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Assessing Internalized Porphyromonas gingivalis in Host Endothelial Cells

作者：

简介：

Overview

This study investigates the interaction between Porphyromonas gingivalis and endothelial cells, focusing on bacterial internalization and survival. The methodology involves assessing the ability of the bacteria to invade and survive within host cells through a series of controlled experiments.

Key Study Components

Area of Science

Microbiology
Infectious Diseases
Cell Biology

Background

Porphyromonas gingivalis is an anaerobic bacterium associated with periodontal disease.
The fimbriae of these bacteria play a crucial role in binding to host cell receptors.
Understanding bacterial survival within host cells is essential for developing therapeutic strategies.
Endothelial cells are significant in the study of bacterial infections due to their role in vascular health.

Purpose of Study

To evaluate the internalization of Porphyromonas gingivalis by endothelial cells.
To determine the survival rate of the bacteria post-internalization.
To establish a reliable method for assessing bacterial invasion in vitro.

Methods Used

Infection of endothelial cells with Porphyromonas gingivalis suspension.
Washing cells with anaerobic buffer to remove non-internalized bacteria.
Using antibiotics to eliminate surface-attached bacteria.
Collecting lysates and plating on blood agar for colony counting.

Main Results

Successful internalization of Porphyromonas gingivalis within endothelial cells was observed.
Colony counts indicated the survival of bacteria after internalization.
The methodology allowed for effective assessment of bacterial invasion.
Results contribute to understanding the pathogenicity of Porphyromonas gingivalis.

Conclusions

Porphyromonas gingivalis can invade and survive within endothelial cells.
The study provides insights into bacterial-host interactions.
Findings may inform future research on therapeutic interventions for periodontal disease.

Frequently Asked Questions

What is Porphyromonas gingivalis?

Porphyromonas gingivalis is an anaerobic bacterium linked to periodontal disease.

How does Porphyromonas gingivalis invade host cells?

It binds to specific receptors on endothelial cells, initiating internalization.

What methods are used to assess bacterial survival?

Colony counting on blood agar plates after lysing endothelial cells.

Why is studying endothelial cells important?

Endothelial cells are crucial for understanding vascular health and infection dynamics.

What are the implications of this research?

It may lead to better therapeutic strategies for managing periodontal disease.

During Porphyromonas gingivalis infection, the fimbriae on the anaerobic bacteria bind to specific receptors on host cells.

This binding initiates intracellular signaling cascades, resulting in actin rearrangement and bacterial internalization within phagosomes.

To assess the survival of internalized Porphyromonas gingivalis within endothelial cells in vitro, take a multi-well plate containing endothelial cells adhered to coverslips.

Add Porphyromonas gingivalis suspension. Incubate to facilitate bacterial internalization.

Remove media and wash using an anaerobic buffer to remove non-internalized bacteria.

Add media containing antibiotics, impermeable to the endothelial cells, to eliminate the surface-attached bacteria.

Incubate with a mild surfactant to lyse the endothelial cells, releasing the bacteria without causing their lysis.

Dilute the collected lysate with media. Prepare serial dilutions of the lysate.

Plate the dilutions on blood agar plates. Incubate under anaerobic conditions for Porphyromonas gingivalis to grow and form visible colonies.

Count the colonies to assess the ability of Porphyromonas gingivalis to invade and survive within endothelial cells.

Following infection of HUVEC cells, wash the cells three times with anaerobic PBS. Next, add 2 milliliters of VEGF medium supplemented with pre-optimized concentrations of antibiotics to the HUVEC cells. Incubate the cells for one hour. Aspirate the medium, and add 2 milliliters of 1% saponin solution in BHI to each well. Incubate for 15 minutes.

Next, scrape the lysed cells, and collect the lysate in a centrifuge tube. Dilute the lysate 1 to 1 with BHI medium, and then prepare serial dilutions starting at 1 to 100. Plate 200 microliters of the suitable concentrations on blood agar plates. Allow the bacteria to grow for seven days in an anaerobic chamber, and then count the colonies.

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