Ang-1基因修饰的内皮祖细胞修复动脉瘤作用及转归的实验研究
发布时间：2018-11-24 20:48
【摘要】：研究背景:颅内动脉瘤(Intracranial aneurysm,ICA)的治疗方法主要有血管内介入治疗和手术夹闭。弹簧圈栓塞后动脉瘤的复发率比手术夹闭高,其与动脉瘤囊增大、弹簧圈压缩及动脉瘤颈部内皮覆盖不全等因素有关。其中,瘤颈部内皮覆盖不全是动脉瘤复发的重要因素,瘤颈部若形成完整的内皮覆盖则有利于减少血流对瘤腔冲击,可明显降低复发的风险。有研究证实动脉瘤患者外周循环中内皮祖细胞(Endothelial progenitor cells,EPCs)数量减少、功能下降。EPCs可分化为内皮细胞参与损伤血管的修复。血管生成素-1(Angiogenin-l,Ang-1)是一种可调节内皮祖细胞功能的分泌型糖蛋白。Ang-1可通过其受体Tie-2发挥作用,而Tie-2可在EPCs高度表达。激活Ang-1/Tie-2可增强EPCs迁移、分化及管腔形成等血管修复功能,而血清中Ang-1升高提示动脉瘤破裂引起的蛛网膜下腔出血患者预后良好。因此,过表达Ang-1基因也许可增强EPCs功能,促进动脉瘤修复过程。此外,前期研究中,我们发现EPCs可加速动脉瘤修复作用,动脉瘤腔内α-SMA阳性,提示瘤腔内存在平滑肌样细胞,但是仍未清楚其是否来源于EPCs。综上所述,本研究欲通过过表达Ang-1基因从而增强EPCs功能,经尾静脉注射过表达Ang-1基因的EPCs,观察其对动脉瘤腔和瘤颈部的修复作用及其在动脉瘤腔内的转归,有望为降低颅内动脉瘤栓塞术后的复发率提供理论基础。第一部分构建血管生成素-1基因修饰的EPCs及其在动脉瘤中的修复作用目的:构建血管生成素-1(Ang-1)基因修饰的EPCs,并验证其对动脉瘤修复效果。方法:EPCs分别转染携带Ang-1和NC基因的慢病毒,通过管腔形成、划痕及MTT实验检测过表达Ang-1对EPCs功能的影响。动脉瘤模型构建完成2周后取动脉瘤组织行HE染色、Masson染色及免疫荧光检测。结果:管腔形成、划痕及MTT实验发现Ang-1基因修饰EPCs可增强其成管、迁移及增殖能力。病理学检测发现Ang-1-EPCs可加速动脉瘤的修复。免疫荧光显示动脉瘤腔内存在平滑肌样细胞。结论:过表达Ang-1基因可增强EPCs的管腔形成、迁移及增殖能力,加速动脉瘤的修复。第二部分平滑肌细胞对EPCs分化的影响及EPCs在动脉瘤中的转归目的:探索平滑肌细胞(SMCs)是否可影响EPCs的分化及EPCs是否可分化为SMCs参与动脉瘤的修复。方法:RT-PCR检测与SMCs共培养后的EPCs中α-SMA,SM22α,CD31和vWF mRNA表达情况。向动脉瘤模型注射携带GFP的EPCs,术后14d及28d取动脉瘤组织行免疫荧光检测EPCs在动脉瘤内的转归。结果:RT-PCR结果示SMCs可增加EPCs中平滑肌特异性基因α-SMA和SM22α的表达量(P0.05),降低vWF表达(P0.05),但CD31表达量增加(P0.05)。免疫荧光示动脉瘤腔内GFP及α-SMA双阳性。结论:SMCs可诱导EPCs向SMCs方向分化,EPCs可分化为SMCs参与动脉瘤的修复。
[Abstract]:Background: Intracranial aneurysms (Intracranial aneurysm,ICA) are mainly treated by intravascular interventional therapy and surgical clipping. The recurrent rate of aneurysm after embolization by coils was higher than that by surgical clipping, which was related to the enlargement of aneurysm sac, the compression of coils and the incomplete coverage of the neck endothelium of aneurysms. The incomplete coverage of the aneurysm neck endothelium is an important factor in the recurrence of aneurysm. If a complete endothelial covering is formed in the tumour neck, it can reduce the impact of blood flow on the lumen of the aneurysm and significantly reduce the risk of recurrence. Some studies have confirmed that the number and function of endothelial progenitor cells (Endothelial progenitor cells,EPCs) in peripheral circulation of patients with aneurysm are decreased. EPCs can differentiate into endothelial cells to participate in the repair of injured blood vessels. Angiopoietin 1 (Angiogenin-l,Ang-1) is a secretory glycoprotein that regulates the function of endothelial progenitor cells (EPCs). Ang-1 acts through its receptor Tie-2 and Tie-2 is highly expressed in EPCs. Activation of Ang-1/Tie-2 could enhance the function of vascular repair such as migration, differentiation and lumen formation of EPCs, while the increase of Ang-1 in serum suggested that the prognosis of patients with subarachnoid hemorrhage caused by aneurysm rupture was good. Therefore, overexpression of Ang-1 gene may enhance the function of EPCs and promote the process of aneurysm repair. In addition, in previous studies, we found that EPCs can accelerate the repair of aneurysms, and 伪-SMA positive in aneurysms, suggesting that smooth muscle cells exist in the lumen of aneurysms, but it is not clear whether it originated from EPCs.. In conclusion, the purpose of this study was to enhance the function of EPCs by overexpressing the Ang-1 gene, and to observe the effect of EPCs, which expressed Ang-1 gene through tail vein, on the repair of aneurysm cavity and neck and its outcome in the aneurysm cavity. It is expected to provide a theoretical basis for reducing the recurrence rate of intracranial aneurysms after embolization. The first part: construction of angiopoietin 1 gene modified EPCs and its repair in aneurysms objective: to construct angiopoietin 1 (Ang-1) modified EPCs, and verify its effect on the repair of aneurysms. Methods: EPCs was transfected into lentiviruses carrying Ang-1 and NC genes, respectively. The effects of Ang-1 expression on EPCs function were detected by tube formation, scratch and MTT experiments. The aneurysm tissues were collected for HE staining, Masson staining and immunofluorescence detection 2 weeks after the construction of the aneurysm model. Results: the results of cavity formation, scratch and MTT showed that Ang-1 gene modified EPCs could enhance its ability of tube formation, migration and proliferation. Pathological examination showed that Ang-1-EPCs could accelerate the repair of aneurysm. Immunofluorescence revealed the presence of smooth muscle like cells in the aneurysm. Conclusion: overexpression of Ang-1 gene can enhance the formation, migration and proliferation of EPCs, and accelerate the repair of aneurysms. The second part is the effect of smooth muscle cells on the differentiation of EPCs and the outcome of EPCs in aneurysms. Objective: to explore whether smooth muscle cell (SMCs) can affect the differentiation of EPCs and whether EPCs can differentiate into SMCs to participate in the repair of aneurysms. Methods: RT-PCR was used to detect the expression of 伪-SMA,SM22 伪, CD31 and vWF mRNA in EPCs co-cultured with SMCs. After 14 days and 28 days of injection of EPCs, carrying GFP into the aneurysm model, the tissue of aneurysm was collected and the outcome of EPCs in the aneurysm was detected by immunofluorescence. Results: RT-PCR results showed that SMCs could increase the expression of smooth muscle specific genes 伪 -SMA and SM22 伪 in EPCs (P0.05), decrease the expression of vWF (P0.05), but increase the expression of CD31 (P0.05). Immunofluorescence showed that both GFP and 伪-SMA were positive in aneurysms. Conclusion: SMCs can induce EPCs to differentiate into SMCs and EPCs can differentiate into SMCs to participate in the repair of aneurysm.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743
本文编号：2354999
[Abstract]:Background: Intracranial aneurysms (Intracranial aneurysm,ICA) are mainly treated by intravascular interventional therapy and surgical clipping. The recurrent rate of aneurysm after embolization by coils was higher than that by surgical clipping, which was related to the enlargement of aneurysm sac, the compression of coils and the incomplete coverage of the neck endothelium of aneurysms. The incomplete coverage of the aneurysm neck endothelium is an important factor in the recurrence of aneurysm. If a complete endothelial covering is formed in the tumour neck, it can reduce the impact of blood flow on the lumen of the aneurysm and significantly reduce the risk of recurrence. Some studies have confirmed that the number and function of endothelial progenitor cells (Endothelial progenitor cells,EPCs) in peripheral circulation of patients with aneurysm are decreased. EPCs can differentiate into endothelial cells to participate in the repair of injured blood vessels. Angiopoietin 1 (Angiogenin-l,Ang-1) is a secretory glycoprotein that regulates the function of endothelial progenitor cells (EPCs). Ang-1 acts through its receptor Tie-2 and Tie-2 is highly expressed in EPCs. Activation of Ang-1/Tie-2 could enhance the function of vascular repair such as migration, differentiation and lumen formation of EPCs, while the increase of Ang-1 in serum suggested that the prognosis of patients with subarachnoid hemorrhage caused by aneurysm rupture was good. Therefore, overexpression of Ang-1 gene may enhance the function of EPCs and promote the process of aneurysm repair. In addition, in previous studies, we found that EPCs can accelerate the repair of aneurysms, and 伪-SMA positive in aneurysms, suggesting that smooth muscle cells exist in the lumen of aneurysms, but it is not clear whether it originated from EPCs.. In conclusion, the purpose of this study was to enhance the function of EPCs by overexpressing the Ang-1 gene, and to observe the effect of EPCs, which expressed Ang-1 gene through tail vein, on the repair of aneurysm cavity and neck and its outcome in the aneurysm cavity. It is expected to provide a theoretical basis for reducing the recurrence rate of intracranial aneurysms after embolization. The first part: construction of angiopoietin 1 gene modified EPCs and its repair in aneurysms objective: to construct angiopoietin 1 (Ang-1) modified EPCs, and verify its effect on the repair of aneurysms. Methods: EPCs was transfected into lentiviruses carrying Ang-1 and NC genes, respectively. The effects of Ang-1 expression on EPCs function were detected by tube formation, scratch and MTT experiments. The aneurysm tissues were collected for HE staining, Masson staining and immunofluorescence detection 2 weeks after the construction of the aneurysm model. Results: the results of cavity formation, scratch and MTT showed that Ang-1 gene modified EPCs could enhance its ability of tube formation, migration and proliferation. Pathological examination showed that Ang-1-EPCs could accelerate the repair of aneurysm. Immunofluorescence revealed the presence of smooth muscle like cells in the aneurysm. Conclusion: overexpression of Ang-1 gene can enhance the formation, migration and proliferation of EPCs, and accelerate the repair of aneurysms. The second part is the effect of smooth muscle cells on the differentiation of EPCs and the outcome of EPCs in aneurysms. Objective: to explore whether smooth muscle cell (SMCs) can affect the differentiation of EPCs and whether EPCs can differentiate into SMCs to participate in the repair of aneurysms. Methods: RT-PCR was used to detect the expression of 伪-SMA,SM22 伪, CD31 and vWF mRNA in EPCs co-cultured with SMCs. After 14 days and 28 days of injection of EPCs, carrying GFP into the aneurysm model, the tissue of aneurysm was collected and the outcome of EPCs in the aneurysm was detected by immunofluorescence. Results: RT-PCR results showed that SMCs could increase the expression of smooth muscle specific genes 伪 -SMA and SM22 伪 in EPCs (P0.05), decrease the expression of vWF (P0.05), but increase the expression of CD31 (P0.05). Immunofluorescence showed that both GFP and 伪-SMA were positive in aneurysms. Conclusion: SMCs can induce EPCs to differentiate into SMCs and EPCs can differentiate into SMCs to participate in the repair of aneurysm.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743
【参考文献】
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1 姬斌;汪求精;孙新林;薛杉;杜谋选;蔡颖谦;唐艳萍;陈镇洲;姜晓丹;;负载VEGF聚合物修饰铂金微弹簧圈栓塞动脉瘤模型的研究[J];中华神经医学杂志;2012年01期
，本文编号：2354999
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