KGF increases SP-A and SP-D mRNA levels and secretion in cultured rat alveolar type II cells
KGF increases SP-A and SP-D mRNA levels and secretion in cultured rat alveolar type II cells
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DOI：
10.1165/ajrcmb.18.2.2824
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发表时间：
1998-02-01
影响因子：
6.4
通讯作者：
Mason, RJ
中科院分区：
文献类型：
--
作者：
Xu, XS;McCormick-Shannon, K;Mason, RJ
肺泡II型细胞分泌表面活性蛋白的研究受到限制，因为这些蛋白的基因表达在原代培养中迅速下降。我们开发了一种培养系统，以调查肺泡II型细胞的脂质和蛋白质分泌的调节和参与这些过程的基因。将大鼠II型细胞接种在用鼠尾胶原包被的膜插入物上，在含有10%胎牛血清（FBS）的培养基中培养1天，然后更换为含有5 ng/ml角质形成细胞生长因子（KGF）和2%血清的培养基培养3天，并更换为含有5% Engelbreth-Holm-Swarm肿瘤基质（EHS）但不含血清的培养基培养2天。从此时起，将细胞置于摇摆平台上，并在四种不同的无血清培养基中在sir-液体界面（A/L）的顶面上用0.4ml培养基培养：基础Dulbecco改良Eagle培养基（DMEM）/F12培养基（DF 12），基础培养基加EHS（DF 12/EHS）、基础培养基加KGF（DF 12/KGF）和基础培养基加EHS和KGF（DF 12/EHS/KGF）。在DF 12和DF 12/EHS中培养的细胞呈现变细、扁平的形态，而在DF 12/KGF和DF 12/EHS/KGF中培养的细胞更立方形，含有许多板层体，并且具有顶端微绒毛。在DF 12和DF 12/EHS中培养的细胞产生表面活性蛋白mRNA（分别为表面活性蛋白[SP]-A、SP-B、SP-C和SP-D）的相对弱的信号，并且SP-A和SP-D的分泌保持低水平。与此相反，在A/L下培养3 d并在KGF存在下培养的细胞显示SP-A、SP-B和SP-D mRNA的强信号，并将SP-A、SP-D和溶菌酶分泌到顶端培养基中。12-O-十四酰基-佛波醇-11-乙酸酯（TPA）和特布他林联合刺激[H-3]磷脂酰胆碱（[H-3]PC）、SP-A和溶菌酶的分泌，但不刺激SP-D。这种原代培养系统应证明是有用的SP-A，SP-D，和表面活性剂脂质的分泌机制的研究。
Studies of secretion of surfactant proteins by alveolar type II cells have been limited because the expression of the genes for these proteins decreases rapidly in primary culture. We developed a culture system to investigate the regulation of lipid and protein secretion by alveolar type II cells and the genes involved in these processes. Rat type II cells were plated an membrane inserts coated with rat-tail collagen in medium containing 10% fetal bovine serum (FBS) for 1 d before being changed to medium containing 5 ng/ml keratinocyte growth factor (KGF) and 2% serum for 3 d and to medium with 5% Engelbreth-Holm-Swarm tumor matrix (EHS) but without serum for 2 d. From this time forward, the cells were placed on a rocking platform and cultured with 0.4 ml medium on the apical surface at the sir-liquid interface (A/L) in four different, serum-free media: basal Dulbecco's modified Eagle's medium (DMEM)/F12 medium (DF12), basal medium plus EHS (DF12/EHS), basal medium plus KGF (DF12/KGF), and basal medium plus EHS and KGF (DF12/EHS/KGF). Cells cultured in DF12 and DF12/EHS assumed an attenuated, flattened morphology, whereas those in DF12/KGF and DF12/EHS/KGF were more cuboidal, contained numerous lamellar bodies, and had apical microvilli. Cells cultured in DF12 and DF12/EHS produced a relatively weak signal for the surfactant protein mRNAs (surfactant proteins [SP]-A, SP-B, SP-C, and SP-D, respectively), and secretion of SP-A and SP-D remained low. In contrast, cells maintained for 3 d at A/L and cultured in the presence of KGF showed strong signals for SP-A, SP-B, and SP-D mRNAs, and secreted SP-A, SP-D, and lysozyme into the apical medium. The combination of 12-O-tetradecanoyl-phorbol-11-acetate (TPA) and terbutaline stimulated secretion of [H-3]phosphatidylcholine ([H-3]PC), SP-A, and lysozyme, but not SP-D. This primary culture system should prove useful for mechanistic studies of the secretion of SP-A, SP-D, and surfactant lipids.