OBJECTIVE: To determine the link between the tumor necrosis factor induced protein 1(TNFAIP1) and TNF-alpha production in response to the P.gingivalis/LPS, and to investigate the mechanism that regulates the effect of the TNFAIP1 gene expression on TNF-alpha synthesis, using a new, simple, yet effective technique that allowed us to apply our methods on human macrophages.
MATERIALS AND METHODS: We performed several experiments including: A culture of mouse RAW cells and human THP-1 cells in RPMI media supplemented with 10% FBS at 37°C in 5% CO2, Polymerase Chain reaction (PCR) using specifically designed primers and DNA cloning of TNFAIP1, transfection of the cloned TNFAIP1 cDNA into macrophages, Western blot analysis, ELISA analysis after treatment in macrophages, and GFP imaging system.
RESULTS:
1. TNFAIP1 expression can reciprocally affect TNF-α production in macrophages.
2. Blocking MAPK, PI3K and JAK downregulates the TNF-alpha production.
3. The transfection of TNFAIP1 in THP-1 using a 1.5 ml Eppendorf tube generated a high transfection efficiency without the PMA treatment.
4. TNFAIP1 induces Caspase 1 gene expression in macrophages.
5. Caspase 1 induced by TNFAIP1 functions downregulation of TNF-alpha via p73.
CONCLUSIONS:
1. MAP kinase, JAK, or PI3K, may be involved in TNF-α gene expression in LPS-dependent pathway.
2. TNFAIP1 expression can induce TNF-alpha production in mouse macrophages and vice versa.
3. TNFAIP1 gene expression in response to LPS is independent of NFkB-mediated signaling pathway.
4. The animal model has been confirmed by using both mouse macrophage-like cells (RAW cells) and a human macrophage-like cells (THP-1 cells).
5. The signaling pathway for the activation of TNF-alpha production in early stages is: LPS/ TNFAIP1/ PI3Kinase/AP-1/ TNF-alpha.
6. TNFAIP1 acts as a suppressor in later stage to down-regulate TNF-α gene expression via the following signaling pathway: LPS/TNF-alpha/ TNFAIP1/ Caspase 1/ Apoptotic genes/ Degradation of TNF-α/ Cell apoptosis. / 2020-05-23T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/35747 |
| Date | 23 May 2019 |
| Creators | Aljahdali, Bushra Hameed |
| Contributors | Tang, Xiaoren |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0067 seconds