Primary cell culture of human type II pneumonocytes: Maintenance of a differentiated phenotype and transfection with recombinant adenoviruses
Primary cell culture of human type II pneumonocytes: Maintenance of a differentiated phenotype and transfection with recombinant adenoviruses
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DOI：
10.1165/ajrcmb.17.6.2858
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发表时间：
1997-12-01
影响因子：
6.4
通讯作者：
Mendelson, CR
中科院分区：
文献类型：
--
作者：
Alcorn, JL;Smith, ME;Mendelson, CR
由于缺乏维持II型细胞表型特性的细胞培养系统，表面活性剂脂蛋白代谢和分泌以及表面活性剂蛋白基因表达的调节研究受到阻碍。我们已经开发了一种原代培养系统，该系统有利于维持II型肺细胞的一些形态学和生化特性长达2周。通过胶原酶消化在双丁酰环腺苷酸（Bt（2）cAMP）存在下在器官培养物中维持5天的妊娠中期人胎肺组织来分离细胞。通过用DEAE-葡聚糖处理，将分离的细胞富集上皮成分，铺在来源于Madin-Darby犬肾（MDCK）细胞的细胞外基质（ECM）上，并在空气/液体界面处在含有Bt（2）cAMP的最少量培养基中孵育。细胞培养物由含有大量致密嗜锇颗粒的圆形上皮样细胞岛组成，具有板层体外观，这是II型肺细胞的显著特征。此外，培养物保持SP-A基因表达水平升高长达2周。糖皮质激素和环腺苷酸以类似于在器官培养的胎肺组织中观察到的方式调节培养细胞中编码SP-A、SP-B和SP-C的mRNA的表达。当细胞在空气/液体界面培养时，分化表型最明显。为了利用培养的II型细胞研究各种蛋白质过表达的影响和用于启动子分析，重要的是将DNA构建体高效地转染到这些细胞中。不幸的是，我们发现细胞对使用常规方法的DNA有效转移是难治的（即，脂质转染、电穿孔或磷酸钙介导的转染）。然而，发现复制缺陷型重组人腺病毒提供了将DNA引入II型肺细胞的高效手段。此外，我们观察到在II型细胞富集培养物感染的重组腺病毒含有lacZ基因的控制下的巨细胞病毒启动子，β-半乳糖苷酶均匀地表达在岛的II型细胞和周围的成纤维细胞。相反，在用含有在SP-A基因启动子和5 ′-侧翼区控制下的人生长激素（hGH）基因的重组腺病毒感染的培养物中，hGH仅在II型细胞中表达。因此，该培养系统提供了鉴定介导II型细胞特异性基因表达的基因组元件的极好手段。
Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained, We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt(2)cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt(2)cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type Il cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type Il cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type Il cells, Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.