human immortalized oral epithelial cell lines  (ATCC)

Journal: Journal of Dental Research

Article Title: Expression and Possible Immune-regulatory Function of Ghrelin in Oral Epithelium

doi: 10.1177/0022034511420431

Figure Lengend Snippet: Concentrations of ghrelin and IL-8 in saliva and GCF. (A) Concentrations of ghrelin protein in GCF and saliva. (B) Concentrations of IL-8 protein in GCF and saliva. The protein levels of IL-8 and the levels of ghrelin in saliva and GCF from 12 healthy volunteers were determined by ELISA. *The concentrations of ghrelin in GCF are significantly different from those in saliva (Student’s t test: p < 0.05).

Article Snippet: Cell Lines and Synthetic Ghrelin Peptides Immortalized human oral epithelial cell lines RT7 and OBA9 were cultured in Keratinocytes-SFM (Invitrogen, Carlsbad, CA, USA), as described previously ( Hosokawa et al ., 2006 ; Ohta et al ., 2008 ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Dental Research

Article Title: Expression and Possible Immune-regulatory Function of Ghrelin in Oral Epithelium

doi: 10.1177/0022034511420431

Figure Lengend Snippet: Expression of ghrelin in oral epithelium. (A) Ghrelin mRNA expression in RT7, OBA9, and OKF6/TERT-2 epithelial cell lines. Total RNA was isolated from each cell line at a confluent culture, after which RT-PCR was performed for ghrelin and β-actin. Human leukemic cell lines HL-60 and THP-1, which were reported to express ghrelin mRNA, were used as positive controls (De Vriese and Delporte, 2007). (B) Ghrelin mRNA expression in human buccal mucosal cells from three healthy volunteers (one man, aged 40 yrs, and two women, ages 35 and 31 yrs). The samples were obtained from healthy individuals after they provided informed consent (Spivack et al., 2004). Total RNA was isolated from each participant, after which RT-PCR was performed for ghrelin and β-actin. (C) Ghrelin immunostaining in gingival epithelium. Periodontal tissues collected from healthy human participants were stained with polyclonal rabbit IgG anti-ghrelin antibodies (a) and control non-immunized rabbit IgG antibodies (b). The gingival epithelium shown in (C) was isolated from the cervical epithelium that faces the inner side of gingival crevice. Original magnification, 200x.

Article Snippet: Cell Lines and Synthetic Ghrelin Peptides Immortalized human oral epithelial cell lines RT7 and OBA9 were cultured in Keratinocytes-SFM (Invitrogen, Carlsbad, CA, USA), as described previously ( Hosokawa et al ., 2006 ; Ohta et al ., 2008 ).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Staining, Control

Journal: Journal of Dental Research

Article Title: Expression and Possible Immune-regulatory Function of Ghrelin in Oral Epithelium

doi: 10.1177/0022034511420431

Figure Lengend Snippet: The effects of ghrelin on intracellular calcium and cAMP levels. (A) Confluent OBA9 cells in 96-well plates were exposed to various concentrations of ghrelin or scrambled ghrelin for 1 hr. Intracellular calcium mobilization was measured by means of a Fluo-8 no-wash calcium assay kit. Relative fluorescence intensity ratios are presented as the mean ± SD of 3 independent experiments relative to that of buffer alone. The ratios among the various concentrations of ghrelin showed statistical differences (ANOVA, p < 0.01). *Significantly different from non-treated cells (Student’s t test: p < 0.05). N.D.: no significant difference. (B) Confluent OBA9 in 6-well plates were exposed to various concentrations of ghrelin or scrambled ghrelin for 2 hrs. Medium was removed, and cells underwent lysis in 0.1 M HCl. Intracellular cAMP levels were determined by means of a cAMP assay kit. The cAMP levels among the various concentrations of ghrelin showed statistical differences (ANOVA, p < 0.01). Data are shown as the mean ± standard deviation of 3 independent experiments. *Significantly different from non-treated cells (Student’s t test: p < 0.05). N.D.: no significant difference.

Article Snippet: Cell Lines and Synthetic Ghrelin Peptides Immortalized human oral epithelial cell lines RT7 and OBA9 were cultured in Keratinocytes-SFM (Invitrogen, Carlsbad, CA, USA), as described previously ( Hosokawa et al ., 2006 ; Ohta et al ., 2008 ).

Techniques: Calcium Assay, Fluorescence, Lysis, cAMP Assay, Standard Deviation

Journal: Journal of Dental Research

Article Title: Expression and Possible Immune-regulatory Function of Ghrelin in Oral Epithelium

doi: 10.1177/0022034511420431

Figure Lengend Snippet: Effects of ghrelin on IL-8 expression from TNF-α- or LPS-stimulated oral epithelial cells. RT7 and OBA9 cells were cultured for 48 hrs in the presence or absence of (A) TNF-α (10 ng/mL), (B) E. coli LPS (50 µg/mL), or (C) P. gingivalis LPS (50 µg/mL) with different concentrations of ghrelin, after which the level of IL-8 in the culture supernatant was measured by ELISA. Data are shown as the mean ± SD of 3 independent experiments. *Significantly different from TNF-α or LPS alone (Student’s t test: p < 0.05). (D) OBA9 cells were stimulated with TNF-α (10 ng/mL), E. coli LPS (50 µg/mL), or P. gingivalis LPS (50 µg/mL) for 48 hrs in the presence or absence of scrambled ghrelin (10 µg/mL). The level of IL-8 in culture supernatant was measured by ELISA. Data are shown as the mean ± SD of 3 independent experiments. N.D.: no significant difference.

Article Snippet: Cell Lines and Synthetic Ghrelin Peptides Immortalized human oral epithelial cell lines RT7 and OBA9 were cultured in Keratinocytes-SFM (Invitrogen, Carlsbad, CA, USA), as described previously ( Hosokawa et al ., 2006 ; Ohta et al ., 2008 ).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay