Dissertation
Dissertation > Medicine, health > Pharmacy > Pharmacology > Experimental Pharmacology

Experimental Studies on Protective Effects of Ulinastatin on Human Pulmonary Vascular Endothelial Cells in Vitro

Author ZouXianBao
Tutor LiXinYu
School Shihezi University
Course Anesthesiology
Keywords pulmonary vascular endothelial cells acute lung injury severe sepsis nuclear factor-κB tumor necrosis factor-α Interleukin-10 intercellular adhesion molecule -1 vascular endothelial growth factor cell viability transmission electron microscope
CLC R965
Type Master's thesis
Year 2011
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Objective:To provide in vitro experimental evidence for UTI treatment of acute lung injury or acute respiratory distress syndrome induced by sepsis through human pulmonary vascular endothelial cells (PMVECs), through the role of UTI on isolated PMVECs stimulation of severe sepsis serum at different time, then investigating the concentrations of tumor necrosis factor-a (TNF-a),interleukin-10(IL-10), intercellular adhesion molecule1 (ICAM-1), vascular endothelial growth factor (VEGF),the expression of NF-κB on PMVECs, and the ultrastructure of PMVECs.Methods:1、PMVECs were cultured in vitro, randomized into 3 groups, normal group (with 10% fetal bovine serum, group N), health serum group (with 10% healthy human serum, group H), patients’ serum group (with 10% severe sepsis patients’serum, group S).The proliferation of PMVECs were Observed by means of MTT, the expression of NF-κB were Observed by means of immunohistochemistry, the ultrastructure were Observed by means of transmission electron,and the concentrations of TNF-a, IL-10, ICAM-1,and VEGF in culture medium were tested at the phase 0,1,2,4,6h among different groups.2、PMVECs were cultured in vitro, randomed into 6 groups, normal group (with 10% fetal bovine serum, group N), health serum group (with 10% healthy human serum, group H), patients’serum group (with 10% severe sepsis patients’serum, group S), UTI group (accoding to the concentrations of UTI in culture medium, cells were divided into 3 groups, group U1 with 100u/ml UTI and 10% severe sepsis patients’serum, group U2with 500u/ml UTI and 10% severe sepsis patients’serum, group U3with 1000u/ml UTI and 10% severe sepsis patients’serum).The concentrations of TNF-a, IL-10, ICAM-1,and VEGF in culture medium were tested at the phase 0,l,2,4,6h among different groups.The expression of NF-κB in PMVECs by means of immunohistochemistry, optical density values (AOD) were tested at the phase of lhrs among different groups, the ultrastructure were Observed by means of transmission electron at the phase of 6hrs among different groups.Results:1、(1)Different OD values of PMVECs could be seen at different time point among 3 groups (P <0.01),the OD values of each time point PMVECs were different trend (P<0.01). Compared with group N and group N, the OD values in group S remained at low level with time went.(2) The AOD values of group S were highest at the 1 hrs, then the AOD values decreased gradually(0.54±0.06,0.52±0.01,0.49±0.00,0.44±0.03). (3) No differences of tendency changes of TNF-a, IL-10, ICAM-1, VEGF between group N and group H (P>0.05). Compared with group N, the concentration of TNF-a at the phase of lhrs,2hrs in group S were higher than that of groups N(p<0.05), the concentration of TNF-a at the phase of 4hrs,6hrs in group S were higher than that of groups N(p<0.01).Compared with group N, the concentration of IL-10, ICAM-1, VEGF at the phase of 2h,4h,6h in group S were higher than that of groups N(p<0.01). (4) No significant ultra-structure changes can be seen in group N and group H. the outer membrane were rich of microvilli, a large number of round or oval Weibel-Palade bodies (WPBs),and a small amount of multivesicular body in the cytoplasm. Membrane was integrity, microvilli and WPBs bodies was slightly reduced endoplasmic reticulum were seen at the phase of 1h in group S. Membrane Incomplete, microvilli and WPBs significantly reduced, mitochondrial vacuolization of changed, ribosome reduced, gap widend between the cells visible vesicle-like change were seen at the phase of 2h in group S. Membrane ruptured, microvilli and WPBs bodies disappeared, nucleolus asided, heterochromatin agglomerate were seen at the phase of 4h in group S. nuclei irregulared, nuclear vacuolesed were seen at the phase of 6h in group S.2> (1) Tendency changes of TNF-α, IL-10, ICAM-l,and VEGF in culture medium were found different among groups (p<0.01), and differences could be seen among groups (p<0.01). (2) TNF-a in culture medium of group U was significantly lower than that of group S (p<0.01).The lhrs of cultured,TNF-a in culture medium of group U2, U3 was significantly lower than that of group U1 (p<0.05; p<0.01). The 2hrs.4hrs,6hrs of cultured, TNF-a in culture medium of group U was significantly lower than that of group S (p<0.01). The 4 hrs TNF-a in culture medium of group U2, U3 was significantly lower than that of group U1 (p<0.05; p<0.01). The 6hrs TNF-a in culture medium of group U3was significantly lower than that of group U1 (p<0.05).(3) The 4hrs of cultured, IL-10 in culture medium of group U2, U3 was significantly higher than that of group S(p<0.01). The 6hrs of cultured,IL-10 in culture medium of group U2, U3 was significantly higher than that of group S(p<0.05, p<0.01). The 4hrs of cultured,IL-10 in culture medium of group U2, U3 was significantly higher than that of group U1 (p<0.05, p<0.01). The 6hrs of cultured,IL-10 in culture medium of group U3was significantly higher than that of group U1 (p<0.01).(3) The lhrs, 2hrs,4hrs of cultured,ICAM-lin culture medium of goup U2 was significantly lower than that of group S(p<0.05). The 6hrs of cultured,ICAM-1in culture medium of goup U2 was significantly lower than that of group S(p<0.01). The 1hrs,2hrs,4hrs,6hrs of cultured,ICAM-lin culture medium of goup U3 was significantly lower than that of group S(p<0.01). The 1hrs,6hrs of cultured,ICAM-lin culture medium of goup U2 was significantly lower than that of group U1(p<0.05).1 hrs,2hrs,4hrs,6hrs of cultured,ICAM-lin culture medium of goup U3 was significantly lower than that of group U1 (p<0.01).(5) The lhrs,2hrs,4hrs,6hrs of cultured,VEGF in culture medium of goup U2, U3 was significantly lower than that of group S (p<0.01). The 4hrs,6hrs of cultured,VEGF in culture medium of goup U2 was significantly lower than thatof group U) (p<0.05;p<0.01). The lhrs,2hrs,4hrs,6hrs of cultured,VEGF in culture medium of goup U3 was significantly lower than that of group U1 (p<0.01). (6) No nuclear expression of NF-κB can be seen in groupN and group H, a majority of positive nuclei were seen in group S. Among 3 groups, the positive nuclei of group U1 was the highest than that of group U2was higher than that of group U3.(7) AOD in group S was significantly higher than that of group N and group H. AOD in group U2, U3 was significantly lower than that of group S (p<0.01). AOD in group U2, U3was significantly lower than that of group U1 (p<0.01).AOD:From modest to serious:group N,group H, group U3, group U2, group U1, group S.(8) Membrane ruptured, microvilli and WPBs bodies disappeared, important structure of the cytoplasm vanished, nucleus irregulared, the nucleus cavity of the change were seen in group S. Membrane incompleted, microvilli and WPBs bodies disappeared, endoplasmic reticulum and mitochondria expansioned, nuclear chromatin margined were seen in group U1. Membrane intact, microvilli and WPBs bodies reduced slightly, stain lighter nuclear were seen in group U2. Membrane integrity, the outer membrane were rich of microvilli, a large number of round or oval Weibel-Palade bodies (WPBs), multivesicular body increased significantly were seen in group U3.Conclusions:1、The severe sepsis patients’serum could reduce the proliferation activity of human pulmonary vascular endothelial cell, and lead to the changes of submicroscopic structure of PMVECs.2、UTI could down-regulate the expression of NF-κB of PMVECs stimulate by severe sepsis serum.3、UTI could reduce the levels of TNF-a, ICAM-1, VEGF and increase the levels of IL-10 from PMVECs culture medium, which were caused by severe sepsis.4、UTI could lighten the effects which were caused by severe sepsis on the submicrostructures of PMVECs.

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