Study On Human Umbilical Vein Endothelial Cell Invasion Mechanism Of Vibrio Vulnificus

Background:Vibrio vulnificus (Vibrio vulnificus, Vv) is the fifth group of Vibrio bacteria which is gram-stain negative, curved, rod-shaped, pleomorphic, activist and with capsules halophilic. Mainly grows inside the bodies of fritillaria and fish in seawater . It can be divided into three kinds of biological type (Ⅰ,Ⅱ,Ⅲ) while type I is the major pathogens of human wound infections and primary septicemia. It usually infected through an open wound contact with bacteria-containing seawater or raw seafood. So Vibrio vulnificus infection is not only an important issue in trauma medicine at sea but also plays a significant role in the hygiene of seafood. Vv infection can cause serious human infections and sepsis. It also caused multiple organ dysfunction syndrome which is the leading cause of death in patients . The mortality rate of Vv infection is more than 50%. Vibrio vulnificus septicemia sepsis is mainly cause by a large number of Vibrio vulnificus invade into the blood while vascular endothelial cell is the first barrier to resist Vibrio vulnificus entering the blood stream . To reveal the pathogenicity of Vibrio vulnificus against the blood vessel and clarify the influence of the basic form of human vascular endothelial cell and biological behavior juring Vibrio vulnificus infection, we conducted this study for clinical diagnosis and treatment of Vibrio vulnificus infection. Methods:The standard strains of Vibrio vulnificus is ATCC27562 which is purchased from the Chinese Academy of Sciences Institute of Microbiology General Microbiology collections management center. Gram stained and observed under light microscopy after recovery culture. Then crossed inoculated on thiosulfate-citrate-bile salts-sucrose agar (thiosulfate-cifrae-bile salt-sucrose, TCBS) agar plate and general nutrition to observe the colony morphology. Finally, cultured the Vv in biochemical identification tube and biochemical strip, machine-readable identification of the results after 24 hours.First, observe the general characteristics of Vibrio vulnificus and the effects on vascular endothelial cell:(1) Human umbilical vein endothelial cells was provides by the Guangzhou General Hospital of Guangzhou Military Region. Immunofluorescence and factor VIII identification on it. Through the previous experimental results of our research group ^ we have defined the growth curve of vascular endothelial cell through the 96-well plate culture experiments and MTT method after the attack of Vibrio vulnificus. We also use cell scratch experiment to measure the influence of cell migration after attack by different concentrations of Vibrio vulnificus. (2) Trypan blue staining was used to detect the cell survival immediately and after 24h after invasion. (3) Flow cytometry was used to determine the influence of the apoptosis rate after different concentration Vv attack on the HU cell. The highest Vv attack experimental concentration is 103cfu /ml. (4) Using hoechest 33342 fluorescent staining to detect the change of HU nucleus and (5) plate colony forming assay to detect the change of cell colony-forming ability. (6) Using 50 high magnification views to detect the separatist number of the normal cells and cells after invasion. Observing the changes in the biological behavior of cells under anti-concentration limit. (7) Measuring the stereological parameters of the cell area (Ac), the perimeter (Cc), the nucleus area (An), cytoplasmic area (Acp) of nuclear cytoplasm ratio (Rnp) and cell and nuclear's long axis (dmaxc, dmaxn), cell and nuclear minor axis (dminc, dminn), axial ratio (Rac, Ran), cell and nuclear shape factor PE (PEc, PEn), cell and nuclear shape factor AR (ARc, ARn), the planning cell and nuclear shape factor RFF (RFFc, RFFn) and irregular shape index of FII (FⅡic, FⅡn) in normal group and Vv attack group (104cf/ml) to test proliferation (8) Morphological changes of the human umbilical vein endothelial cells under the attack of Vv.Second:Vibrio vulnificus endotoxin extraction, identification, as well as affect on vascular endothelial cells.To take fresh culture of Vibrio vulnificus and use phenol-water to extract endotoxin then centrifugation it. Ues TAL to identify endotoxin according to the 2005-published "Chinese Pharmacopoeia" and draw the concentration curve.Select the median lethal concentration of intestinal bacteria endotoxin to HU cells. Select the domain of its upper and lower concentration of total 4 concentrations on the human umbilical vein endothelial cells for MTT, plate cloning and scratches to determine the migration changes of the cells. Use flow cytometry to determine the apoptosis rate. Measuring the stereological parameters of the cell area (Ac), perimeter (Cc), the nucleus area (An), circumference (Cn), cell volume (V), cell volume density (Vv), surface density (Sv) and numerical density (Nv) in normal group and endotoxin attack group (104EU/ml) to test proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of endotoxin and judge the damage of HU.Third:Cytolytic toxin of Vibrio vulnificus identification and determination of the concentration and its effects on vascular endothelial cells.Our research team has been pre-recombinanted pET-32a-vvhA/E.coliBL21 (DE3) and for blue-white screening. We select the white plaque for PCR amplification of Vibrio vulnificus cytolysin toxin (rVVC). We also use SDS-PAGE for verification and bicichoninicacid (BCA) to quantitative protein, then render the standard curve and calculate protein concentration.50mg/kg injected into the SPF level BALB/c mice. Execute the mouse and then anatomy immediately. Take the main damage organs and tissues to produced pathological slice and observe it under light microscope.Select the median lethal concentration of cytolytic toxin to HU cells. Select the domain of its upper and lower concentration of total 4 concentrations on the human umbilical vein endothelial cells for MTT, plate cloning and scratches to determine the migration changes of the cells. Use flow cytometry to determine the apoptosis rate. Measuring the stereological parameters of the cell area (Ac), perimeter (Cc), the nucleus area (An), circumference (Cn), cell volume (V), cell volume density (Vv), surface density (Sv) and numerical density (Nv) in normal group and cytolytic toxin attack group (3EU/ml) to test proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of cytolytic toxin and judge the damage of HU.Result:Vibrio vulnificus ATCC27562 grows in TCBS plate green colonies, shiny and shows a typical mucous colony. It also appears yellow white translucent colony mucus in ordinary nutrient agar plates. Biochemical Identification Results:The Vibrio vulnificus%id (coincidence rate) is 98probablitity.First:The proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of Vv(1) Through 96 plate culture method and the MTT method to detect the cell growth curve and the determination of absorbance of vascular endothelial under the attack of different concentrations of Vibrio vulnificus invasion. The cells growth inhibition rates after 24h of 10/ml group,102cfu/ml group,103cfu/ml group and 104 cfu/ml were:(10.27±7.17)%, (16.13±14.33)%, (26.01±6.45)%, (37.46±5.06) % and after 48h were:(13.12±8.60)%, (26.63±5.42)%, (34.20±7.92)%, (42.63±3.58)%while after 72h were:(16.50±5.92)%, (28.93±5.48)%, (38.93±4.94)%, (49.62±20.35)% and after 96h were:(25.44±8.15)%, (36.41±15.31)%, (45.37±14.51)%, (52.8U10.51)%. (2) Trypan blue staining method detects the cell survival rate immediately and after 24h after invasion of different concentration. The immediately survival rates were 100%,100%,99.48%, 99.38%,98.55% and the 24h cell survival rates were 100%,96.90%,88.48%,76.78%, 57.65%. (3) Flow cytometry detect result shows the apoptosis rate in Vv attack group is significantly higher than the control group. (4) Hoechest 33342 fluorescent staining of HU nucleus shows the endothelial cell nuclei of blank control group appears uniform blue fluorescence. The cells in low concentration group 10cfu/ml begins to apoptosis 24h after Vv attack while the 104cfu/ml group has the most apoptotic cells and appears round or oval-shaped bright blue fluorescence. We can see a clear chromatin condensation. (5) Colony formation assay cloning efficiency is 63.73% in the control group and 39.60% in the 10/ml group.37.86% decrease compared with Control group. The 104cfu/ml group is 15.53%,91.17% decrease compared with Control group and 60.79% decrease compared with 10cfu/ml group. (6) Under 50 high-power field of vision the division of nuclear in normal cells were 1.48±0.182 and the 104cfu/ml group was 3.02±0.638. (7) The cell parameters of the control group and the Vv attack group whose concentration is 104cfu/ml were as follows:long-axis(μm) are 36.17±14.34 and 30.24±8.21, short-axis(μm) are 15.64±7.32 and 22.37±5.73, cell area(μm2) are 431.26±296.37 and 537.64±239.285, cell circumference (μm) are 82.20±26.10 and 132.69±30.66, axial ratio are 2.61±1.19 and 1.39±0.38, PE are 0.33±0.11 and 0.44±0.13, AR are 0.90±0.12 and 0.97±0.05, RFF are 0.64±0.12 and 0.66±0.10, FII are 1.80±0.30 and 1.56±0.28, nuclear area(μm2) are 133.70±26.23 and 143.07±27.85, cytoplasmic area (μm2) are 312.72±268.04 and 371.34±237.87, nuclear cytoplasm ratio are 0.25±0.10 and 0.75±0.55. (8)The human umbilical vein endothelial cells in normal group appear spindle, cytoplasm stained pink, round or oval nucleus and clear nucleolus. But the cells in Vv attack group appear unclear structure, lightly stained cytoplasm, small vacuoles formed, light nuclear staining, nucleolar fragmentation.Second:Vibrio vulnificus endotoxin extraction, identification, as well as affect on vascular endothelial cells.Detect the extracted endotoxin under a single wavelength of 405nm. According to the standard curve lgT= y-xlgC, (T is the color, C is the concentration), the concentration of the LPS is 20434EU/ml.(1)Through MTT and pre-test we determine the cell growth curve and absorbance of HU under the attack of LPS with different concentrations. The inhibition rate in groups of 2500EU/ml,5000EU/ml,7500EU/ml and 10000EU/ml after 24hwere (1.554±0.409)%, (4.762±0.690)%, (4.028±0.743)%and (47.440±4.716)%. After 48h were (1.262±0.494)%, (2.893±1.166)%, (15.498±6.786)% and (59.591±5.696)%. After 72h were (1.459±0.458)%, (6.649±0.588)%, (14.074±4.088)% and (48.525±2.159)% while after 96h were (4.248±1.312)%, (8.977±2..089)%, (22.929±3.696)% and (22.929±3.696)%. (2) Colony formation assay cloning efficiency is 72.73% in the control group and 40.13% in the 104EU/ml group. 44.82% decrease compared with Control group. (3) We use FCM to detect the affect of LPS attack over HU and the result shows the apoptosis rate in LPS attack group is higher than control group. (4)The cell parameters of the control group and the LPS group whose final concentration is 104EU/ml were as follows:An(μm2) are 135.80±26.37 and 150.43±32.69, Cn(μm) are 32.00±6.08 and 70.67±13.87, Ac(μm2) are 415.43±72.00 and 442.26±69.74, Cc(μm) are 141.06±30.75 and 133.10±17.55, cytoplasmic area(μm2) are 93.55±20.31 and 303.50±61.33, nuclear cytoplasm ratio are 0.44±0.09 and 0.65±0.15, Vv are 0.35±0.44 and 0.30±0.04, Nv are 0.00016±0.000042 and 0.00014±0.000045, Sv are 39.05±8.21 and 81.93±16.17.Third:Cytolytic toxin of Vibrio vulnificus identification and determination of the concentration and its effects on vascular endothelial cells.Put the pre-synthesized exotoxin of Vibrio vulnificus under SDS-PAGE electrophoresis to verify the fusion protein rVVC. It shows protein bands on 70KD of the gelatin (Vibrio vulnificus cytolysin toxin molecular weight of about 50KD, carrier protein is about 20KD). According to the standard protein concentration and its corresponding absorbance values we obtained the standard calculated the corresponding concentration of the protein samples is 4000.282ug/ml. Observing of pathological results under optical microscope we can see there is hemorrhagic injury in the lung of mouse. We can also find part of spotty or sheet necrosis abscess-like change in the liver. But there is no injury in other organs.(1)Through MTT and pre-test we determine the cell growth curve and absorbance of HU under the attack of VVC with different concentrations. The growth inhibition rates in 2EU/ml group,2.5EU/ml group,3EU/ml group and 3.5EU/ml group after 24h were (3.31±5.61)%, (3.61±8.22)%, (50.31±2.82)% and (55.11±4.52)%. After 48h were (3.22±14.21)%, (3.74±7.91)%, (50.72±1.52)% and (58.71±50.71)%. After 72h were(6.33±1.91)%, (9.14±10.72)%, (54.23±5.71)% and (55.74±2.13)% while after 96h were (4.21±4.23)%, (5.92±11.82)%, (50.11±0.7)% and (54.33±0.9)%. (2)Colony formation assay cloning efficiency is 67.87% in the control group and 33.47% in the 3EU/ml group.52.10% decrease compared with Control group. (3)We use FCM to detect the affect of WC attack over HU and the result shows the apoptosis rate in VVC attack group is higher than control group. The normal cells rate in control group,3EU/ml group and 3.5EU/ml group were 97.43%, 47.98% and 35.07% and the apoptosis index were 1.77%,51.21% and 61.30%. (4)The cell parameters of the control group and the WC group whose concentration is 3EU/ml were as follows:Nucleus area (μm2) are 135.80±26.37 and 147.77±40.62, nuclear perimeter(μm) are 32.00±6.08 and 91.99±18.30, cell area(μm2) are 415.43±72.00 and 240.80±77.77, cell circumference (μm) are 141.06±30.75 and 125.83±26.77, cytoplasmic area (μm2) are 93.55±20.31 and 70.62±30.11, the nuclear cytoplasm ratio are 0.42±0.08 and 2.40±3.59, bulk density are 0.35±0.04 and 0.66±0.16, number density are 0.00016±0.000042 and 0.00029±0.00013, surface density are 39.05±8.21 and 214.19±62.76.Conclusion:All Vibrio vulnificus, Vibrio vulnificus lipopolysaccharide (LPS) and cytolytic toxin of Vibrio vulnificus (VVC) have significantly inhibition on proliferation of human umbilical vein endothelial cell line (HU). Its inhibitory ability has something to do with the concentration and action time of Vibrio vulnificus. It also has something to do with the concentration of LPS and WC. And all of them go through the change of the structure of vascular endothelial cells as well as the injury through the vessel wall into the bloodstream then cause severe infection and sepsis.