| BackgroundWith the development of social economy and the improvement of liviing standards.the prevalence of thoracicAortic dissection(TAD)has been increasing year After year.The structural and mechanical properties ofAortic wall have been changed during the development of TAD,which makes the medial layer easier to lear and form true and false lumens.The proliferation and migration of Vascular Smooth Muscle Cell(VSMC).an important component of the mesangium,and the alteration of its cytoskeleton structure arc one of the important pathological bases,but the molecular mechanism remains to be elucidated.Platelet-derived growth factor(PDGF)is an important mitogen.which can induce Vascular Smooth Muscle Cells to migrate from the mesangium to the intima.PDGF is one of the main factors causing many vascular diseases,including TAD.It is also the strongest chemoattractant of vascular smooth muscle cells in vitro.Studies have shown that a certain dose of ultrasound irradiation can inhibit the proliferation and migration of VSMC,and it has been confirmed that ultrasound cavitation effect is the basis of its role,and its specific mechanism is not clear.Although ultrasound microbubbles have shown their important value and potential application prospects in many fields such as gene transfection,drug release,open cell harrier and cancer therapy,few studies have focused on the effects of cavitation on cell biological behavior.Therefore,exploring the effects of ultrasound microbubbles on the biological behavior of VSMC,such as proliferation and migration,and their molecular mechanisms will provide new ideas for the basic research on the occurrence and development of TAD,which is of great significance for the prevention and treatment of TAD.Part Ⅰ:The effects of Ultrasound Microbubbles on the proliferation and migration of Human Aortic Vascular Smooth Muscle Cells and the most significant parametersObjectiveIn this research,Human Aortic Vascular Smoooth Muscle was cultured in vitro to investigate the effects of ultrasound irradiation alone and combined with microbubble contrast agent on the PDGF-BB-mediated proliferation and migration of HA-VSMC,and to explore the most significant parameters affecting the proliferation and migration of HA-VSMC.MethodsHuman Aortic Vascular Smooth Muscle were cultured in vitro.The migration ability of HA-VSMC was detected by Transwell chamber,the proliferation ability of HA-VSM(was detected by CCK-8 test Wc treated HA-VSMCs in the following two ways.Firstly,HA-VSMCs were exposed to 1MHz continuous waves ultrasound for 20s,30s,60s,90s,120s,150sAt intensity of 0.35 W/cm2,0.50W/cm2,0.75w/cm2,1W/cm2 with or without microbubbles.Secondly,the migration of HA-VSMCs were detected by Transwell chamber zapparatus and the proliferation of HA-VSMCs were detected by CCK-8 Test.SPSS-22.0 Was used to analyze all the data.The measurement data were expressed as(x-±s).One-way ANOVA was used to compare the results of proliferation,migration and cell proliferation cycle.Dunnett-t test was used to compare the results of each group with those of the control group.P<0.05 showed significant difference,P<0.01 showed very significant difference.Results1.PDGF-BBstimulate the migration off HA-VSMC.2.Ulti*asound sonication for 90sAt intensity of 0.75W/cm2 And 1W/cm2 without microbubbles inhibited the migration of HA-VSMC mediated by PDGF-BB(P<0.05).while migration of HA-VSMC mediated by PDGF-BB were inhibited by the sonication for just 60s at intensit of 0.75W/cm2 and 1W/cm2 with microbubbles(P<0.01).As the time prolonged,the extent of these effects increased too,the greatesl effect of inhibiting migration,was attained at 1W/cm2,150s(P<0.01).3.PDGF-BB stimulate the proliferation of HA-VSMC.when HA-VSMCs Are irradiated by ultrasound without microbubbles,except for the conditions of 0.5W/cm2 irradiation for 20s and 120s.the other conditions could significantly inhibit the proliferation of HA-VSMC mediated by PDGF-BB(P<0.01),and the strongest inhibitory effect on the proliferation of VSMC was observed at the conditions of 0.75 W/cm2 and 90s(P<0.01);When combined with microbubbles,the condition of no significant inhibition on HA-VSMC was 0.5W/cm2 irradiation for 20s,30s And 150s.Similarly,other conditions significantly inhibited the proliferation of HA-VSMC(P<0.01).and the strongest inhibitory effect was observed at 0.75W/cm2 and 60s(P<0.01).Conclusion:Ultrasound irradiation and combined ultrasound contrast agent irradiation can inhibit the migration of HA-VSMC and increase with the prolongation of irradiation time.The migration inhibition of HA-VSMC was the most obvious under the conditions of 1W/cm2 and 150s,and the degree of inhibition in contrast group was greater than that in ultrasound group.At the same time,the effect of contrast agent combined with ultrasound irradiation on migration ability of HA-VSMC was better than that of ultrasound irradiation alone.Ultrasound irradiation with or without echo-ontrast agent can inhibit the proliferation of HA-VSMC.Under the conditions of 0.75W/cm2,90s and(0.75 W/cm2,60s,ultrasound combined with or without microbubbles had the most obvious effect on the prolifcration of HA-VSMC.Under most conditions,the proliferation of f HA-VSMC was more signifcantly affected by ultrasound irradiation combined with microbubbles at the same inensity and the duration of irradiation.Part Ⅱ:The effects of Ultrasound Microbubbles on the proliferation and migration of Human Aortic Vascular Smooth Muscle Cells and its mechanismOb.jectiveIn this research,Human Aortie Vascular Smooth Muscle Cells was cultured in vitro to iiIvestigate the effects of ultrasound irradiation(1W/em2、150s)and combined with mierobubble contrast agent on the PDGF-BB-mediated proliferation and migration of HA-VSMC,and to explored its relationship with PI3/Akt pathxway and cvtoskeleton.MethodsHuman Aortic Vascular Smooth Muscle Ceils(HA-VSMCs)were cultured in vitro.HA-VSMC was irradiated with ultrasound(1MHz,1W/cm2)with or without microbubbles for 150sto observe the corresponding changes;the migration ability of HA-VSMC was detected by Transwell chamber,the proliferation ability of HA-VSMC was cdetected by CCK-8 test,cell proliferation cycle was measured by flow cytometry,and effects of PDGF-BB on HA-VSMC proliferation and migration ability were observed;the expressions of PI3,P-PI3,Akt and P-Akt were measured by Western blot test,respectively;The cytoskeleton of HA-VSMC was obscervced by laser searnning confocal microscopy.SPSS-22.0 was used to analyze all the data.The measurement data were expressed as(x±s).One-way ANOVA was used to compare the results of proliferation,migration and cell proliferation cycle.Dunnett-t test was used to compare the results of each group with those of the control group.P<0.05 showed signillcant difference,P<0.01 showed very significant difference.ResultsPDGF-BB can promote the proliferation and migration of HA-VSMC.Continuous wave ultrasound irradiation(1W/cm2、150s)with or without microbubbles could inhibit the proliferation and migration of HA-VSMC and increase the proportion of G0-G1 phase(P<0.01).After microbubbles treatment,the expression of P-PI3 and P-Akt in HA-VSMC decreased significantlyConclusion:Ultrasound Microbubbles can inhibit the proliferation and migration of HA-VSMC and enlarge the proportion of G0-G1 phase(P<0.01).The molecular mechanism of Ultrasound microbubbles is related to the inhibitioin of phosphorylation of PI3 and Akt,but has little relationship with cytoskeleton... |
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