Dataset Information

Expression of c-kit and Cytokeratin 5 in the Submandibular Gland after Release of Long-Term Ligation of the Main Excretory Duct in Mice.

ABSTRACT: Long-term submandibular duct obstruction is thought to cause irreversible atrophy and dysfunction of the submandibular gland. As an atrophic gland may be induced clinically by chronic or recurrent infection, it is generally removed surgically. However, the regenerative capacity of atrophic submandibular glands after long-term obstruction is not completely understood. We studied the regenerative capacity of the mouse submandibular duct using an aneurysm clip ligation model. We documented changes in the size, histologic structure and gene expression of the submandibular gland after 2 months of ligation, and 1 week, 1 month and 2 months after clip removal. Two months of ligation caused atrophy, particularly in the acinar portion. In the 2 months after clip removal, we observed a steady and significant increase in the expression of the acinar cell precursor gene cytokeratin 5 (CK5), and a significant decrease in the expression of the stem cell marker c-kit. These findings suggest that the submandibular gland retains some capacity for regeneration even after long-term obstruction, and that CK5 could serve as a marker of this regenerative process.

SUBMITTER: Watanabe H

PROVIDER: S-EPMC5519490 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Expression of c-kit and Cytokeratin 5 in the Submandibular Gland after Release of Long-Term Ligation of the Main Excretory Duct in Mice.

Watanabe Hiroshi H Takahashi Haruka H Hata-Kawakami Miyuki M Tanaka Akira A

Acta histochemica et cytochemica 20170622 3

Long-term submandibular duct obstruction is thought to cause irreversible atrophy and dysfunction of the submandibular gland. As an atrophic gland may be induced clinically by chronic or recurrent infection, it is generally removed surgically. However, the regenerative capacity of atrophic submandibular glands after long-term obstruction is not completely understood. We studied the regenerative capacity of the mouse submandibular duct using an aneurysm clip ligation model. We documented changes ...[more]

PMID: 28744028

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Project description:BACKGROUND:Organ replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown. METHODS:Isolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin-eosin staining, Alcian blue-periodic acid-Schiff staining, and Masson's trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo. RESULTS:The isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland-specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (-) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion. CONCLUSION:FGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.

Project description:Male Sprague-Dawley rats weighing 250-300 g were purchased from Japan SLC Co. (Shizuoka, Japan). The animals were housed under a daily controlled 12-h light and 12-h dark cycle at 23 °C with free access to rat chow (Japan SLC Co.) and water for 1 week prior to the experiments. Rats were laparotomized under ether anesthesia in each experiment, and the common bile duct of each rat was ligated between the liver hilus and small intestine. Control animals underwent a sham operation with exposure but without ligation of the common bile duct. After the CBDL or sham operation, animals were allowed access to food and water ad libitum. Groups of three rats were sacrificed under ether anesthesia at 1, 3 and 12 hr after the CBDL or sham operation. At the time of sacrifice, the right lateral liver lobe was removed and flash-frozen in liquid nitrogen and stored at -80 °C. Total RNA isolated from frozen livers using TRIzol reagent (Invitrogen Co., Carlsbad, CA) was mixed to minimize variation among animals. Poly(A) RNA was purified using Oligotex-dT30 (Takara Shuzo Co., Ltd., Kyoto, Japan) in accordance with manufacturer’s instructions. Fluorescence-labeled probes were prepared by reverse transcription using a superscript II reverse transcriptase (Invitrogen Co.) and cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has undergone CBDL and from those that has undergone a sham operation were labeled with cyanine-3 and cyanine-5, respectively, and vise varsa. Fluorescence-labeled probes were purified using a MinElute PCR Products Purification Kit (Quiagen GmbH, Hilden, Germany) in accordance with manufacturer’s instructions. Each purified probe was suspended in hybridization buffer containing 1.6 mg/mL poly(A) (Roche Diagnostics, Basel, Switzerland) and yeast tRNA (Roche Diagnostics), 0.67 mg/mL herring sperm, 16% 20 x SSC and 0.3% SDS and applied to a cDNA microarray containing 1,800 rat genes on a slide glass (Asahi Technoglass Co., Tokyo, Japan). Three house-keeping genes (GAPDH, HPRT and b-actin) and rat unrelated traits (Lambda DNA) were also spotted on the slide as internal positive and negative controls, respectively. Hybridization was carried out twice to eliminate any dye bias. In one experiment, duplicate slides were hybridized with probes derived from CBDL and control rats that had been labeld with cyanine-3 and cyanine-5, respectively. In a replicate experiment, other duplicate slides were hybridized with probes derived from CBDL rats and control rats that had been labeled with cyanine-5 and cyanine-3, respectively (color swap). Then, the slides were cover-slipped and incubated in a sealed chamber (Asahi Technoglass Co.) for 16 hrs under a humidified (65 °C) condition. After being washed in low-stringency buffer (2 x SSC and 0.1% SDS), high-stringency buffer (0.2 x SSC and 0.1% SDS) and 0.2 x SSC and then rinsing with 99.5% ethanol, the slide was dried by centrifugation at low speed and used for scanning. Fluorescence was scanned by using a ScanArray 4000 (Packard BioChip Technologies, Billerica, MA) and quantified by using QuantArray Software (Packard BioChip Technologies, Billerica, MA). Keywords: time-course

Dataset Information

Expression of c-kit and Cytokeratin 5 in the Submandibular Gland after Release of Long-Term Ligation of the Main Excretory Duct in Mice.

Publications

Expression of c-kit and Cytokeratin 5 in the Submandibular Gland after Release of Long-Term Ligation of the Main Excretory Duct in Mice.

Similar Datasets

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