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Cell Journal، جلد ۱۶، شماره Suppl ۱، صفحات ۵۹-۵۹
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عنوان انگلیسی Ps-27: Construction of A Recombinant Eukaryotic Expression Vector Containing Human Toll-Like Receptor 4 (TLR4) and MD2 Genes and Its Expression in HEK Cells, as A Whole-Cellular Reporter to Monitor The Inflammatory Condition of Cells
چکیده انگلیسی مقاله Objective: Toll-like receptor 4 (TLR4) is a transmembrane pattern-recognition receptor (PRR) that initiates signals in response to diverse pathogen-associated molecular patterns (PAMPs), especially LPS. The key roles of toll-like receptor 4 (TLR4) as a mediator of the detection and responses of immune cells to invading pathogens are well known. Until recently, TLRs have been examined predominantly for their contribution to immune-related disorders. However, cumulative evidence suggests that TLR4 not only contributes to pathophysiology, but also plays a vital role in facilitating neurodegenerative conditions. Recently, there has been an increasing number of studies about the role of TLRs in the pathogenesis of brain disorders such as ischemic stroke, Alzheimer’s disease and multiple sclerosis, as well as the therapeutic potential of TLR intervention in such diseases. However, most of the evidence for the role of TLR4 is observational and little is known of the molecular mechanism. In the current study, to assess the molecular mechanisms of inflammation in a cell reporter moed we have constructed a recombinant eukaryotic vector expressing the TLR4 and MD2 encoding sequences. By implementing aforementioned vector we have established a stably transformed human embryonic kidney (HEK) cell line for studying the molecular events behind the inflammation. Materials and Methods: Using genetic engineering approaches, we successfully constructed a recombinant pBudCE4.1 (+) containing CDS of TLR4 and MD2. This vector was transfected into the HEK cells. The stable transformants were isolated by addition of pouromycin. Resistant cells were isolated for further experiments. Results: Expression of MD2 and TLR4 was confirmed in isolated stable cells. Moreover the functional activity assessment using LPS indicated increased expression of TNFα, iNOS and TLR4 in these cells. These results indicate that transfection of expression vector encoding human TLR4 and MD2 into the HEK cells is sufficient for the activation of the TLR4 by LPS. Conclusion: We have established a stable cell line appropriate for study of inflammation. This system is approved to be feasible for future studies especially as a cell model to chase the inflammation processes.
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/353
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