CN114235773B - A Raman imaging method for dynamic monitoring of cell membrane repair processes - Google Patents
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Abstract
Description
一、技术领域1. Technical field
本发明涉及一种用于细胞膜修复过程动态监测的拉曼成像方法。The invention relates to a Raman imaging method for dynamic monitoring of cell membrane repair processes.
二、背景技术2. Background technology
生物膜孔广泛存在于细胞表面,与免疫系统、炎症或其它相关疾病中细胞的死亡息息相关。膜孔可由NK细胞或T细胞分泌的内毒素-穿孔素聚集产生,也可由外毒素,如胆固醇依赖的细胞溶素(CDC)家族中的细菌外毒素-链球菌溶血素(SLO)连至细胞膜后聚集形成。膜孔的形成机制已被很好研究,然而由于相关技术的匮乏,难以进行细胞膜上膜孔修复过程的监测以及修复机制的研究。目前,膜孔的可视化主要通过在固定后的死细胞或者人造磷脂双分子层上借助电子显微镜和原子力显微镜实现。此外,一些基于指示流经膜孔的Ca2+或者荧光标记穿孔单体来监测膜孔修复的荧光方法,由于荧光信号读出与实际修复过程不能同步,而且灵敏度也不足,不能指示活细胞上膜孔修复的真实过程。因此,在该领域迫切需要发展一种灵敏的测试方法,实现活细胞上膜孔修复过程的动态监测与修复机制研究。Biofilm pores are widely present on cell surfaces and are closely related to cell death in the immune system, inflammation or other related diseases. Membrane pores can be produced by the accumulation of perforin, an endotoxin secreted by NK cells or T cells, or by exotoxins, such as streptococcal hemolysin (SLO), a bacterial exotoxin in the cholesterol-dependent cytolysin (CDC) family that is attached to the cell membrane. After aggregation is formed. The formation mechanism of membrane pores has been well studied. However, due to the lack of relevant technologies, it is difficult to monitor the repair process of membrane pores on cell membranes and study the repair mechanism. Currently, the visualization of membrane pores is mainly achieved by electron microscopy and atomic force microscopy on fixed dead cells or artificial phospholipid bilayers. In addition, some fluorescence methods based on indicating Ca 2+ flowing through the membrane pores or fluorescently labeled perforated monomers to monitor membrane pore repair cannot indicate the presence of membrane pores on living cells because the fluorescence signal readout cannot be synchronized with the actual repair process, and the sensitivity is insufficient. The real process of membrane hole repair. Therefore, there is an urgent need to develop a sensitive testing method in this field to achieve dynamic monitoring of the membrane pore repair process and study of the repair mechanism in living cells.
该专利设计了一种成孔蛋白诱导的动态拉曼成像方法,通过修复过程中表面增强拉曼(SERS)信号的降低,实现对活细胞膜修复过程的动态成像。This patent designs a pore-forming protein-induced dynamic Raman imaging method to achieve dynamic imaging of the repair process of living cell membranes by reducing the surface-enhanced Raman (SERS) signal during the repair process.
三、发明内容3. Contents of the invention
本发明的目的是:基于金纳米星(AuNSs)的聚集产生SERS信号,设计了由成孔蛋白-SLO在细胞膜上聚集,诱导SERS探针在膜孔上聚集而产生的SERS信号。通过细胞膜修复过程中,SLO及其偶联的SERS探针不断地从细胞表面脱落,导致细胞膜上拉曼信号的降低,实现细胞膜修复过程的动态监测,并阐明修复机制,为细胞膜修复相关疾病的治疗提供新的技术支撑。The purpose of the present invention is to generate SERS signals based on the aggregation of gold nanostars (AuNSs), and to design a SERS signal generated by aggregation of pore-forming protein-SLO on the cell membrane and inducing the aggregation of SERS probes on the membrane pores. During the cell membrane repair process, SLO and its coupled SERS probe are continuously shed from the cell surface, resulting in a decrease in the Raman signal on the cell membrane. This enables dynamic monitoring of the cell membrane repair process and elucidates the repair mechanism, providing insights into cell membrane repair-related diseases. Treatment provides new technical support.
本发明提出的细胞膜修复的动态SERS成像策略如图1所示。首先合成由二苄环辛基-磺基-N-羟基琥珀酰亚胺酯(DBCO-sulfo-NHS ester)修饰的SLO(SLO-DBCO),并用SLO-DBCO在细胞膜上聚集形成膜孔。同时,将拉曼信标分子(对巯基苯甲酸)和叠氮-聚乙二醇(PEG-N3)同时修饰在AuNSs表面获得SERS探针。SERS探针与细胞膜孔上SLO-DBCO的点击化学反应使其在膜孔周围聚集产生SERS信号,实现膜孔的拉曼成像。在细胞膜修复过程中,膜孔逐渐变小,SLO及其偶联的SERS探针不断地从细胞表面脱落,使SERS信号逐渐降低,直至膜孔完全修复,通过SERS信号的变化实现整个修复过程的动态监测。The dynamic SERS imaging strategy for cell membrane repair proposed by the present invention is shown in Figure 1. First, SLO (SLO-DBCO) modified by dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester (DBCO-sulfo-NHS ester) was synthesized, and SLO-DBCO was used to aggregate on the cell membrane to form membrane pores. At the same time, Raman beacon molecules (p-mercaptobenzoic acid) and azide-polyethylene glycol (PEG-N 3 ) were simultaneously modified on the surface of AuNSs to obtain a SERS probe. The click chemical reaction of the SERS probe with SLO-DBCO on the cell membrane pores causes them to gather around the membrane pores to generate SERS signals, thereby achieving Raman imaging of the membrane pores. During the cell membrane repair process, the membrane pores gradually become smaller, and SLO and its coupled SERS probes continue to fall off the cell surface, causing the SERS signal to gradually decrease until the membrane pores are completely repaired. The entire repair process is realized through changes in the SERS signal. Dynamic Monitoring.
本发明通过以下技术方案来实现:The present invention is realized through the following technical solutions:
1)将成孔蛋白SLO与DBCO-sulfo-NHS ester在室温下反应2小时,形成具有成孔活性的成SLO-DBCO偶联物。1) React the pore-forming protein SLO with DBCO-sulfo-NHS ester at room temperature for 2 hours to form a SLO-DBCO conjugate with pore-forming activity.
2)将拉曼信标分子(对巯基苯甲酸)和叠氮-聚乙二醇(PEG-N3)共修饰在AuNSs表面获得SERS探针。2) Co-modify the Raman beacon molecule (p-mercaptobenzoic acid) and azide-polyethylene glycol (PEG-N 3 ) on the surface of AuNSs to obtain a SERS probe.
3)活细胞在含SLO-DBCO的培养液中温育,可使SLO-DBCO在细胞膜上聚集形成大的膜孔。3) Incubation of living cells in culture medium containing SLO-DBCO can cause SLO-DBCO to aggregate on the cell membrane to form large membrane pores.
4)如图1所示,上述组成膜孔的SLO-DBCO通过与SERS探针的点击反应,使SERS探针在膜孔上方聚集,从而产生SERS信号。4) As shown in Figure 1, the SLO-DBCO that constitutes the membrane pores causes the SERS probes to gather above the membrane pores through a click reaction with the SERS probe, thereby generating a SERS signal.
5)如图1所示,随着细胞膜孔的修复,膜孔逐渐变小,SLO及其偶联的SERS探针不断地从细胞表面脱落,使SERS信号逐渐降低,从而实现活细胞膜孔修复的动态拉曼成像。5) As shown in Figure 1, as the cell membrane pores are repaired, the membrane pores gradually become smaller, and SLO and its coupled SERS probes continue to fall off from the cell surface, causing the SERS signal to gradually decrease, thus realizing the repair of membrane pores in living cells. Dynamic Raman imaging.
与现有技术相比,本发明具有以下特点:Compared with the existing technology, the present invention has the following characteristics:
本发明中设计的监测方法,具有简单、无损、灵敏、原位动态追踪的特点。膜孔在活细胞上形成之后,不需对细胞进行固定后再成像,信号产生策略对细胞以及膜孔没有损伤,且基于金纳米粒子固有的信号增强效应,实现对信号放大,以满足本发明原位动态监测膜孔修复过程的需求。The monitoring method designed in the present invention has the characteristics of simple, non-destructive, sensitive and in-situ dynamic tracking. After membrane pores are formed on living cells, there is no need to fix the cells before imaging. The signal generation strategy does not damage the cells or membrane pores, and based on the inherent signal enhancement effect of gold nanoparticles, signal amplification is achieved to meet the needs of the present invention. The need for in-situ dynamic monitoring of the membrane pore repair process.
四、附图说明4. Description of drawings
图1.细胞膜修复过程动态监测的表面增强拉曼光谱方法示意图Figure 1. Schematic diagram of surface-enhanced Raman spectroscopy method for dynamic monitoring of cell membrane repair process.
五、具体实施方式5. Specific implementation methods
实施例1:SLO-DBCO偶联物的合成与细胞穿孔实验Example 1: Synthesis and cell perforation experiment of SLO-DBCO conjugates
用二硫苏糖醇(DTT)将成孔蛋白SLO在37℃下活化2小时后,取SLO溶液(0.55μM)与DBCO-sulfo-NHS ester(47μM)混合,在垂直旋转仪上反应2小时。用30kDa的超滤管超滤(14000g,4℃)5次除去反应体系中多余的DBCO-sulfo-NHS ester,制得具有穿孔活性的SLO-DBCO偶联物。After activating the pore-forming protein SLO with dithiothreitol (DTT) at 37°C for 2 hours, mix the SLO solution (0.55 μM) and DBCO-sulfo-NHS ester (47 μM) and react on a vertical rotator for 2 hours. Use a 30kDa ultrafiltration tube to ultrafiltrate (14000g, 4°C) five times to remove excess DBCO-sulfo-NHS ester in the reaction system to prepare a SLO-DBCO conjugate with perforation activity.
以MCF-7乳腺癌细胞系为细胞模型,MCF-7细胞在37℃培养箱中温育过夜,用Hank’s平衡盐缓冲液(HBSS,含有2mM CaCl2)洗3次后,与SLO-DBCO(200U mL-1)在37℃培养箱中温育20分钟,即可在细胞膜上形成SLO-DBCO聚集的孔。MCF-7 breast cancer cell line was used as a cell model. MCF-7 cells were incubated overnight in a 37°C incubator, washed three times with Hank's balanced salt buffer (HBSS, containing 2mM CaCl 2 ), and then incubated with SLO-DBCO (200U mL -1 ) and incubate it in a 37°C incubator for 20 minutes to form pores where SLO-DBCO aggregates on the cell membrane.
实施例2:SERS探针的合成Example 2: Synthesis of SERS probe
首先将5mL 1%柠檬酸三钠加入100mL沸腾的HAuCl4溶液反应20分钟制得15-nm金种,冷却至室温后滴入8.6mL PVP(25.6g L-1,MW=10000)过夜,再与15mL含0.12mM HAuCl4和10mM PVP的DMF溶液反应15分钟,制得金纳米星(AuNSs)。将AuNSs分散在500μL水中后,依次加入10μL10mM拉曼报告分子MBA的乙醇溶液和490μL新制备的HS-PEG-N3(2mM)溶液,在室温下摇匀过夜。在8000rpm下离心洗涤两次,得到SERS探针(AuNSs-MBA/PEG-N3),并悬浮于500μL水中备用。First, 5 mL of 1% trisodium citrate was added to 100 mL of boiling HAuCl 4 solution and reacted for 20 minutes to obtain 15-nm gold seeds. After cooling to room temperature, 8.6 mL of PVP (25.6 g L -1 , MW=10000) was added dropwise overnight, and then Gold nanostars (AuNSs) were prepared by reacting with 15 mL of DMF solution containing 0.12mM HAuCl 4 and 10mM PVP for 15 minutes. After the AuNSs were dispersed in 500 μL of water, 10 μL of the 10 mM Raman reporter MBA ethanol solution and 490 μL of the newly prepared HS-PEG-N 3 (2 mM) solution were added in sequence, and the mixture was shaken overnight at room temperature. Centrifuge and wash twice at 8000 rpm to obtain the SERS probe (AuNSs-MBA/PEG-N 3 ), which is suspended in 500 μL of water for later use.
实施例3:结合图1,SERS探针在细胞膜上的聚集产生SERS信号Example 3: Combined with Figure 1, the aggregation of SERS probes on the cell membrane generates SERS signals
将实施例1中获得的细胞与SERS探针(0.2nM)在37℃培养箱中温育30分钟,通过-PEG-N3与-DBCO的点击反应,可将SERS探针聚集在细胞膜上由SLO形成的孔上,产生SERS信号,实现对细胞膜孔的拉曼成像。The cells obtained in Example 1 were incubated with the SERS probe (0.2nM) in a 37°C incubator for 30 minutes. Through the click reaction of -PEG-N 3 and -DBCO, the SERS probe can be gathered on the cell membrane by SLO. On the formed pores, SERS signals are generated to realize Raman imaging of cell membrane pores.
实施例4:结合图1,活细胞膜修复过程的动态拉曼成像监测Example 4: Combined with Figure 1, dynamic Raman imaging monitoring of living cell membrane repair process
将实施例3中经SERS探针聚集的细胞转至含10%胎牛血清的1640培养液中,在37℃培养启动细胞的修复过程。每隔10分钟,取出修复不同时间的细胞,用不含胎牛血清的1640培养液轻洗3次进行拉曼成像,直至完成整个修复行为,即可实现对活细胞修复过程的动态监测。The cells aggregated by the SERS probe in Example 3 were transferred to 1640 culture medium containing 10% fetal calf serum, and cultured at 37°C to initiate the repair process of the cells. Every 10 minutes, cells that have been repaired for different times are taken out and washed three times with 1640 culture medium without fetal bovine serum for Raman imaging until the entire repair behavior is completed, thus enabling dynamic monitoring of the repair process of living cells.
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