CN112964682A - Method for visually and quantitatively marking aggregated functional protein in cells - Google Patents
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Abstract
本发明涉及生物探针和蛋白检测技术领域，提供了一种可视化定量标记细胞中聚集型功能蛋白的方法，通过构建和表征聚集诱导发光探针AIE‑cRGD，测定探针AIE‑cRGD的吸收峰和荧光发射峰以及适用浓度，检测确定探针的靶向蛋白为整联蛋白αvβ3，基于poly‑HEMA铺板法建立悬浮存活细胞模型，诱导悬浮存活细胞模型内细胞在脱离粘附的环境下生长，模拟细胞的抗凋亡状态，利用探针标记细胞中的整联蛋白并进行成像，当整联蛋白内化进入内涵体后，确定整联细胞在脱离粘附细胞内的分布情况，检测磷酸化粘着斑激酶p‑FAK与活化整联蛋白在内涵体上的共定位，确定细胞的抗凋亡能力。本发明可直接用于活细胞的染色，为生理功能的可视化奠定了基础，有利于高品质成像及高灵敏度的在线传感监测。
The invention relates to the technical field of biological probes and protein detection, and provides a method for visually and quantitatively labeling aggregated functional proteins in cells. and fluorescence emission peaks and applicable concentrations, the target protein of the probe was determined to be integrin αvβ3, a suspension survival cell model was established based on the poly-HEMA plating method, and the cells in the suspension survival cell model were induced to grow in a disengaged environment. Simulate the anti-apoptotic state of cells, use probes to label integrins in cells and perform imaging, when integrins are internalized into endosomes, determine the distribution of integrins in detached cells, and detect phosphorylation Co-localization of the focal adhesion kinase p-FAK with activating integrins on endosomes determines the anti-apoptotic capacity of cells. The invention can be directly used for the staining of living cells, lays a foundation for the visualization of physiological functions, and is beneficial to high-quality imaging and high-sensitivity online sensing monitoring.
Description
Technical Field
The invention relates to the technical field of biological probes and protein detection, in particular to a method for visually and quantitatively marking aggregated functional protein in cells.
Background
Integrins are widely distributed on the cell surface, consist of two subunits, alpha and beta, which constitute transmembrane heterodimers, and are a class of transmembrane receptor glycoproteins whose ligands are mainly extracellular matrix (ECM) proteins, such as vitronectin, laminin, and the like. Integrins mostly have a longer extracellular domain, a transmembrane region and a shorter cytoplasmic domain, with different domains having different functions. In the case of the extracellular domain, it can mediate cell-to-extracellular matrix, cell-to-cell adhesion signals by recognizing a specific sequence of extracellular matrix proteins, i.e., arginine-glycine-aspartic acid (RGD sequence); the cytoplasmic domain is combined with a cytoskeleton to form an extracellular matrix-integrin-cytoskeleton transmembrane complex, participates in bidirectional conduction of signals inside and outside cells, regulates survival and proliferation of cells, invasion and metastasis of tumors and the like, and can further understand the occurrence of cell adhesion and regulation of related signals by researching the structural function and expression regulation of integrin.
Studies have shown that integrins regulate the pro-survival signals of tumor cells by regulating their downstream signaling molecules and pathways, including FAK (Focal attachment Kinase), Src, ILK (Integrin-linked Kinase), PI3K/Akt, MAPK. FAK is a non-receptor tyrosine kinase that acts as one of the downstream activation site molecules of integrin signaling pathways and mediates cell adhesion, migration, proliferation, and the like. FAK contains a plurality of tyrosine sites, integrins are combined with extracellular matrix, FAK is recruited through beta subunits on adhesion sites formed by the FAK and is phosphorylated at Y397 site to form a compound with downstream Src kinase, Src kinase is activated, phosphorylation of other FAK sites is activated, PI3K-Akt and MAPK-Erk signaling pathways are activated, and apoptosis is inhibited.
In 2015, Jonna Alanko et al found that integrin-mediated pro-cell growth signals were not limited to the adhesion sites of cells to the extracellular matrix, but also appeared on endosomes. When the cells are connected with the extracellular matrix, the integrin can recruit downstream signal molecules FAK at an adhesion site, conduct integrin mediated signals, lose the adhesion of the cells, and endocytose the integrin into an endosome, and conduct integrin mediated cell growth signals through the endosome, so that the endosome can be used as an integrin mediated FAK cell growth promotion signal platform except for a plasma membrane adhesion site, prolongs survival signals depending on the extracellular matrix, and maintains the survival of tumor cells. The integrin endocytosis has been proved to promote FAK phosphorylation on the endosome, and the phosphorylated FAK signal on the endosome can inhibit tumor cell apoptosis, support the non-anchoring growth of tumor cells and promote tumor metastasis. Thus, the viability of cells after cell detachment can be tested by testing the p-FAK signaling platform established on endosomes by the activated integrin bundle markers internalized into endosomes.
Fluorescence imaging, as an intuitive, in situ detection technique, can be used to detect expression and distribution of molecules. At present, the research on integrins mostly focuses on imaging analysis through antibody incubation expression, the concentration of traditional organic fluorescent molecules is high, aggregation-induced quenching (ACQ) effect can occur in an aggregation state, the working concentration of the fluorescent molecules needs to be reasonably controlled in the using process, and the application range of the fluorescent molecules is greatly limited.
Aggregation-induced emission (AIE) molecules are used as a special fluorescent molecule, and active groups contained in the AIE molecule perform relative motion (such as vibration and rotation), so that the molecules in an excited state consume in the form of converting light energy into heat energy and the like through the relative motion, and the proportion of the output energy in the form of light is low; the AIE molecules are gathered, the relative motion of the molecules is limited, the proportion of energy output in the form of light energy is greatly improved, so that the phenomenon of fluorescence enhancement is shown, even a jump from dark to bright occurs, and the visual qualitative analysis and quantitative detection of an induction source are realized, therefore, gathering induced emission (AIE) molecules provides possibility for the visualization of the physiological function of biomacromolecules, and the method is favorable for high-quality imaging and high-sensitivity online sensing monitoring.
Disclosure of Invention
The invention provides a method for visually and quantitatively marking aggregation type functional proteins in cells, aiming at the problems that protein signals are provided by using antibody recognition protein sites through fluorescence amplification in the current protein detection process, most antibodies are connected with FITC, TRITC and other traditional organic fluorescent molecules, fluorescence quenching is easy to occur after long-time exposure, and fluorescence molecule aggregation quenching effect is generated in a protein aggregation area.
The invention specifically adopts the following technical scheme:
a method for visually and quantitatively marking aggregated functional protein in cells adopts an aggregation-induced emission probe AIE-cRGD, and specifically comprises the following steps:
Mixing 1, 2-di (4-carboxyphenyl) -1, 2-styrene and cRGD according to the same molar ratio, adding a catalyst EDC-NHS for catalysis, reacting to obtain an aggregation-induced emission probe AIE-cRGD, preparing a test system consisting of dimethyl sulfoxide and water, measuring an ultraviolet absorption spectrum and a fluorescence spectrum of the aggregation-induced emission probe AIE-cRGD in the test system, and determining an absorption peak and a fluorescence emission peak of the aggregation-induced emission probe AIE-cRGD;
Changing the concentration of the aggregation-induced emission probe AIE-cRGD, detecting the cell activity under the condition of different concentrations of the aggregation-induced emission probe AIE-cRGD by using a cell counting reagent CCK-8, and determining the applicable concentration of the aggregation-induced emission probe AIE-cRGD for living cell imaging;
step 3, detecting the target protein of the aggregation-induced emission probe AIE-cRGD
Respectively diluting bovine serum albumin BSA, Trypsin Trypsin, Glutamine Glutamine, Cysteine, Cystine Cysteine, matrix metalloproteinase MMP9 and integrin alpha v beta 3 to 100 mu g/mL, diluting the concentration of aggregation-induced emission probe AIE-cRGD to be within an applicable concentration range, respectively adding each protein solution and the aggregation-induced emission probe AIE-cRGD into 300 mu L of phosphate buffer solution according to the volume ratio of 1:1 for mixing, measuring the ultraviolet absorption degree of the aggregation-induced emission probe AIE-cRGD at an absorption peak aiming at each protein solution, determining the integrin alpha v beta 3 as a target protein of the aggregation-induced emission probe AIE-cRGD, and staining integrins in cells by using the aggregation-induced emission probe AIE-cRGD;
step 4, establishing a suspension survival cell model based on a poly-HEMA plating method, inducing cells in the suspension survival cell model to grow in an adhesion-free environment, and simulating the anti-apoptosis state of the cells;
and 5, marking the integrin of the cells by using an aggregation-induced emission probe AIE-cRGD in a suspension survival cell model, imaging, determining the distribution condition of the integrin in the detached adhesion cells after the integrin is internalized into an endosome, detecting the co-localization of phosphorylated focal adhesion kinase p-FAK and activated integrin on the endosome, and determining the anti-apoptosis capacity of the cells.
Preferably, the aggregation-inducing luminescent probe AIE-cRGD contains an aggregation luminescent molecule and an integrin-targeting polypeptide.
Preferably, in step 1, the ratio of dimethyl sulfoxide to water in the test system is 1: 100.
Preferably, in step 2, the concentration of the aggregation-inducing luminescent probe AIE-cRGD is adjusted to 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL, respectively.
Preferably, in the step 5, a quantitative multi-parameter image analysis platform is programmed based on MATLAB software, and the shape and position of each endosome in the suspension survival cell model are determined by segmenting the image.
Preferably, in step 5, phosphorylated focal adhesion kinase p-FAK increases with integrin increase, and the fluorescence intensity of the aggregation-inducing luminescent probe AIE-cRGD shows integrin activation for detecting integrin-mediated endonexin p-FAK signaling platform.
Preferably, in the step 5, the relationship between the fluorescence intensity of the aggregation-inducing luminescent probe AIE-cRGD and the fluorescence intensity of the phosphorylated focal adhesion kinase p-FAK is N (p-FAK) ═ 186.06+0.16N (AIE-cRGD), where N (p-FAK) represents the fluorescence intensity of the phosphorylated focal adhesion kinase p-FAK and N (AIE-cRGD) represents the fluorescence intensity of the aggregation-inducing luminescent probe AIE-cRGD.
The invention has the following beneficial effects:
the invention adopts the aggregation-induced emission probe AIE-cRGD to dye the integrin, has better biocompatibility compared with an antibody dyeing method, can directly dye and evaluate in living cells, does not need to dye fixed cells, has simple operation, only needs one-time incubation, greatly shortens the detection time, simultaneously adopts the aggregation-induced emission probe AIE-cRGD to induce the integrin in the cells to emit fluorescence, avoids the occurrence of aggregation quenching effect, can directly analyze the protein aggregation condition according to the change of the fluorescence intensity of the probe, accurately evaluates the content of protein molecules, realizes the imaging of the cells and the determination of the integrin distribution condition in the cells, and is favorable for researching the anti-apoptosis condition of the cells.
Drawings
FIG. 1 shows the results of the biocompatibility test of the aggregation-induced emission probe AIE-cRGD.
FIG. 2 shows the absorbance of the aggregation-inducing luminescent probe AIE-cRGD after mixing with each protein solution.
FIG. 3 shows fluorescence intensities of the aggregation-inducing luminescent probe AIE-cRGD after mixing with each protein solution.
Detailed Description
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings:
example 1: construction and characterization of aggregation-induced emission probe AIE-cRGD
The aggregation-induced emission probe AIE-cRGD contains polypeptide of aggregation luminescent molecules and targeting integrin, 1, 2-di (4-carboxybenzene) -1, 2-styrene and cRGD are mixed according to the same molar ratio, and then catalyst EDC-NHS is added for catalysis, and the aggregation-induced emission probe AIE-cRGD is obtained through overnight reaction;
preparing a test system by dimethyl sulfoxide (DMSO) and water according to a ratio of 1:100, and measuring ultraviolet absorption spectra and fluorescence spectra of aggregation-induced emission probes AIE-cRGD with different concentrations in the test system, wherein the ultraviolet absorption spectra show that the aggregation-induced emission probes AIE-cRGD with different concentrations all have absorption peaks at 230nm, and the fluorescence spectra show that the aggregation-induced emission probes AIE-cRGD with different concentrations all have fluorescence emission peaks at 425nm, namely determining that the absorption peaks of the aggregation-induced emission probes AIE-cRGD are 230nm and the fluorescence emission peaks are 425 nm.
Example 2: concentration of aggregation-induced emission Probe AIE-cRGD
Taking breast tumor cells MCF-7 as an example, determining the concentration of an aggregation-induced emission probe AIE-cRGD; the concentration of aggregation-inducing luminescent probe AIE-cRGD was set to 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL and 100. mu.g/mL, respectively, and MCF-7 cells were placed in aggregation-inducing luminescent probe AIE-cRGD solutions of respective concentrations, respectively, and the activities of MCF-7 cells in the aggregation-inducing luminescent probe AIE-cRGD solutions of different concentrations were measured using cell counting reagent CCK-8, and by analyzing the influence of the concentration of aggregation-inducing luminescent probe AIE-cRGD on the cell activities, it was found that when the concentration of aggregation-inducing luminescent probe AIE-cRGD was 1. mu.g/mL, 10. mu.g/mL and 50. mu.g/mL, no significant change in the cell activities occurred, and when the concentration of aggregation-inducing luminescent probe AIE-cRGD was 100. mu.g/mL, the cell activities were decreased, as shown in FIG. 1, therefore, it was determined that the concentration of the aggregation-inducing luminescent probe AIE-cRGD should be no more than 100. mu.g/mL, and that the aggregation-inducing luminescent probe AIE-cRGD can be used for living cell imaging when the concentration of the aggregation-inducing luminescent probe AIE-cRGD is no more than 100. mu.g/mL.
Example 3: target protein for detecting aggregation-induced emission probe AIE-cRGD
Bovine serum albumin BSA, Trypsin Trypsin, Glutamine Glutamine, Cysteine, Cystine Cystine, matrix metalloproteinase MMP9 and integrin α v β 3 were each diluted to 100 μ g/mL, the concentration of aggregation-inducing luminescent probe AIE-cRGD was diluted to 100 μ g/mL, each protein solution and aggregation-inducing luminescent probe AIE-cRGD solution were each added to 300 μ L of phosphate buffer salt solution at a volume ratio of 1:1 and mixed, for each protein solution, the change in ultraviolet absorption intensity of aggregation-inducing luminescent probe AIE-cRGD at 230nm was measured, the response of aggregation-inducing luminescent probe AIE-cRGD to each protein was analyzed, as shown in FIGS. 2 and 3, the absorbance of the aggregation-inducing luminescent probe AIE-cRGD solution at 230nm was 0.0027, and the aggregation-inducing luminescent probe AIE-cRGD was added to the integrin α v β 3 solution, the absorbance at 230nm was 0.31, the absorbance was significantly increased, which was 115 times the absorbance at 230nm of the aggregation inducing luminescent probe AIE-cRGD solution, and the absorbance of the aggregation inducing luminescent probe AIE-cRGD mixed with bovine serum albumin BSA, Trypsin, Glutamine Glutamine, Cysteine, Cystine, and matrix metalloproteinase MMP9 was lower than the absorbance of the aggregation inducing luminescent probe AIE-cRGD mixed with integrin α v β 3, and these protein solutions enhanced the absorption intensity of the aggregation inducing luminescent probe AIE-cRGD but were lower than the absorption intensity caused by integrin α v β 3, so that the responsiveness of the aggregation inducing luminescent probe AIE-cRGD to integrin α v β 3 was significantly higher than the specific binding of bovine serum albumin, Trypsin, Glutamine Glutamine, Cysteine, Cystine, matrix metalloproteinase MMP9, and the aggregation inducing luminescent probe AIE-cRGD to integrin α v β 3, integrin α v β 3 is a targeting protein of aggregation-induced emission probe AIE-cRGD, and can stain integrin in cells using aggregation-induced emission probe AIE-cRGD.
Example 4: establishing a suspension survival cell model
Establishing a suspension survival cell model based on a poly-HEMA plating method, inducing cells in the suspension survival cell model to grow in an environment without adhesion, simulating the anti-apoptosis state of the cells, taking cells cultured by a low adhesion plate as a control, finding out through comparison that the cells in the suspension survival cell model are aggregated to grow in a cluster, and transferring the cells in the suspension survival cell model to other matrixes to ensure that the cells can grow and proliferate again. Meanwhile, comparing the binding capacity of both the aggregation-inducing luminescent probe AIE-cRGD and the integrin alphavbeta 3 antibody to integrin, labeling MCF-7 cells and MDA-MB-231 cells with the aggregation-inducing luminescent probe AIE-cRGD and the integrin alphavbeta 3 antibody, respectively, and investigating the fluorescent co-localization of the aggregation-inducing luminescent probe AIE-cRGD and the integrin alphavbeta 3 antibody with integrin in the cells, respectively, using a confocal fluorescence microscope, it was found that in MCF-7 cells and MDA-MB-231 cells, the fluorescent spots of the aggregation-inducing luminescent probe AIE-cRGD and the integrin alphavbeta 3 antibody substantially coincide with each other, the co-localization coefficient of both MCF-7 cells is 0.954, the co-localization coefficient of both MDA-MB-231 cells is 0.837, and the co-localization coefficient represents the overlapping degree of luminescent spots, the higher the co-localization coefficient, the higher the degree of overlap of the luminescence spots, and it was found that the aggregation inducing luminescence probe AIE-cRGD was able to specifically bind to integrin, the integrin recognition ability of the aggregation inducing luminescence probe AIE-cRGD was comparable to that of integrin α v β 3 antibody, and further the comparison of the fluorescence intensities of the two labels revealed that the fluorescence signal of the aggregation inducing luminescence probe AIE-cRGD was stronger than that of integrin α v β 3 antibody, and it was presumed that it was possible that the aggregation inducing luminescence probe AIE-cRGD, after binding to integrin, restricted the rotation of the benzene ring within the molecule and greatly enhanced the fluorescence signal, and therefore, the aggregation inducing luminescence probe AIE-cRGD was more suitable for staining of aggregation type integrin than integrin α v β 3 antibody.
Example 5: determining the apoptotic capacity of cells
A quantitative multi-parameter image analysis platform is compiled based on MATLAB software and is used for displaying the imaging result of an endosome in the suspension survival cell model, in a suspension survival cell model, an aggregation-induced emission probe AIE-cRGD is used for marking the integrin of cells, and then a marker protein Rab5 and a marker protein Rab7 of an endosome are marked by an integrin alpha v beta 3 antibody, after imaging, analyzing the co-localization of the aggregation-induced emission probe AIE-cRGD and the marker proteins Rab5 and Rab7, determining the distribution of the integrin in the detached adhesion cells after internalization, the co-localization rate of the aggregation-induced emission probe AIE-cRGD and the marker protein Rab5 is higher than that of the marker protein Rab7, when the cell is in a state of being detached from the adhesion, the integrin is internalized into early endosome, and establishing a signal platform for maintaining cell survival on the early endosome, wherein the cells in a suspension state are supported by the signal platform of the endosome to survive and grow.
The phosphorylation focal adhesion kinase p-FAK is increased along with the increase of the integrin, the fluorescence intensity of the aggregation-induced emission probe AIE-cRGD shows the activation condition of the integrin, and the fluorescence intensity is used for detecting an integrin-mediated endosome p-FAK signal platform, detecting the fluorescent co-localization of the phosphorylation focal adhesion kinase p-FAK and the activated integrin on an endosome and determining the anti-apoptosis capacity of the cell.
Taking MDA-MB-231 cells as an example, in a suspension survival cell model, an aggregation-induced emission probe AIE-cRGD is used for marking integrin in detached adhesion cells, p-FAK and early endosome are respectively marked by fluorescent antibodies against p-FAK and Rab5, and after measuring the fluorescent co-localization of the integrin marked by the aggregation-induced emission probe AIE-cRGD with the p-FAK and Rab5, the co-localization rate of the integrin marked by the aggregation-induced emission probe AIE-cRGD with the p-FAK and Rab5 is high, and the intensity of the aggregation-induced emission probe AIE-cRGD has certain correlation with the expression of the p-FAK and Rab5 in cells which survive after detachment of viscosity.
Example 6: co-localization analysis of aggregation-induced emission probe AIE-cRGD and p-FAK in early endosome
The co-localization fluorescent spots of aggregation-induced emission probes AIE-cRGD, p-FAK and Rab5 are less in MDA-MB-231 cells, a large number of co-localization fluorescent spots of aggregation-induced emission probes AIE-cRGD, p-FAK and Rab5 are displayed in a suspension survival cell model, the co-localization rate statistics is carried out on two co-localization fluorescent spots of the three, namely the co-localization rates of the aggregation-induced emission probes AIE-cRGD and p-FAK, the aggregation-induced emission probes AIE-cRGD and Rab5 and the p-FAK and Rab5 are respectively carried out, the co-localization rates in the suspension survival cell model are higher than that in the MDA-MB-231 cells, so that the MDA-MB-231 cells establish an early endosome signal growth platform, have stronger survival capability, activated integrin bundles separated from adherent cells are internalized and enter endosomes, the method comprises the steps of recruiting and activating FAK on early endosomes, establishing a signal system capable of maintaining cell growth and survival, then selecting cells such as U87-MG, KB, HeLa, HepG2, DU-145, Caco-2, A549, 143B and the like to perform experiments, determining the survival capability of the cells after the cells are detached from adhesion, and finding out that the co-localization rate of each cell is different but lower than the capability of an endosome signal platform established in a suspension survival cell model through comparison, so that the aggregation-induced emission probe AIE-cRGD can be used for detecting the establishment of the integrin street endosome p-FAK signal platform in suspension cells, and supporting the growth and survival of the cells.
Example 7
Comparing the fluorescent signal distribution, the fluorescent intensity and the spot ratio of the fluorescent signal of the aggregation-induced emission probe AIE-cRGD, the p-FAK and the Rab5, analyzing the correlation of the fluorescent signal intensity between the aggregation-induced emission probe AIE-cRGD and the p-FAK, finding that there is a significant positive correlation between the two signals, determining the fluorescent signal intensity correlation between the aggregation-induced emission probe AIE-cRGD and the p-FAK as N (p-FAK) ═ 186.06+0.16N (AIE-cRGD) by fitting the images, wherein N (p-FAK) represents the fluorescent intensity of the phosphorylated focal adhesion kinase p-FAK, N (AIE-cRGD) represents the fluorescent intensity of the aggregation-induced emission probe AIE-cRGD, the signal intensity of the probe shows the bundle activation of the integrin, the phosphorylated focal adhesion kinase p-FAK increases with the increase of the integrin, indicating that a signal platform supporting the survival and growth of cells is established in the endosome; and then, a quantitative multi-parameter image analysis platform developed based on MATLAB software is utilized, the shape and the position of each endosome are described by segmenting an image, a pixel group of a fluorescence co-localization area is selected, after the fluorescence intensity is analyzed, the aggregation-induced emission probe AIE-cRGD can be used for activating an integrin bundle-mediated endosome FAK signal platform, and the survival capability of the cells after detachment can be determined according to the change of the fluorescence signal intensity of the aggregation-induced emission probe AIE-cRGD.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art may make modifications, alterations, additions or substitutions within the spirit and scope of the present invention.
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