[Detection of keratin mRNA and human origin of xenotransplanted human tracheobronchial epithelium by in situ hybridization].
An in situ hybridization method was applied to detect keratin mRNA expression and human origin of xenotransplanted human tracheobronchial epithelium into nude mice. Tissues from eight tracheas repopulated with cells from five different donors were used. A K6 (56 kd) human keratin cDNA (KA-1) and a K14 (50 kd) cDNA (KB-2) were radiolabeled with 3H-dATP/TTP and used as probes to study changes in keratin mRNA expression in various lesions of the tracheobronchial epithelium. To identify human tissues xenotransplanted into nude mice, high-molecular-weight DNAs extracted from human or mouse tissues were sonicated, nick-translated with 32p-dCTP, and used as probes. Dot-blot hybridization of 32p-labeled DNA probes revealed clear species-specific signals and cells of human origin were distinguishable by in situ hybridization with sonicated human DNA probe. In situ hybridization with either KA-1 or KB-2 probe showed similar localization of silver grains in all histologic types in consecutive tissue lesions of xenotransplanted tracheobronchial epithelia. Very few grains were seen over cells of simple, pseudostratified, or stratified epithelia two to three cell layer thick. Nonkeratinizing stratified hyperplastic epithelia of more than three cell layers showed uniform localization of grains throughout the lesions. In contrast, epidermoid metaplasias exhibit a dense and localized pattern of grains on the basal and parabasal cell layers with a decrease in grain density toward the surface layers. In situ hybridization with sonicated DNA probes provided a powerful tool for verifying the human origin of xenotransplanted tissues in nude mice, showing several advantages, simple preparation of probes, high sensitivity, and applicability to all types of tissues and cells. With KA-1 and KB-2 probes, the different localization pattern of keratin mRNAs in different epithelial lesions were demonstrated. It was of particular interest that an ordered program of keratin mRNA expression proportional to the level of cellular differentiation was observed in epidermoid metaplasias. Both of these probes serve as keratinization markers of human tracheobronchial epithelial lesions.
Obara
T
T
Third Department of Internal Medicine, Asahikawa Medical College, Japan.
D000818:Animals; D001980:Bronchi / Q000637:transplantation*; D015342:DNA Probes; D004848:Epithelium / Q000637:transplantation; D015870:Gene Expression; D006801:Humans; D007633:Keratins / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger; D014132:Trachea / Q000637:transplantation*; D014183:Transplantation, Heterologous
D015342:DNA Probes; D012333:RNA, Messenger; D007633:Keratins