The objective of my study so far has been to establish the cytokines produced by human fibroblasts from healthy gingival tissue.
The specific aims of these studies were to first determine if there is a steady change in cytokine expression by fibroblasts during their passages in vitro. In this study, cytokine expression was assessed in fibroblasts following each of 10 passages. Secondly, we wanted to determine if there is a difference in IL-6 production by different fibroblast cell lines which were obtained from healthy gingival tissue. Also, we want to determine the effect of bacterial lipopolysaccharide (LPS) and cytokines on regulating IL-6 production by both healthy and diseased human gingival fibroblasts (HGF). The HGF cell lines used in these studies are established by an explant technique from healthy gingival tissue from donors undergoing periodontal surgery. HGF cells (passage 5) were grown to confluency and then stimulated with E. coli LPS, Porphyromonas gingivalis LPS, TNF-a, IL-1-a (100 ng/ml), IL-lb, or IL-la + TNF-a for an additional 24 hrs. Following incubation, supernatants were collected and assessed for cytokine production by ELISA. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the ribonucleotide protection assay (RPA). After comparison of constiutive IL-6 production of several different fibroblast cell lines, a time course and dose response study was done to determine the optimal conditions for stimulation of IL-6 production by a healthy gingival fibroblast cell line. HGF cells (passage 4) were grown to confluency and then stimulated with E. coli LPS, Porphyromonas gingivalis LPS, TNF-a, IL-1-a (100 ng/ml), IL-lb, or IL-la + TNF-a at several increasing doses and at five different times of incubation for each of the doses. Following incubation, supernatants were collected and assessed for IL-6 production by ELISA. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 nRNA by the ribonucleotide protection assay (RPA). This same experiment was also done with a second healthy gingival fibroblast cell line using specific doses and a 24 hour incubation time. Similar patterns of IL-6 stimulation were seen for both cell lines. Immunofluorescence of healthy human gingival fibroblast has also been done to look for expression of CD14.
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