Objective: Heat shock proteins (Hsps) are expressed in the cells under unstressed conditions as well as under the stressed conditions induced by heat shock and other physiological stresses. The 27kDa Hsp (Hsp27) has been suggested to work at crucial cellular checkpoints for proliferation, and differentiation, and cell death in vivo. Our previous work demonstrated that Hsp27 regulates acinar cell differentiation in postnatal development of the rat submandibular gland. In this study, we have examined the chronological immunolocalization of Hsp27 in the regenerating acinar cells after release of duct ligation in the rat submandibular gland. Methods: Male Wister rats of 8 weeks old were anesthetized and the right main excretory duct of submandibular gland were ligated by small clips and the left is control. After 1 week, clips were removed to release the duct ligation. Submandibular glands were fixed with 4% paraformaldehyde solution after 0, 1, 3, 5, 7, 10, 14 and 28 days after clip removal. They were treated for immunohistochemistry of Hsp27 and PCNA. Results: In the control and normal submandibular glands, Hsp27-immunoreactivitv was not observed in the acinar and duct cells. Three days after the clip removal, small number of Hsp27-immunopositive cells was found in the distal end of regenerating acinar portion. Hsp27-immunoreacitvity was intensely localized in the cytoplasm of small and round-shaped epithelial cells. Hsp27-positive cells were increased in number until 7 days after clip removal, but no positive cells were recognized in the acini after 14 days of clip removal. Histologically, normal acinar cells were recognized after 14 days. Hsp27-positive epithelial cells were not colocalized with PCNA-immunoreactivity during the acinar regeneration. Conclusion: These results suggest that differentiation of acinar cells occur during 3-7 days after clip removal, and Hsp27 regulates the initial differentiation of acinar cells during submandibular gland regeneration.

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