Ang-1基因修飾的內(nèi)皮祖細(xì)胞修復(fù)動(dòng)脈瘤作用及轉(zhuǎn)歸的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-11-24 20:48
【摘要】：研究背景:顱內(nèi)動(dòng)脈瘤(Intracranial aneurysm,ICA)的治療方法主要有血管內(nèi)介入治療和手術(shù)夾閉。彈簧圈栓塞后動(dòng)脈瘤的復(fù)發(fā)率比手術(shù)夾閉高,其與動(dòng)脈瘤囊增大、彈簧圈壓縮及動(dòng)脈瘤頸部?jī)?nèi)皮覆蓋不全等因素有關(guān)。其中,瘤頸部?jī)?nèi)皮覆蓋不全是動(dòng)脈瘤復(fù)發(fā)的重要因素,瘤頸部若形成完整的內(nèi)皮覆蓋則有利于減少血流對(duì)瘤腔沖擊,可明顯降低復(fù)發(fā)的風(fēng)險(xiǎn)。有研究證實(shí)動(dòng)脈瘤患者外周循環(huán)中內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs)數(shù)量減少、功能下降。EPCs可分化為內(nèi)皮細(xì)胞參與損傷血管的修復(fù)。血管生成素-1(Angiogenin-l,Ang-1)是一種可調(diào)節(jié)內(nèi)皮祖細(xì)胞功能的分泌型糖蛋白。Ang-1可通過(guò)其受體Tie-2發(fā)揮作用,而Tie-2可在EPCs高度表達(dá)。激活A(yù)ng-1/Tie-2可增強(qiáng)EPCs遷移、分化及管腔形成等血管修復(fù)功能,而血清中Ang-1升高提示動(dòng)脈瘤破裂引起的蛛網(wǎng)膜下腔出血患者預(yù)后良好。因此,過(guò)表達(dá)Ang-1基因也許可增強(qiáng)EPCs功能,促進(jìn)動(dòng)脈瘤修復(fù)過(guò)程。此外,前期研究中,我們發(fā)現(xiàn)EPCs可加速動(dòng)脈瘤修復(fù)作用,動(dòng)脈瘤腔內(nèi)α-SMA陽(yáng)性,提示瘤腔內(nèi)存在平滑肌樣細(xì)胞,但是仍未清楚其是否來(lái)源于EPCs。綜上所述,本研究欲通過(guò)過(guò)表達(dá)Ang-1基因從而增強(qiáng)EPCs功能,經(jīng)尾靜脈注射過(guò)表達(dá)Ang-1基因的EPCs,觀察其對(duì)動(dòng)脈瘤腔和瘤頸部的修復(fù)作用及其在動(dòng)脈瘤腔內(nèi)的轉(zhuǎn)歸,有望為降低顱內(nèi)動(dòng)脈瘤栓塞術(shù)后的復(fù)發(fā)率提供理論基礎(chǔ)。第一部分構(gòu)建血管生成素-1基因修飾的EPCs及其在動(dòng)脈瘤中的修復(fù)作用目的:構(gòu)建血管生成素-1(Ang-1)基因修飾的EPCs,并驗(yàn)證其對(duì)動(dòng)脈瘤修復(fù)效果。方法:EPCs分別轉(zhuǎn)染攜帶Ang-1和NC基因的慢病毒,通過(guò)管腔形成、劃痕及MTT實(shí)驗(yàn)檢測(cè)過(guò)表達(dá)Ang-1對(duì)EPCs功能的影響。動(dòng)脈瘤模型構(gòu)建完成2周后取動(dòng)脈瘤組織行HE染色、Masson染色及免疫熒光檢測(cè)。結(jié)果:管腔形成、劃痕及MTT實(shí)驗(yàn)發(fā)現(xiàn)Ang-1基因修飾EPCs可增強(qiáng)其成管、遷移及增殖能力。病理學(xué)檢測(cè)發(fā)現(xiàn)Ang-1-EPCs可加速動(dòng)脈瘤的修復(fù)。免疫熒光顯示動(dòng)脈瘤腔內(nèi)存在平滑肌樣細(xì)胞。結(jié)論:過(guò)表達(dá)Ang-1基因可增強(qiáng)EPCs的管腔形成、遷移及增殖能力,加速動(dòng)脈瘤的修復(fù)。第二部分平滑肌細(xì)胞對(duì)EPCs分化的影響及EPCs在動(dòng)脈瘤中的轉(zhuǎn)歸目的:探索平滑肌細(xì)胞(SMCs)是否可影響EPCs的分化及EPCs是否可分化為SMCs參與動(dòng)脈瘤的修復(fù)。方法:RT-PCR檢測(cè)與SMCs共培養(yǎng)后的EPCs中α-SMA,SM22α,CD31和vWF mRNA表達(dá)情況。向動(dòng)脈瘤模型注射攜帶GFP的EPCs,術(shù)后14d及28d取動(dòng)脈瘤組織行免疫熒光檢測(cè)EPCs在動(dòng)脈瘤內(nèi)的轉(zhuǎn)歸。結(jié)果:RT-PCR結(jié)果示SMCs可增加EPCs中平滑肌特異性基因α-SMA和SM22α的表達(dá)量(P0.05),降低vWF表達(dá)(P0.05),但CD31表達(dá)量增加(P0.05)。免疫熒光示動(dòng)脈瘤腔內(nèi)GFP及α-SMA雙陽(yáng)性。結(jié)論:SMCs可誘導(dǎo)EPCs向SMCs方向分化,EPCs可分化為SMCs參與動(dòng)脈瘤的修復(fù)。
[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 偽-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 偽-SMA,SM22 偽, 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 偽 -SMA and SM22 偽 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 偽-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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743
本文編號(hào)：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 偽-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 偽-SMA,SM22 偽, 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 偽 -SMA and SM22 偽 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 偽-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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 姬斌;汪求精;孫新林;薛杉;杜謀選;蔡穎謙;唐艷萍;陳鎮(zhèn)洲;姜曉丹;;負(fù)載VEGF聚合物修飾鉑金微彈簧圈栓塞動(dòng)脈瘤模型的研究[J];中華神經(jīng)醫(yī)學(xué)雜志;2012年01期
，本文編號(hào)：2354999
本文鏈接：http://sikaile.net/yixuelunwen/shenjingyixue/2354999.html
最近更新
教材專著
