title: [Investigation of rat submandibular gland cells and antheraea pernyi silk fibroin films co-culture in vitro].
abstract: OBJECTIVE
  In this study, the morphology, attachment, growth and secretion of rat submandibular gland cells (RSMGs) cultured on the antheraea pernyi silk fibroin(ApSF) films were observed in vitro.
  
  
  METHODS
  RSMGs were seeded on ApSF scaffolds and bombyx mori silk fibroin (BmSF) scaffolds respectively. RSMGs which were seeded on tissue culture plastic were treated as a negative control group. With the method of immunocytochemistry (CK8 and amylase), the phenotype of cells were identified. The co-cultivation of RSMGs and two kinds of scaffolds were observed by using SEM and fluorescence microscopy. The adherence rate of cells attaching to each experimental material was estimated. MTT assay was performed to determine the proliferation of RSMGs cultured on different scaffolds. The secretion function of cultured cells was evaluated by assay of amylase activity. All the data was analyzed by SPSS13.0 software package.
  
  
  RESULTS
  The immunohistochemical staining showed that the cultured epithelial cells of RSMGs were positive for the specific antibody of CK8 and the acinar cells were positive for the specific antibody of amylase. SEM showed that the cultured cells with microvillus anchored well to ApSF films and extended pseudopods to the scaffolds. Fluorescence microscopy showed that with the extension of incubation time, the amount of cells that attached to scaffolds increased. The attachment of RSMGs on ApSF films was almost the same as that on BmSF films after 1 h culture(P>0.05). The adherence rate of RSMGs on ApSF films was higher than that on BmSF films (post-seeding 4,8,12 h, P<0.05). There was no significant difference between ApSF group and BmSF group after 24 h culture (P>0.05), Proliferation of RSMGs cultured on ApSF films for 3 to 5 days was remarkable and that of RSMGs cultured on ApSF films for 7 days reached peak. The proliferation rate of RSMGs cultured on ApSF scaffolds was higher than that on BmSF scaffolds (post-seeding 3,5,7 d, P<0.05), ApSF group and BmSF group were higher than the negative control group (P<0.05). Amylase content of RSMGs cultured on ApSF films was higher than that on BmSF films (post-seeding 3,5,7 d, P<0.05) , ApSF group and BmSF group were higher than the negative control group (P<0.05).
  
  
  CONCLUSIONS
  ApSF films can support RSMGs attachment, growth, secretion function maintenance and phenotypic maintenance; and has better biocompatibility for RSMGs in vitro culture.
authors.affiliation: Department of Oral Anatomy and Physiology, School of Stomatology, China Medical University, Shenyang 110002, Liaoning Province, China. liuyan5124047@163.com
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Xue-xin
authors.initials: XX
authors.lastname: Tan
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Yi
authors.forename: Li-ping
authors.initials: LP
authors.lastname: Liu
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D005349:Fibroins; D066298:In Vitro Techniques; D009036:Moths; D051381:Rats; D047011:Silk; D013363:Submandibular Gland; D023822:Tissue Engineering
chemical_list: D047011:Silk; D005349:Fibroins