。结论弹性蛋白酶作用温和,不损伤胞膜,分离所得细胞活力好;IgG免疫粘附纯化法操作简单,纯化效率高。电镜、BCIP/NBT、巴氏染色、单宁酸染色及免疫组化染色等鉴定方法稳定可靠,特异性高。

Objective To establish a stable method for isolation, purification, primary culture and identification of alveolar epithelial cells type Ⅱ （AEC Ⅱ ）. Methods The lungs were instilled with 4.2U/ml elastinase solution via the tracheal cannula to isolate AEC Ⅱ cells from the lung tissue . The cell suspensionwas seeded on a rat IgG-coated Petri dish to purify AEC Ⅱ. The primary culture cells were identified by electron microscopy, improved papanicolaou staining, tannic acid staining, alkaline phosphatase （BCIP/ NBT）staining and immunohistochemical staining. Results The primary cultures presented the island-like growth feature under the inverted microscope. The lamellar bodies were observed by electron microscopy, and they were presented as blue or black granules in the cytoplasm by BCIP/NBT staining, or improved papanicolaou staining and tannic acid staining respectively. The cells reacted specifically to surfactant protein A antibody with immunohistochemieal staining as well. Conclusion Elastinase functions gently and keeps the cell membrane intact during the separation process, so the gained cells have good viability. Immunological attachment with rat IgG can efficiently increase the purity of AECⅡ from cell suspensions. All of the above used assays are stable and specific to identify isolated AEC Ⅱ.