Method for performing ASGPR imaging detection and assembly change on cell surface through super-resolution fluorescence microscope

Abstract

The invention discloses a method for detecting the assembly change of an ASGPR (asialoglycoprotein receptor) on the surface of a hepatocyte, which comprises the following steps: carrying out specific labeling on the ASGPR on the surface of the hepatocyte through an antibody, and carrying out fluorescence imaging on the ASGPR on the surface of a cell membrane, the purpose of detecting ASGPR assembly change on the liver cell membrane is achieved by statistically analyzing the number of ASGPR protein positioning points on the surface of the cell membrane and aggregation parameters. According to the method, the distribution and aggregation condition of the ASGPR on the cell surface is measured through the nanoscale resolution, the change of the ASGPR in the cell membrane assembly relation is detected, damage to sample cells is small, the time cost of the experimental process is small, protein positioning is accurate, and as the ASGPR is an endocytosis receptor specifically existing on the surface of hepatocellular carcinoma cells, the detection result is accurate, and the detection accuracy is high. Efficient treatment can be achieved through a drug delivery system formed by targeting ASGPR, ASGPR can be better known by observing the assembly difference of ASGPR on the surface of a cell membrane from the nanoscale, and the method has wide application prospects in the field of medical detection and diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of small molecule labeling methods for cell fluorescence imaging, and more specifically, to the preparation of an antibody fluorescent probe that can be used for super-resolution fluorescence imaging to observe protein assembly distribution, which is of great significance to the biomedical field. Background Art

[0002] The asialoglycoprotein receptor (ASGPR) is a receptor specifically expressed by hepatocytes. It belongs to the C-type lectin family and performs specific recognition of ligands in a calcium-dependent manner. Its molecular weight is 42KDa. The molecular structure of ASGPR can be divided into four functional domains: cytoplasmic domain, transmembrane domain, stem domain, and sugar recognition domain. It is an efficient endocytic receptor that can specifically bind to sugar molecules or sugar complexes such as asialoglycoprotein, galactose, galactosamine, and N-acetylgalactosamine with high affinity. In recent years, a variety of ligands have been selected as modified molecules for targeted drug delivery, such as N-acetylgalactosamine, by utilizing the high affinity of specific ligands to ASGPR. Studying the assembly morphology of ASGPR on the cell membrane is of great significance for drug delivery.

[0003] The main physiological function of ASGPR is believed to be to bind, internalize and subsequently remove glycoproteins containing terminal galactose or GlcNAc residues from the circulation. As a multifunctional membrane receptor, it is also involved in the clearance of apoptotic cells, the disposal of cellular fibronectin, and the clearance of circulating IgA. The main research at home and abroad is on ASGPR as a drug delivery receptor targeting hepatocytes, where ligands are linked to drug molecules or nanocarriers are coated with drugs for drug delivery, and drugs are brought into cells through the endocytosis of ASGPR for release and recycling. Although there have been many reports that the application and treatment prognosis of drug delivery combined with ASGPR have been significantly improved, people have used a variety of biochemical methods to prove that ASGPR can be used as an advantageous target for the diagnosis and treatment of liver cancer, and have used fluorescence quantitative analysis or cell viscosity experiments to reveal the relationship between ASGPR and the occurrence and development of cancer. However, at the molecular level, the assembly and distribution of ASGPR on the cell membrane surface and the revelation of the mechanism of cancer occurrence and endocytosis have been rarely involved.

[0004] Currently, many researchers use cancer-related tumor markers to diagnose and evaluate through immunofluorescence detection. However, these reported methods are limited by the resolution of traditional optical microscopes, making it difficult to obtain detailed spatial assembly information of tumor markers. More sophisticated means are needed to diagnose cell surface protein assembly. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention proposes a method for single-molecule localization analysis of ASGPR protein on the cell membrane surface based on super-resolution fluorescence microscopy to analyze the assembly morphology of ASGPR on the surface of hepatocellular carcinoma cells.

[0006] The present invention is achieved through the following technical solutions: A method for observing the assembly morphology of ASGPR on the cell surface by imaging with a super-resolution fluorescence microscope, characterized in that the steps are as follows:

[0007] (1) Fix cells with a confluence of ~60% with 4% paraformaldehyde (PFA) at room temperature for 20 min and wash three times with PBS.

[0008] (2) Block with 3% BSA for 20 min.

[0009] (3) After removing the blocking buffer, cells were stained with Alexa647-conjugated ASGPR antibody at 4°C overnight. Samples were washed five times with PBS and the dye was added.

[0010] (4) Remove the Alexa 647-antibody staining solution and wash the samples 5 times with PBS for dSTORM imaging experiments.

[0011] (5) Using an inverted fluorescence microscope and an oil-immersion objective, the sample was excited with a 639 nm laser and 8000 raw frames were acquired by combining ImageJ software.

[0012] (6) Perform ThunderSTORM analysis on the raw data, reconstruct the ThunderSTORM image, and obtain the localization information of each single molecule.

[0013] (7) The distribution of ASGPR protein per unit area was calculated using the localization information, and the localization coordinates of each single molecule were imported into SR-Tesseler to analyze the aggregation of ASGPR.

[0014] The present invention is based on a super-resolution fluorescence microscope that breaks the optical diffraction limit, so that the lateral resolution can reach tens of nanometers, and can obtain detailed single-molecule localization information and assembly distribution of subcellular structure ASGPR cell membrane proteins, thereby providing a very suitable tool for solving traditional diagnostic problems.

[0015] Compared with the prior art, the present invention has less damage to the sample, less changes in the physiological activities of the cell itself, and can calculate the possibility of cell migration with less time cost and quickly complete the analysis process; because the membrane protein has a small size, the super-resolution fluorescence microscope has a higher spatial resolution and less damage to the sample. The present invention has the advantages of high reliability of results, less time required, and high accuracy of results. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0017] Figure 1 Schematic diagram of antibody-probe binding according to an embodiment of the present invention.

[0018] Figure 2 This is the distribution of ASGPR assembly on the surface of normal liver cells L-02 according to an embodiment of the present invention.

[0019] Figure 3 This is the distribution of ASGPR assembly on the surface of Hep-G2 liver cancer cells in the example of the present invention.

[0020] The present invention will be further described below in conjunction with embodiments: Example 1: Super-resolution imaging of normal human liver cells L-02: Normal human liver L-02 cells with a confluence of ~60% were fixed with 4% paraformaldehyde at room temperature for 20 min and washed three times with PBS. Cells were blocked with 3% BSA for 20 min. After removing the blocking buffer, cells were stained with Alexa647-conjugated ASGPR antibody at 4°C overnight. Samples were washed with PBS five times and dyes were added. The staining solution Alexa647-antibody was removed, and samples were washed with PBS five times. Using an inverted fluorescence microscope and an oil-immersion objective, the samples were excited with a 647 nm laser, and 8000 raw frames were obtained by combining ImageJ software for imaging the target. The raw data were analyzed by ThunderSTORM, and ThunderSTORM images were reconstructed to obtain the localization information of each single molecule. The distribution number of ASGPR proteins per unit area was calculated based on the localization information, and the coordinates of the localization points were imported into SR-Tesseler software to calculate its clustering area.

[0021] Detection: Human normal liver cells L-02 were used as the object, and the ASGPR fluorescence localization points after the experiment in Example 1 were analyzed using a super-resolution fluorescence microscope. The results showed that the average ASGPR localization point density on the basement membrane of normal liver cells L-02 was relatively high, and the result after quantitative analysis was 358 N / μm 2 , see Figure 2 .

[0022] Example 2: Super-resolution imaging of Hep-G2 liver cancer cells after ACM treatment: Human hepatoma Hep-G2 cells with a confluence of ~60% were fixed with 4% paraformaldehyde at room temperature for 20 min and washed three times with PBS. Cells were blocked with 3% BSA for 20 min. After removing the blocking buffer, cells were stained with Alexa647-conjugated ASGPR antibody at 4°C overnight. Samples were washed with PBS five times and dyes were added. The staining solution Alexa647-antibody was removed, and samples were washed with PBS five times. Using an inverted fluorescence microscope and an oil-immersion objective, the samples were excited with a 647 nm laser, and 8000 raw frames were obtained by combining ImageJ software for imaging the target. The raw data were analyzed by ThunderSTORM, and ThunderSTORM images were reconstructed to obtain the localization information of each single molecule. The distribution number of ASGPR proteins per unit area was calculated based on the localization information, and the coordinates of the localization points were imported into the SR-Tesseler software to calculate its clustering area.

[0023] Detection: Human liver cancer cell Hep-G2 cells were used as the object, and the ASGPR fluorescence localization points after the experiment in Example 2 were analyzed using a super-resolution fluorescence microscope. The results showed that the average ASGPR localization point density on the basement membrane of liver cancer cell Hep-G2 cells was relatively high, and the result after quantitative analysis was 1154 N / μm 2 , see Figure 3 .

[0024] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for imaging and detecting the assembly changes of cell surface ASGPR by super-resolution fluorescence microscopy, characterized in that: Here are the steps: (1) Fix cells with a confluence of ∼60% with 4% paraformaldehyde (PFA) for 20 min at room temperature and wash three times with PBS; (2) Block with 3% BSA for 20 min; (3) After removing the blocking buffer, cells were stained with Alexa647-conjugated ASGPR antibody at 4°C overnight, samples were washed five times with PBS, and the dye was added; (4) Remove the staining solution Alexa647-antibody, wash the samples 5 times with PBS, and use them for dSTORM imaging experiments; (5) Using an inverted fluorescence microscope and an oil-immersion objective, the sample was excited with a 639 nm laser, and 8000 raw frames were obtained by imaging the target in combination with ImageJ software; (6) Perform ThunderSTORM analysis on the raw data, reconstruct the ThunderSTORM image, and obtain the localization information of each single molecule; (7) The distribution amount of ASGPR protein per unit area was calculated based on the localization information, and the localization coordinates of each single molecule were imported into SR-Tesseler to analyze the aggregation of ASGPR.

2. The method for super-resolution fluorescence imaging according to claim 1, characterized in that: The experimental method can observe the distribution of ASGPR on the cell membrane surface.

3. The antibody probe imaging for super-resolution fluorescence imaging according to claim 1, characterized in that: The glycoprotein is an important membrane protein that exists specifically on liver cells and liver cancer cells and is widely distributed on the surface of cancer cells.

4. The specific antibody probe imaging for super-resolution fluorescence imaging according to claim 1, characterized in that: The antibody can be coupled to a fluorescent dye to locate the protein by fluorescence.

5. The specific antibody probe for super-resolution fluorescence imaging according to claim 1, characterized in that: The fluorescent dye molecule is Alexa532, Alexa647, Cy3, Cy5, Cy5.5, Cy7, Atto532, Atto488 or TAMRA.

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