Change Of Endoplasmic Reticulum Molecular Chaperone In The Process Of Escherichia Coli Invasion To Human Brain Microvascular Endothelial Cells

ObjectiveThe blood - brain barrier ( BBB) is formed by the complex tight junctions between the endothelial cells of the brain capillaries. This structure results in the capillary wall that behaves as a continuous lipid bi - layer controlling the access of solutes and toxic substances to the central nervous system ( CNS) so as to maintain brain homeostasis. So, study of human brain microvascular endothelial cell ( HBMEC ) will lead us to understand the regulative mechanism of BBB homeostasis, and pathogenesis of some CNS disorders.Escherichia coli K1 is the most common gram - negative pathogen of neonatal meningitis. Invasion of HBMEC is essential to the crossing over BBB by E. coli Kl.And the invasion process of E. coli to HBMEC is a severe stress process. To explore the mechanisms of E. coli Kl invasion. So we use HBMEC, the main content of BBB, as the model, we investigated the role and the change of Endoplasmic Reticulum Molecular Chaperone in the process of E. coli invasion to HBMEC , and, to know the mechanism in the process of the invasion, we investigated the change of vinculin, a cytoskeletal protein that plays an adherence role in the invasion, hoping to accumulate some scientific materials for the mechanism of the invasion to the HBMEC by E. coli and the function of BBB.Methods1. detection of GRP78 GRP94 mRNA by RT - PCR during the process of the invasion of E. coli to the HBMEC.2. detection of GRP78 GRP94 protein by Western Blot during the process of the invasion of E. coli to the HBMEC.3. detection the distribution of GRP78 GRP94 by immunofluorescence during the process of the invasion of E. coli to the HBMEC.4. detection of vinculin protein by Western Blot during the process of the invasion of E. coli to the HBMEC.Results1. Basal expressions of GRP78 GRP94 are detected in HBMEC.2. Levels of both mRNA and protein of GRP78 of HBMEC in pre - invasion and post - invasion are of non - difference.3. Levels of both mRNA and protein of GRP94 of HBMEC in pre - invasion and post - invasion are of evident difference. Levels in post - invasion are far lower than in pre - invasion.4. Distribution and content of GRP78 of HBMEC in pre - invasion and post - invasion are of non - difference by means of immunofluorescence. While Distribution and content of GRP94 of HBMEC in pre - invasion and post - invasion are of evident difference by means of immunofluorescence. The content is decreased with the invading duration and the distribution is focusing on the circumference of the cell.5. Levels of protein of vinculin of HBMEC in pre - invasion and post - invasion are of evident difference. Levels in post - invasion are far higher than in pre - invasion.Conclusion1. GRP94 involves in the invasion of E. coli to HBMEC. And its mechanism is likely to be relevant to the expression of vinculin.2. GRP78 does not involve in the invasion of E. coli to HBMEC.