The Role And Mechanism Of Bone Marrow-derived Mesenchymal Stem Cells In The Process Of Tissue Repair Of Abdominal Aortic Aneurysm

Chapter 1.Establishment of rodent abdominal aortic aneurysm model and the value of the the circumferential strain of ultrasonicObjective AngiotensinⅡ（AngⅡ）with the mini-pump perfusion method was applied to establish the rodent abdominal aortic aneurysm（AAA）model,and the doppler ultrasound was used to monitor the whole pathological process,so as to explore the feasibility of the model and relevant characteristics,and evaluate the application value of the the circumferential strain of ultrasound.Methods A total of 25 APOE^-/-male mice were applied and mini-pump implantation was adopted to establish the model.And 5 mice were implanted with mini-pump perfusion of saline（Con group）and the other 20 mice were micropump perfusion of AngⅡ（AngⅡ group）.All the mice were tested with doppler ultrasound at 1 day（D0）before surgery,3days（D3）after surgery,1 week（D7）after surgery,2 weeks（D14）after surgery and 4weeks（D28）after surgery,And the 150%of normal mouse arteries were used as the aortic aneurysm-forming standard at D28,then the AngⅡ group could be divided into the AngⅡ+AAA and the AngⅡ+NoAAA group.In addition to the application of B-mode（brightness mode）measurement of aortic diameter changes,and the systolic and diastolic arterial diameters（D_S,D_D）were measured in M-mode（motion mode）to calculate the circumferential strain;The vascular spot tracing technique was used to observe the elastic trajectory of vessels.The diameter,time and location of aortic aneurysm formation were observed and compared,and the rate of aortic aneurysm formation,mortality and circumferential cyclic strain were calculated;Then the aortic samples of the mice were taken and sectioned at 4 weeks after implantation.ResultsAt the end point of the study,The Con group mice,with the final survival rate of 100%,were formed with no abdominal aortic aneurysms;The survival rate and aortic aneurysm formation rate of the AngⅡ group were 80%and 62.5%,respectively;And the mean maximum diameter of abdominal aorta was significantly higher than Con group;The diameter of the aorta in the AngⅡ group showed an increasing trend.The maximum diameter of abdominal aorta with D7 was significantly larger than that the early stage;At the same time,the maximum diameter of the aorta with D28 was significantly increased compared with D14,but the aneurysm formation rate has no change.The circumferential cyclic strain of AngⅡ group was showed a trend of gradual decline,and the cyclic strain of AngⅡ group with D3 was significantly decreased with D0,but no aneurysm was formed.And the difference was also obvious compared with the Con group at D14;According to the criteria of aneurysm formation at D28,it could be found that the cyclic strain of AngⅡ+AAA group showed a downward trend and decreased significantly at D3,moreover it was significantly different from the other two groups.However,the cyclic strain of the Con group and AngⅡ+NoAAA group had not significant change during the whole process;The ultrasound vascular spot tracking technique found that AngⅡ+AAA mice showed no significant changes in vascular morphology and diameter at D3,but the vascular elasticity was decreased with the cardiac cycle.The gross specimens showed that the abdominal aorta in the upper segment of renal artery of the AngⅡ+AAA group was significantly dilated,while the vessels with the Con group and the AngⅡ+NoAAA group werenormal without obvious dilation.The HE stain of arterial sections with the AngⅡ+AAA mice showed significant thickening of the aorta wall compared with the other two groups and theelastic fiber layer is interrupted or even partially disappeared;Even some artery structure of the AngⅡ group were unrecognizable.Conclusion The model of rodent with the perfusion of AngⅡ was an relatively ideal animal model of AAA,which with a relatively simple operation and the stable rate of AAA formation,But there were also still a certain rate of death;Ultrasonic monitoring could be easy to operate,and monitor the dynamic viewing with the process aneurysm formation,And the circumferential cyclic strain has a good application value in predicting occurrence and development of aneurysms.Chapter 2.TThe Role and Mechanism of Bone Marrow-derived Mesenchymal Stem Cells in the Process of Tissue Repair of Abdominal Aortic Aneurysm Objective The mice with AAA,which was defined by ultrasound,were performed by tail vein injection of BM-MSCs;To observe whether BM-MSCs could participate in the repair process of AAA by means of migration to the injured vessel,as well as the specific therapeutic mode and related mechanisms.Methods A total of 28 APOE-/-mice,5 of which were given saline with minipump implantation（Con group）,another 23 were given Ang Ⅱ with minipump implantation,Two weeks after the implantation,ultrasound examination was performed to screen for the mice with AAA,according to normal diameter × 150%.And 12 mice met the criteria;All mice with AAA were randomly divided into the Ang Ⅱ+Msc group and Ang Ⅱ group,and there was no significant difference between the two groups in the maximum diameter of aorta and the circumferential cyclic strain.For the Ang Ⅱ group,saline was injected through the tail vein.The Ang Ⅱ+Msc group was given BM-MSCs with self-contained fluorescent label.And the second injection was performed after 4 weeks of the implantion;The mice were sacrificed after ultrasound examination at the 6th week after the implantion.Then the related detection means,such as immunofluorescence,ELISA,Western Blotting,were applicated to detect whether there was a difference between the three groupsResults There were no significant difference in the maximum diameter between the Ang Ⅱ group and the Ang Ⅱ+Msc group;But the maximum diameter of the aneurysm in the Ang Ⅱ group was significantly increased at 6th week compared with at 2th week,but there was no significant difference in the Ang Ⅱ+Msc group;The circumferential cyclic strain had no significant difference between the two groups or among the group.Immunofluorescence showed that MSCs had the characteristics of migration to lesions,but its mainly were detected in the intima and media,not in the adventitia.The CD68+ staining showed that MSCs could reduce invasion of macrophages on the vessel wall;The apoptosis rate of the Ang Ⅱ+Msc group was better than that of the Ang Ⅱ group.The HE staining and EVG staining of aortic sections showed that the vascular lumen of the Con group was normal and the media elastic fiber layer was intact,and the elastic fiber layer of the media was damaged in the other two groups;But the Ang Ⅱ+Msc group had a structure with continuous cell arrangement in the vascular lumen and more elastin-positive fibers with a layered arrangement in the media,while the Ang Ⅱ group had the intraluminal thrombus in the lumen.The CD31 staining showed that MSCs had the ability to differentiate into endothelial-like cells,while the continuously arranged CD31+ cells were also detected in the lumen in the Ang Ⅱ+Msc group.The immunohistochemical staining of α-SMA/OPN in the aorta section:The VSMCs within the elastic fiber layer were mainly stained with α-SMA,but OPN was almost non-expressed in the Con group.And the VSMCs of the Ang Ⅱ+Msc group were still spindle-shaped with more α-SMA+ stained,but the VSMCs were mainly in the shape of a circle or a triangle with more OPN+ stained in the Ang Ⅱ group.And for the part of the media without elastic fiber layer,the layered fibers could be observed in the Ang Ⅱ+Msc group,not in the Ang Ⅱ group.And the cells in the Ang Ⅱ+Msc group were spindle-shaped with more α-SMA+ stained,not in the Ang Ⅱ group.MSCs could express insulin-like growth factor-1（IGF-1）,vascular endothelial growth factor（VEGF）and platelet-derived factor-B（PDGF-B）with detection of immunofluorescence;The ELISA detected related inflammatory factors and found that MSCs intervention could reduce the levels of IL-6,MCP-1 and TNF-α in aneurysm tissues.Finally,the Western Blotting showed that the expression of MMP-2 and MMP-9 in the Ang Ⅱ+Msc group was less in the Ang Ⅱ group,and the expression level of α-SMA in the Ang Ⅱ group and the Ang Ⅱ+Msc group were both decreased;Although the Ang Ⅱ+Msc group is higher than the Ang Ⅱ group,but the difference is not obvious;OPN was not expressed in the Con group,and the level in the Ang Ⅱ+Msc group was significantly lower than that in the Ang Ⅱ group.Conclusion MSCs could also inhibit the development of aneurysm-formed AAA models.It could not only reduce the invasion of the inflammatory cells on the vessel wall and the apoptosis rate of VSMC,inhibite the activity of related inflammatory factors and MMPs,secrete some related growth factors but also reduce the occurrence intraluminal thrombus in the lumen by promoting the repair of vascular endothelial cell layer,and play an important role in maintaining the balance of function and quantity of VSMC by regulating VSMC dedifferentiation.The role of the above effects together to achieve the role of inhibiting the further development of AAA.Chapter 3.Verification and Possible Mechanism of Bone Marrow Mesenchymal Stem Cells Regulating Smooth Muscle Cell PhenotypeObjective To confirm the role of MSCs in the regulation of VSMC apoptosis and phenotype by cell co-culture,And explore the role of Notch channel in phenotypic regulation.Methods Vascular smooth muscle cell cultured alone and VSMC direct mixing or indirect Transwell mixing with MSCs were performed,which were counted as the VSMC group,the VSMC-MSC group,and the Tra-VSMC-MSC group respectively.Immunofluorescence and Western Blotting were used to detect the differences,and the differences between the groups were compared.Results The proportion of α-SMA+ VSMC/VSMC in the VSMC-MSC group was significantly higher than that in the VSMC group;And the proportion of OPN+VSMC/VSMC in the VSMC group was more than that of the VSMC-MSC group,and the difference between two groups was significant;But there was no significant difference between the Tra-VSMC-MSC group and the VSMC group;After 200 um H₂O₂ stimulation for 24 hours,the apoptotic rate of VSMC in the VSMC-MSC group was lower than that of the VSMC group;And the apoptotic rate of VSMC in the Tra-VSMC-MSC group was also lower than that of the VSMC group.But there was no difference between the VSMC-MSC group and the Tra-VMSC-MSC group.The ratio of NICD+VSMC in the VSMC-MSC was higher than that in VSMC group;The detection of Western Blotting with the same number of the VSMC and MSC : MSCs expressed more Jagged-1 than VSMC.Conclusion In addition to inhibiting VSMC apoptosis,MSCs could regulate phenotypic in VSMC through direct mixing;And the Jagged-1-Notch channel might play an important role in this process,which might be one of the possible mechanisms of MSCs inhibited the occurrence and development of the AAA.The proliferation,phenotype and function of VSMC in pathological process might be a complex process;And there was an intrinsic connection,but they were not completely synchronized;Maintaining the balance of VSMC structure and function might be more significant;The activation of Notch channel in the AAA might be a double-edged sword,which might be harmful in aggravating inflammatory response.And it might be beneficial to inhibit the AAA for the regulation of VSMC phenotype,anti-apoptosis,maintenance of the number of VSMC and functional balance.And these effects might be related to the microenvironment of the vessel wall.