【作者】 李子付；

【导师】 刘建民；

【作者基本信息】 第二军医大学 ， 神经外科（专业学位）， 2013， 博士

【摘要】 蛛网膜下腔出血的主要原因是颅内动脉瘤破裂，临床上具有高死亡率、高致残率等特点，给社会和家庭带来严重的经济负担。随着医学的发展，尤其是介入医学和再生医学的发展，给予颅内动脉瘤的治疗带来新的希望。介入治疗从早期单纯的弹簧圈栓塞发展到血流导向装置。与传统支架不同，血流导向装置具有更致密的网孔，也就是更高的金属覆盖率，通过提高金属覆盖率达到改善血流动力学的目的。血流导向装置通过重建载瘤动脉，提供内膜修复的支架支撑，让瘤颈口内膜重新修复，最终达到让动脉瘤与管腔完全隔离，从而治愈动脉瘤。再生医学的发展，尤其内皮祖细胞（Endothelial progenitor cells，EPCs）的发现，为动脉瘤的再生医学治疗提供了诱人的前景。EPCs主要来自于骨髓，在血管损伤后能够动员并参与血管损伤后的修复。前期我们研究发现，骨髓来源的内皮祖细胞参与动脉瘤壁的修复以及动脉瘤支架治疗后的瘤颈处的内膜修复。在本系列研究中，本课题将以内皮祖细胞趋化因子SDF-1α为切入点研究SDF-1α调控EPCs参与动脉瘤内膜修复中的作用及其机制，观察外源性补充SDF-1α对兔囊状动脉瘤支架治疗后的内皮化影响。SDF-1α对兔骨髓来源内皮祖细胞的趋化作用摘要目的研究SDF-1α对EPCs的趋化作用及粘附功能影响。方法从新西兰大白兔骨髓提取单个核细胞层，分离后原代培养；使用boyden小室观察不同SDF-1α浓度及SDF-1α/CXCR4通道阻断剂下EPCs的迁移率；不同SDF-1α浓度下，使用Western blot检测各粘附蛋白表达。结果骨髓来源的经分离培养后可以获得符合生物学特性的EPCs，CD133、CD34、VEGFR-2抗原阳性表达，具有吞噬ac-LDL和UEA-1lectin的内皮细胞功能；不同梯度的SDF-1α迁移结果不同，500ng/ml的迁移效果最强，SDF-1/CXCR4阻断剂可减弱迁移效果；在SDF-1α浓度增加后，粘附分子VE-cadherin表达最高。结论兔骨髓可获得符合生物学要求的EPCs；SDF-1α对EPCs具有趋化作用；在使用趋化因子后，EPCs粘附分子VE-cadherin表达升高。SDF-1α可促进EPCs迁移及粘附功能。SDF-1α在早期兔囊性动脉瘤内膜的表达摘要目的研究猪胰弹力酶诱导的兔囊性动脉瘤模型早期SDF-1α表达变化。方法采用猪胰弹力酶制作兔弹力酶诱导的囊性动脉瘤模型，在术后3、7、4、21天分别取囊性动脉瘤标本，利用Western blot检测内膜SDF-1α蛋白表达变化，荧光定量PCR检测SDF-1α基因表达，免疫组化检测SDF-1α蛋白表达部位。结果免疫组化显示早期动脉瘤瘤壁内膜表达SDF-1α，在第14天表达最高；荧光定量PCR显示SDF-1α在术后第7天动脉瘤内膜处表达最高；Western blot结果显示SDF-1在术后14天SDF-1α表达最高。结论SDF-1α在兔囊性动脉瘤模型制作后早期表达在内膜表面，并呈现动态变化，在术后7至14天，SDF-1α表达最高。rhSDF-1α对兔囊性动脉瘤血流重建治疗后的内膜修复影响摘要目的研究rhSDF-1α对兔囊性动脉瘤血流导向支架治疗后内膜修复的影响。方法20只新西兰大白兔，采用猪胰弹力酶制作兔囊性动脉瘤，模型制作后3周后置入血流导向支架Tubridge；支架治疗后模型随机分为2组，一组使用rhSDF-1α50ug/mg/d耳缘静脉注射，一组使用生理盐水为对照.术后2周及4周时间点，使用荧光定量PCR检测KDR、Tie2、CD34、VE-cadherin等内皮相关分子表达;采用扫描电镜观察瘤颈口新生内膜中内皮样细胞个数。结果荧光定量PCR检测结果表明rhSDF-1组中瘤颈口Tie2、KDR、VE-cadherin表达较对照组明显增高（P<0.05）；扫描电镜显示rhSDF-1α组和对照组相比，2周和4周瘤颈和载瘤动脉上具有较多的内皮样细胞。瘤颈处两组2周内皮样细胞分别为55个及13个每高倍视野(P<0.01)，4周内皮样细胞分别为104个及60个每高倍视野（P<0.05）。结论rhSDF-1α能够促进兔囊性动脉瘤支架治疗后内膜修复，从而促进内膜内皮化。AMD3100对兔囊性动脉瘤血流重建治疗后的内膜修复影响摘要目的AMD3100即可作为SDF-1/CXCR4轴阻断剂亦可从骨髓动员EPCs入血，研究其对动脉瘤支架治疗后内膜修复的影响。方法20只新西兰大白兔，采用猪胰弹力酶制作兔囊性动脉瘤，模型制作后3周后置入血流重建支架Tubridge；支架治疗后模型随机分为2组，一组使用AMD31003ug/mg/d皮下注射，一组使用生理盐水，术后2周及4周时间点，采用硬组织切片技术及Masson染色观察内膜厚度，扫描电镜观察瘤颈及载瘤动脉内皮样细胞数量及荧光定量PCR检测瘤颈口及其周围组织内膜相关分子标志表达（KDR、VE-cadherin、CD34、Tie2）。结果扫描电镜显示AMD3100组和对照组相比，2周和4周瘤颈和载瘤动脉上具有较多的内皮样细胞。瘤颈处两组2周内皮样细胞分别为40个及13个(P<0.01)每高倍视野，4周内皮样细胞分别为92个及60个（P<0.05）每高倍视野；硬组织切片Masson染色，显示AMD3100组4周瘤颈处内膜更为明显，使用AMD3100相对于对照组瘤颈口内膜更为完整。荧光定量PCR检测结果表明AMD3100组中瘤颈口Tie2、KDR、VE-cadherin表达较对照组明显增高。结论AMD3100同样能够促进兔囊性动脉瘤支架治疗后内膜修复，促进内膜内皮化。更多还原

【Abstract】 The rupture of intracranial aneurysm is the main cause of subarachnoid hemorrhage, whichhas the clinical features of high mortality and morbidity. The disease has brought greateconomic burden to the family and society. With the development of medical sciences, theadvancement of interventional and regenerative medicine had shed new lights on thetreatment of intracranial aneurysms. In the intervention field, the endovascular treatmenthad moved from the beginning of singular coiling to the current treatment with flowdiversion devices. Flow diverter can reconstruct the lesional lumen， alter thehemodynamics of aneurysm, and provide a scaffold for intima growth across the aneurysmneck in order to isolate the aneurysms. In the regenerative field, use of endothelialprogenitor cells has brought a prosperous future for the aneurysm treatment. In our earlierexperiments, we found that bone marrow-derived EPCs contributes to the aneurysm repairafter establishement of elastase-induced saccular aneurysm model and participate in theneointima formation in the aneurysm neck and parent artery after flow diverter treatment.In the successive research, we will investigate the mechanism of endothelial repair of EPCsfrom the aspect of chemokine stromal derived factor-1α(SDF-1α) and investigate therole of SDF-1/CXCR4axis during the process of neointima formation in rabbit saccularaneurysm after flow diverter treatment in different time points.The role and mechanism of SDF-1αin homing of EPCsAbstract Objective To investigate the role and mechanism of SDF-1α in the homing ofEPCs. Methods Bone marrow mononuclear cells of ten rabbits were isolated cultured withEGM-2MV medium in flasks coated with fibronectin. Migration assay was tested withBoyden Chamber with different concerntration of SDF-1α. Western blot was used toanalysed the expression of protein including VE-cadherin, E-selectin and P-selectin.Results The cultured EPCs confirm the cells with biological behavior with took upDil-ac-LDL and combined with FITC-UEA-1lectin and showed positive CD34, CD133,and VEGFR-2with immunocytochemistry. The migration ability was different withdifferent conserntration of SDF-1α. With the increase of conserntration of SDF-1α,moreEPCs migrate into the other side and the concerntration of500ng/ml had the highestmigration ability. The expression of VE-adherin increased with the conserntration ofSDF-1α. Conclusion EPCs with biological behavior can be acquired from bone marrow. SDF-1α can attract the EPCs and stimulate EPCs to express VE-cadherin. Expression of SDF-1α in the wall of saccular aneurysm inrabbitsAbstract Objective To investigate SDF-1expression at the early stage of modelestablishement. Methods Saccular aneurysm was established with porcine pancreaticelastase, Sdf-1α protein was investigated in the aneurysm wall with Western blot andSDF-1α mRNA was investigated with realtime PCR;Location of protein expression in thetissue was investigated with immunohistochemistry(IHC). Results IHC results show thatSDF-1α was expressed in the neointima and most significantly on day14after modelestablishement; realtime PCR results showed that SDF-1αmRNA was expressed stronglyon day7, Western blot results showed that SDF-1α expressed most highly on day14.Conclusion SDF-1α can be expressed in the wall of saccular aneurysm in the rabbits, andexpressed significantly on day7to14. rhSDF-1α accelerates reendothelialization of neointimaformation in saccular aneurysm after flow diverter treatmentAbstract Objective To investigate the role of rhSDF-1α in the neointima repair ofsaccular aneurysm in rabbits after flow diverter treatment. Metholds Twenty New ZealandWhite Rabbits were used to establish saccular model with porcine pancreatic elastase.Three weeks later, flow diverter of Tubridge was implanted. All the treated models weredivided into two groups, one using rhSDF-1αintravenously50ug/mg per day and the otherusing saline as control group. Morphology was investigated with SEM on neointimaformation2weeks and4weeks after FD treatment.Expressions of Tie2, CD34,KDR,VE-cadherin were examined with realtime PCR. Results SEM showed that in therhSDF-1αgroup,there are more endothelial-like cells on the neointima at week2and4weeks after flow diverter treatment. In the experiment group and control group, theendothelial-like cells reched40and13per high power field two weeks after flow diverter treatment and reached104and60cells per high power field4weeks after FD treatmentunder electron scanning microscope. There are significant differences between theendothelial markers of Tie2, KDR and VE-cadherin（P<0.05）. Conclusion Systemicdelivery of rhSDF-1αpromotes the endothelial repair and facilitates the endothelializationof aneurysm neck after flow diverter treatment. AMD3100accelerates reendothelialization of neointimaformation in saccular aneurysm after flow diverter treatmentAbstract Objective AMD3100acts as antagonist of CXCR4, this section aims toinvestigate the role of AMD3100in the neointima repair. Metholds Ten New ZealandWhite Rabbits were used to establish saccular model with porcine pancreatic elastase.Three weeks later, flow diverter of Tubridge was implanted. All the treated models weredivided into two groups, one using AMD3100subcutaneously3ug/mg per day and theother using saline as control group. Morphology was investigated with Masson and hardtissue section on neointima formation2weeks and4weeks after FD treatment.ResultsSEM showed that in the AMD3100group, there are more endothelial-like cells on theneointima at week2and4weeks after flow diverter treatment. In the experiment groupand control group, the endothelial-like cells reched40and13per high power field twoweeks after flow diverter treatment and reached92and60cells per high power field4weeks after FD treatment under electron scanning microscope. The intima in theexperiment group was more intact than that in the control group. Conclusions Interval useof AMD3100promotes the endothelial repair and facilitates the endothelialization ofaneurysm neck after flow diverter treatment.更多还原