CRISPR介导的 PECAM-1胞内段的原位删除对其边界定位和内皮细胞边界完整性影响的研究

发布时间：2018-06-01 01:40

本文选题：CRISPR + PECAM-1　；参考：《华中科技大学》2016年博士论文

【摘要】：目的：PEC AM-1是一个分子量为130k的蛋白,属于免疫球蛋白超家族的成员之一,主要在造血细胞系上以及单层内皮细胞—细胞间隙处表达。有研究表明,PEC AM-1的胞内段在内皮细胞中有多重调控功能。PEC AM-1的胞内段是内皮细胞机械应答反应的必要条件,还对凋亡刺激起到细胞保护作用,并与细胞连接处其他的粘附蛋白和细胞骨架分子相互作用。而以上功能不依赖于位于胞内段的免疫受体抑制性基序。鉴于胞内段的碱基长度,可以推测其他碱基可能在上述过程中起到作用。所以本课题的研究目的是探究PEC AM-1的胞内段是否参与内皮细胞-细胞边界定位和内皮屏障功能的维持,以维持血管的完整性。方法：用CRISPR基因编辑技术建立PEC AM-1敲除的永生化人脐静脉内皮细胞系,称为PE-KO;通过两条sgRNA以在原位切除PEC AM-1胞内段的基因,以得到表达ΔCD-PECAM-1的细胞,称为ΔCD-iHUVECs。因为内皮细胞有转染效率低的特点,我们用含有两条sgRNA和Cas9核酸酶cDNA的慢病毒感染iHUVECs。在用嘌呤霉素选择性培养慢病毒感染的细胞后,iHUVECs被流式细胞仪分选为单个克隆。随后分别用PECAM-1胞外段和胞内段特异性的抗体,通过流式细胞术分析鉴定所得克隆细胞。为测定ΔCD-PECAM-1的功能,用共聚焦免疫荧光观察其在内皮细胞中的边界定位,用电细胞—基质阻抗传感仪评估屏障功能,用流式细胞术测定PECAM-1/IgG结合能力以确定同嗜性结合能力。结果：我们成功建立并鉴定了两个独特的iHUVECs细胞系：一个彻底敲除了PEC AM-1表达,另一个细胞系表达ΔCD-PECAM-1。共聚焦显微镜显示ΔCD-PECAM-1可定位于融合内皮细胞的边界。与表达WT PECAM-1的细胞相比,表达ΔCD-PECAM-1的iHUVECs有更高的静息阻抗,而PECAM-1敲除的iHUVECs的静息阻抗则显著下降。用凝血酶破坏内皮细胞屏障功能后,表达ΔCD-PECAM-1的细胞则能更快的恢复屏障,而PE-KO的恢复速率却显著低于表达WT-iHUVECs。最后,与表达WT PECAM-1的细胞相比,在iHUVECs上表达的ΔCD-PECAM-1与PECAM-1/IgG的亲和性则明显下降。结论：PECAM-1这一细胞粘附信号受体,在内皮细胞—细胞边界处聚集的能力,并不依赖于其胞内段的表达。而ΔCD-PECAM-1与其它PECAM-1分子有较低的同嗜性结合亲和力,这导致表达ΔCD-PECAM-1的内皮细胞有更高的静息屏障功能,以及在凝血酶刺激后,能更快的重建内皮细胞边界的完整性。综上所述,以上结果表明PECAM-1胞内段可能与内皮细胞骨架蛋白相互作用,从而限制了它在内皮细胞膜平面的移动性。而缺乏胞内段的PECAM-1得到释放,有更大的移动性,从而能更好的维持内皮细胞—细胞间隙的完整性。
[Abstract]:Objective: AM-1 is a 130k protein, which belongs to the immunoglobulin superfamily. It is expressed mainly in hematopoietic cell lines and the endothelial cell-cell intercellular space of monolayer. Studies have shown that the intracellular segment of PEC AM-1 has multiple regulatory functions in endothelial cells. The intracellular segment of PEC AM-1 is a necessary condition for the mechanical response of endothelial cells, and also plays a cytoprotective role in apoptosis stimulation. It also interacts with other adhesion proteins and cytoskeleton molecules at cell junctions. These functions are independent of the immunoreceptor inhibitory motifs located in the intracellular domain. Given the length of the base in the intracellular segment, it can be speculated that other bases may play a role in the above process. Therefore, the purpose of this study is to explore whether the intracellular segment of PEC AM-1 is involved in endothelial cell-cell boundary localization and endothelial barrier function maintenance, in order to maintain the integrity of blood vessels. Methods: the immortalized human umbilical vein endothelial cell line (PE-KOO) with PEC AM-1 knockout was established by CRISPR gene editing technique, and 螖 CD-iHUVECs was obtained by in situ excision of PEC AM-1 intracellular segment by two sgRNA. Because of the low transfection efficiency of endothelial cells, lentivirus containing two sgRNA and Cas9 nuclease cDNA was used to infect iHUVECs. After selective culture of lentivirus infected cells with purine mycin, iHUVECs were separated into a single clone by flow cytometry. The cloned cells were identified by flow cytometry with specific antibodies of extracellular and intracellular segments of PECAM-1. In order to determine the function of 螖 CD-PECAM-1, the boundary localization of 螖 CD-PECAM-1 in endothelial cells was observed by confocal immunofluorescence. The barrier function was evaluated by electrocell-matrix impedance sensor and the binding ability of PECAM-1/IgG was determined by flow cytometry. Results: we successfully established and identified two unique iHUVECs cell lines: one completely knocked out the expression of PEC AM-1 and the other expressed 螖 CD-PECAM-1. Confocal microscopy showed that 螖 CD-PECAM-1 could locate the boundary of fused endothelial cells. Compared with the cells expressing WT PECAM-1, the iHUVECs expressing 螖 CD-PECAM-1 has a higher resting impedance, while the PECAM-1 knockout iHUVECs has a significantly lower resting impedance. After thrombin was used to destroy endothelial cell barrier function, the cells expressing 螖 CD-PECAM-1 could recover the barrier more quickly, while the recovery rate of PE-KO was significantly lower than that of WT-iHUVECs. Finally, compared with the cells expressing WT PECAM-1, the affinity of 螖 CD-PECAM-1 expressed on iHUVECs to PECAM-1/IgG decreased significantly. Conclusion the ability of the cell adhesion signal receptor: 1 PECAM-1 to gather at the endothelial cell-cell boundary does not depend on the expression of its intracellular segment. However, 螖 CD-PECAM-1 has lower affinity to other PECAM-1 molecules, which leads to higher resting barrier function of endothelial cells expressing 螖 CD-PECAM-1, and faster reconstruction of endothelial cell boundary integrity after thrombin stimulation. In conclusion, the above results suggest that the intracellular segment of PECAM-1 may interact with the endothelial cytoskeleton protein, thus limiting its mobility in the endothelial cell membrane plane. However, PECAM-1, which lacks the intracellular segment, is released and has greater mobility, thus maintaining the integrity of the endothelial cell-cell space.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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