JP3200607B2 - Cell diagnostic labeling agent and cell diagnostic system using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a labeling agent for cytodiagnosis, particularly to a labeling agent comprising a synthetic polymer having a sugar chain to which a fluorescent substance is immobilized. Further, the present invention relates to a cell diagnostic system by a labeling method using the labeling agent.

[0002]

2. Description of the Related Art When cells undergo malignant dedifferentiation, changes in the molecular level occurring inside and outside the cells can be monitored by electrophoresis of cell-derived substances, observation of pathological tissues, electron microscopic observation, or A labeling method of labeling a specific cell membrane protein with a probe compound such as a fluorescent or radiolabel compound has been used. However, the methods other than the labeling method are complicated because they require a long time for sample preparation and require the skill of an operator to perform accurate diagnosis.

[0003] On the other hand, the labeling method uses a monoclonal antibody having high specificity for a differentiation marker on the cell surface,
Using a labeling agent to which a radioisotope such as ¹²⁵ I or a fluorescent molecule is bound, the degree of binding of the cells to the labeling agent is mechanically quantified using a scintillation counter or a flow cytometer. According to this labeling method, the amount of an antibody bound to a differentiation marker such as a glycoprotein expressed on the cell surface is evaluated, whereby the type of the differentiation marker (phenotype) on the cell surface can be identified. Cells can be classified.

[0004] However, there has been a problem that the conventional labeling agent using a monoclonal antibody is troublesome in preparation, and is very expensive and therefore difficult to obtain. Furthermore, this labeling method is a method for diagnosing a disease condition from the type of cell itself or the abundance of specific cells in tissue or blood, based on determining the presence or absence of a differentiation marker expressed on the cell surface. Therefore, it has been difficult to precisely monitor, for example, a cell activation process and a malignant dedifferentiation process. Therefore, it was often found that the result of the cell diagnosis by the labeling method using such an antibody did not correlate with the actual condition of the patient.

[0005] Therefore, the final diagnosis of a pathological condition is currently based on visual judgment of a tissue section or a smeared specimen, and the prediction of the occurrence of the disease state, the degree of the disease state, or the prognostic state, etc. It has been desired to develop a new cell diagnostic system that can be easily and objectively monitored.

On the other hand, there has been a remarkable progress in sugar chain engineering in recent years. For example, biomacromolecules having sugar chains include proteoglycans in the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cell interactions. Glycoproteins involved in recognition are listed, but these high-molecular sugar chains gradually substitute for, assist, amplify, regulate, or inhibit each other. It is being revealed.

Further, if the relationship between such sugar chains and differentiation and proliferation of cells, cell adhesion, immunity, and canceration of cells is clarified, this sugar chain engineering, medicine, cell engineering, or organ engineering Can be expected to be closely related to the development of new developments.

As an example, sugar chains on the cell surface and sugar chains
-Research on the occurrence of diseases due to abnormal interaction between receptors, and the role of sugar chains in viral infections such as AIDS, etc., has been active. Research on the involvement of sugar chains in cell-cell interaction and cell-matrix interaction has also become important for understanding biological reactions.

In view of the above circumstances, the present inventors have studied in detail the interaction between a sugar chain and a cell, and as a result, the degree of the interaction between the sugar chain and a cell is an index indicating various states of the cell. It has been found that the present invention has been accomplished.

[0010]

That is, an object of the present invention is to provide a labeling agent for cytodiagnosis utilizing a specific interaction between a sugar chain and a cell, and to provide a labeling method using the labeling agent. Measure the degree of specific interaction between sugar chains and cells,
An object of the present invention is to construct a cell diagnostic system consisting of analysis.

[0011]

Accordingly, the present invention provides a labeling agent for cytodiagnosis comprising a fluorescent substance immobilized on a synthetic polymer having a sugar chain, and the degree of interaction between the labeling agent and cells. To provide a cell diagnostic system. More specifically, the labeling agent of the present invention is characterized in that a fluorescent substance is immobilized on a sugar chain polymer in which various sugar chains are bound to a synthetic polymer. The term “interaction between a sugar chain and a cell” as used herein refers to specific sugar chain recognition by various cells,
And interactions based on specific sugar chain recognition by sugar chain recognition receptors such as asialoglycoprotein receptors and lectin-like proteins present on various cells.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The labeling agent of the present invention will be described below in detail with reference to the drawings. FIG. 1 is a diagram showing an example of the labeling agent of the present invention, in which a plurality of sugar chain polymers are fixed around a fluorescent substance 2 with the sugar chain 3 outside and the labeling agent 1 is formed.

In the labeling agent of the present invention, as the fluorescent substance,
Fluorescent molecules such as fluorescein isothiocyanate (abbreviated as FITC), tetramethylrhodamine isothiocyanate, phycoerythrin, or Texas Red, or fluorescent beads in which fluorescent molecules are supported on beads of polystyrene, polyethylene, or the like can be used. . Above all, it is easy to manufacture and easy to obtain,
Moreover, it is preferable to use fluorescent beads which have advantages such as relatively high fluorescence intensity and easy adsorption to sugar chain polymers.

The synthetic polymer constituting the sugar chain polymer forming the labeling agent of the present invention is not particularly limited as long as a sugar chain can be introduced as a side chain and can be polymerized by an ordinary method. When using the above fluorescent beads as
It is preferable to use a hydrophobic polymer such as polystyrene, polyethylene, polypropylene, or a derivative thereof. In particular, it is more preferable to use a synthetic polymer composed of polystyrene or a derivative thereof, which is widely used as a material for the fluorescent beads, since the adsorptivity to the fluorescent beads is improved.

The sugar chains constituting the sugar chain polymer include glucose, galactose, lactose, mannose, mannobiose, N-acetylglucosamine, laminaribiose, uronic acid-related substances, and monosaccharides such as sulfated sugars.
Any oligosaccharide or the like can be used, and is not particularly limited as long as it can retain a sugar residue that specifically interacts with cells in a state of being bonded to the synthetic polymer as a side chain.

The sugar chain polymer of the present invention comprises the above-mentioned synthetic polymer as a main chain, and the above-mentioned sugar chain is bonded to the main chain as a side chain. The method of bonding to the chain is preferably a covalent bond such as an amide bond or an ester bond. For example, it is preferable to form an amide bond between the amino group of p-aminomethylstyrene and the terminal carbonyl group of the lactonized sugar. In addition, the synthetic polymer and the sugar chain may be polymerized after bonding the monomer constituting the synthetic polymer and the sugar chain molecule, or may be formed by polymerizing a monomer having a bondable functional group. They may be combined. However, since the number of sugar chains introduced into one molecule can be controlled, it is preferable to polymerize a monomer having sugar chains bonded thereto.

The sugar chain polymer is preferably a homopolymer obtained by homopolymerizing a monomer having a sugar chain bonded thereto, but may be a copolymer with another monomer.
The molecular weight of the sugar chain polymer is not particularly limited as long as it forms micelles when the sugar chain polymer is dissolved in an aqueous solution and is solubilized. In this case, about 30,000 to 50,000 are usually used.
However, for example, in the case of a copolymer with a monomer having a hydrophilic group, there are those that maintain water solubility even if the hydrophilic group has a molecular weight outside the above range,
Such a sugar chain polymer is also included in the scope of the present invention.

In the labeling agent of the present invention, among the above sugar chain polymers, for example, the following formulas (1) to (10)
The sugar chain polymer represented by is particularly preferably used.

[0019]

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[0020]

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[0021]

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[0022]

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[0023]

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[0024]

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[0025]

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[0026]

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[0027]

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[0028]

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The poly (Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 →) represented by the above formula (1)
4) -D-Gluconamide]) (hereinafter abbreviated as PV-LA) is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and lactose,
Has a β-galactose residue.

The poly (Np-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4)-) represented by the above formula (2)
D-Gluconamide]) (hereinafter abbreviated as PV-MA)
Is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and maltose, and has a glucose residue.

The poly (Np-vinylbenzyl- [O-β-D-mannopyranosyl- (1 → 4)-) represented by the above formula (3)
D-mannanamide]) (hereinafter abbreviated as PV-Man)
Is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and mannobiose, and has a mannose residue.

The poly (Np-vinylbenzyl- [O-α-D-galactopyranosyl- (1 →
6) -D-Gluconamide]) (hereinafter abbreviated as PV-MeA) is synthesized from p-aminomethylstyrene and O-α-D-galactopyranosyl- (1 → 6) -D-glucose. It is obtained by homopolymerizing the obtained monomer and has a galactose residue.

The poly (N-p-vinylbenzyl- [O-6-carboxymethyl-β-D-galactopyranosyl- (1 → 4) -OD-6-) represented by the above formula (5) Carboxymethyl-gluconamide]) (hereinafter abbreviated as PV-LACOOH) is a PV-LA obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and lactose.
Having a carboxymethylated galactose residue.

The poly (3-O-4 ′) represented by the above formula (6)
-Vinylbenzyl-D-glucose) (hereinafter abbreviated as PV-G) is obtained by homopolymerizing a monomer synthesized from p-chloromethylstyrene and glucose, and has a glucose residue.

The poly (N-p-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-) represented by the above formula (7)
Glucopyranosyl- (1 → 4) -OD-2-acetamide-
2-deoxy-β-D-glucopyranosyl- (1 → 4) -O-
D-2-acetamido-2-deoxy-β-D-gluconamide]) and poly (Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-) represented by the above formula (8). D-glucopyranosyl- (1 → 4) -OD-2-acetamido-2-
Deoxy-β-D-gluconamide]), or a mixture thereof (hereinafter, abbreviated as PV-GlcNac) both have an N-acetylglucosamine residue.

Poly (Np-vinylbenzyl-D-gluconamide) represented by the above formula (9) (hereinafter abbreviated as PV-GA) is obtained by opening D-glucose to form p-aminomethyl It is obtained by homopolymerizing a monomer combined with styrene.

The poly (Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 →) represented by the above formula (10)
3) -D-Gluconamide]) (hereinafter abbreviated as PV-Lam) is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and laminaribiose, and β1 → 3 glucose Has residues.

The sugar chain polymers of the above formulas (1) to (10) also preferably have a molecular weight of about 30,000 to 50,000, but other molecular weights can also be used. These sugar chain polymers may be used alone or in combination of two or more, and can be appropriately selected according to the purpose of use.

The labeling agent of the present invention is formed by immobilizing a fluorescent substance on the above sugar chain polymer. The fixing method may be a physicochemical method such as adsorption or a chemical method such as a covalent bond. For example, when a commercially available polystyrene fluorescent bead to which a fluorescent molecule is introduced in advance is generally used, a sugar chain polymer having a polystyrene chain as in the above formulas (1) to (10) is used as the fluorescent bead. Preferably, it is adsorbed. In this case, the labeling agent of the present invention can be easily obtained only by treating the fluorescent beads with the aqueous sugar chain polymer solution.

A specific method for treating the fluorescent beads with the aqueous sugar chain polymer solution is 1 to 500 μg / ml, preferably 1 to 500 μg / ml.
00 to 300 µg / ml, more preferably 150 to 2
Fluorescent beads are immersed in a 50 μg / ml aqueous sugar chain polymer solution, and left at room temperature for 10 minutes or more, preferably 20 minutes or more, more preferably 1 hour or more. The amount of the sugar chain polymer adsorbed on the fluorescent beads can be adjusted by the size, material and surface state of the fluorescent beads, the concentration of the sugar chain polymer aqueous solution, and the like. For example, beads having a diameter of 5.0 μm are converted to PV-
When treated with an aqueous LA solution at room temperature for 2 hours, PV-
When the amount of adsorbed LA is represented by the amount of binding of Allo-A, a lectin that recognizes β-galactose, it is about 24 ng per mg of beads.

The size of the fluorescent beads used in the labeling agent of the present invention is not particularly limited, but may be 0.00 in diameter.
Those having a thickness of about 1 to 5.0 μm, preferably about 0.1 to 1.0 μm are used.

When a fluorescent molecule such as FITC is directly immobilized, it is preferable to covalently bond to a sugar chain polymer. For example, the sugar chain polymer is added to a solvent containing a small amount of pyridine. After dissolution, FITC is added to the solution together with a catalyst such as dibutyltin dilaurate and heated. The labeling agent of the present invention can be obtained by recrystallizing the obtained reaction mixture several times in ethanol and filtering the resultant. The content of FITC is not particularly limited, but it can be detected with high sensitivity when the content is increased. Preferably, about 1 molecule of FITC is used for 20 to 200 sugar chains, and more preferably, about 40 to 60 molecules of FITC are used for FITC.
C1 molecule.

The present invention also relates to a cell diagnostic system by a labeling method using the above labeling agent. This cell diagnostic system comprises a labeling means for mixing the labeling agent and the cell and labeling the cell with the labeling agent, a measuring means for measuring the fluorescence intensity of the labeled cell, and an analysis of the measured fluorescence intensity. And analysis means.

The labeling means constituting the cell diagnostic system of the present invention comprises a labeling agent of the present invention in which the above-mentioned fluorescent substance is immobilized on a sugar chain polymer, and blood cells such as erythrocytes and lymphocytes, hepatocytes and the like. And a cell to be diagnosed such as a phagocytic cell such as a macrophage or the like, and fluorescently labeling the cell.

The labeling agent used in this labeling means is
The labeling agent is selected from the labeling agents of the present invention. The type of labeling agent to be selected is determined in consideration of the type of cell to be diagnosed, the sugar chain recognition, or the type of information to be detected.

Illustrating the specificity of sugar chain recognition by various cells, liver parenchymal cells selectively recognize galactose (Gal) and mannose (Man), whereas non-hepatic parenchymal cells recognize Man, glucose (Glc). , N-acetylglucosamine (GlcNAc). Serum tissue was Man, GlcNAc, and N-acetylmannosamine (ManNAc), lymph tissue was Ma
It specifically recognizes nNAc and GlcNAc.

In addition, sugar chain-recognizing proteins such as asialoglycoprotein and lectin-like protein on macrophages present on various cell surfaces also selectively recognize sugar chains. For example, a protein with a molecular weight of 175K on alveolar macrophages is known to be Man, Fucose, and Gl.
cNAc, 180K molecular weight protein on peritoneal macrophages Man, Fuc, and GlcNAc, 30K molecular weight protein on rat Kupffer cells, 42K molecular weight protein on peritoneal macrophages, and 45-60K molecular weight on activated macrophages The proteins are Gal and N-acetylgalactosamine (GalN
Ac) are each specifically recognized.

As an example, the PV represented by the above formula (1)
-LA adsorbed on fluorescent beads (PV-L
(Abbreviated as A labeling agent) is used to label hepatocytes. This PV-LA labeling agent has a galactose residue, and specifically binds to an asialoglycoprotein receptor present on the surface of a hepatocyte, and exhibits a very high affinity for hepatocytes via the binding. Detecting the activity of this asialoglycoprotein receptor is very important in diagnosing metabolic functions.

In order to label the asialoglycoprotein receptor on the surface of hepatocytes with this PV-LA labeling agent, for example, first, the PV-LA labeling agent is added to 0.1% bovine serum albumin (abbreviated as BSA) and 0.1% bovine serum albumin. Dissolved in phosphate buffer (abbreviated as PBS) containing 1% NaN ₃ and 10 ng / ml
Is prepared. As a sample, a PBS in which 100 × 10 ⁴ hepatocytes concentrated by centrifugation are suspended is prepared. A solution of 50 μl of the above-mentioned labeling agent may be added to the sample, and the mixture may be incubated under cooling (4 ° C.) for about 15 minutes with occasional stirring. Labeled cells are, for example, 0.1%
After being washed about twice with PBS containing NaN ₃ and, if necessary, diluted, it is provided to the measuring means. The mixing ratio between the cells and the labeling agent was 10 ng for 100 × 10 ⁴ hepatocytes.
It is preferable that the concentration of the labeling agent aqueous solution be about 50 to 100 μl, but it is not particularly limited.

The fluorescence intensity of the fluorescently labeled cells is measured using a measuring means. The measurement means used in the present invention is not particularly limited as long as it is a means capable of measuring the fluorescence intensity of each cell, such as a flow cytometer and a fluorescence spectrophotometer. It is particularly preferable because the fluorescence intensity of each cell can be measured, and cells can be sorted based on the measurement results. Furthermore, in the measurement using a flow cytometer, in addition to the fluorescence intensity indicating the biological activity of the cell, forward scattered light that reflects the size of the cell in the axial direction of the laser beam applied to the cell, This is preferable because the amount of information obtained is large, such as detection of two types of light, side scattered light that reflects the state of the cytoplasm in the direction.

Further, the cell diagnostic system of the present invention has an analyzing means for analyzing the biological activity and the disease state of the cells using the data of the fluorescence intensity measured by the measuring means.
For example, the activity of a receptor that specifically recognizes a sugar chain can be known from the measurement results of the fluorescence intensity of cells labeled with a labeling agent having one type of sugar chain. When the measurement is performed using a flow cytometer, information on the size of the cell, the state of the cytoplasm, and the like can be obtained by detecting forward scattered light and side scattered light.

For example, in the case of hepatocytes labeled with only the PV-LA labeling agent exemplified above, the activity, alteration, density, etc. of the asialoglycoprotein receptor present on the hepatocyte surface can be quantitatively determined, and Effective information for function diagnosis can be obtained. In addition, we have determined that this P
When a V-LA labeling agent is used for labeling liver cancer cells, it emits fluorescence of different intensity depending on the type of liver cancer.
They also found that the type of cancer could be identified.

However, in the cytodiagnosis system of the present invention, it is particularly preferable to measure the same kind of cells by labeling them with at least two, preferably five to ten different labeling agents having different sugar chains. preferable. By comparing the fluorescence intensities measured for cells labeled with a plurality of different labeling agents in this manner, the state of the cells can be known in more detail.

For example, when cells of the same kind are respectively labeled with seven kinds of labeling agents each having seven kinds of sugar chains A to H, and the fluorescence intensity of those cells is measured by a flow cytometer. Even in cells of the same kind, different fluorescence intensities are obtained according to the respective sugar chains. The result is shown in FIG.
Can be combined into a histogram as shown in FIG. From this fluorescence pattern, a large number of active sugar chain recognition molecules having specific affinity for sugar chains E and G exist on the cell surface, and almost no sugar chain recognition molecules specific for sugar chains C and D are found. Absent or inactivated,
The other sugar chain recognition molecules specific to sugar chains A, B, and H are found to have an intermediate number or activity between the two, and the phenotype of the cell can be identified in detail.

Therefore, the cell diagnostic system of the present invention preferably further comprises storage means such as a memory for storing the above-mentioned fluorescent pattern. By storing a large number of fluorescent patterns in such a storage means and storing them in a database, for example, a fluorescent pattern obtained by analyzing an unknown cell with the cytodiagnosis system of the present invention can be converted into a large number of fluorescent data in the database. By comparing with the pattern,
The phenotype of the cell can be easily identified. Therefore, the cell diagnostic system of the present invention preferably includes a logical operation means for performing such a comparison.

The fluorescent pattern according to the present invention is not limited to the above-mentioned histogram, but may be displayed as a graph such as a line graph, a pie graph, or a numerical value. However, in order to easily determine the similarity of the phenotypes, it is preferable to display them in a graphic such as a graph.

[0057]

EXAMPLES Examples are shown below to specifically explain the labeling agent and the cell diagnostic system of the present invention. (Example 1) (1) Synthesis and coating of sugar chain polymer The sugar chain polymers represented by the above formulas (1) to (5), (7), and (9) were prepared by a known method (Ichi Kobayashi) Seira, polymer
Journal, Vol. 17, pp. 567-575, 198
5 years), the respective sugars constituting each sugar chain polymer are lactonized, the lactonized saccharide is reacted with p-aminomethylstyrene to synthesize a monomer, and the monomer is homopolymerized. Synthesized. The sugar chain polymer represented by the above formula (6) was prepared by a known method (Kobayashi Kazunori et al., Macromolecules, vol. 13, page 234, 1980).
), Glucose was reacted with p-chloromethylstyrene to synthesize a monomer, and the monomer was homopolymerized to synthesize the monomer.

Next, polystyrene latex beads (5 μm in diameter) containing no fluorescent substance were added to each of 200 μg / m 2.
1) The above-mentioned sugar chain polymer aqueous solution was treated at room temperature for 2 hours to be adsorbed, thereby preparing eight kinds of sugar chain polymer-coated polystyrene beads.

(2) Confirmation of Coating of Polystyrene Beads with Sugar Chain Polymer The above-mentioned polystyrene beads coated with a sugar chain polymer were characterized using lectin which is a sugar chain recognizing protein. 4 kinds of lectins labeled with FITC,
That is, a lectin for human natural killer cell separation (E
CL), peanut agglutinin (PNA), lentil agglutinin (LCA), and pea agglutinin (PSA). Of these, ECL and PNA are galactose-recognizing, and LCA and PSA are mannose-recognizing.

[Labeling with Fluorescent Lectin] ECL, P
20 μl of a 20 ng / ml PBS suspension of NA, LCA and PSA, and PBS containing 10 ng / ml of each of the eight types of sugar chain polymer-coated polystyrene beads prepared above
The mixture was mixed with 50 μl of the solution, incubated at room temperature for 30 minutes, and then washed several times with PBS.

The fluorescence intensity of the sugar chain polymer-coated polystyrene beads treated with the fluorescent lectin as described above was measured with a flow cytometer (FACScan, manufactured by Becton Dickinson Immunocytometry System). The results are shown in FIG. From FIG. 3, for example, in beads coated with PV-LA having a galactose residue, PNA and ECL bind preferentially, and in beads coated with PV-Man having a mannose residue,
PSA and LCA are preferentially bound.

From the above results, in the polystyrene beads coated with the above sugar chain polymer, the specificity of the coated sugar chain polymer is clearly expressed. Therefore, it can be seen that the labeling agent of the present application in which fluorescent polystyrene beads are coated with a sugar chain polymer can specifically label cells or the like that interact with the sugar chain.

(3) Preparation of Labeling Agent Using Fluorescent Beads Fluorescent beads having a diameter of 0.5 μm (Fluo
Spheres ^R , manufactured by Molecular Probes). The fluorescent beads are obtained by supporting a yellow-green fluorescent dye on polystyrene beads, and are provided in the form of a latex having a solid content of 2% of the beads. The fluorescent beads are used for 200
The resultant was treated with 2 μg / ml of each of the above sugar chain polymer aqueous solutions at room temperature for 2 hours and adsorbed to prepare eight kinds of labeling agents.

Example 2 (1) Labeling of Cells with the Labeling Agent of the Present Invention Rat spleen cells and red blood cells were labeled with the eight labeling agents of Example 1 in the following manner. First, each of the above eight kinds of labeling agents was added to 0.1% BSA and 0.1% NaN.
Suspended in PBS containing ₃ to prepare a 10 ng / ml labeling agent solution. Next, a concentrated cell suspension is prepared by centrifuging PBS in which 100 × 10 ⁴ rat spleen cells (mainly lymphocytes) are suspended at 1500 rpm for 10 minutes and removing the supernatant. A solution of 50 μl of the above-mentioned labeling agent solution was added to the cell suspension, and the mixture was incubated under cooling (4 ° C.) for about 15 minutes with occasional stirring.

Then, PBS containing 0.1% NaN ₃
2 ml was added, and the mixture was centrifuged at 1500 rpm for 10 minutes to precipitate the cells, and the supernatant was removed to remove the labeling agent not bound to the cells.
1 ml of PBS containing 1% NaN ₃ was added and diluted to obtain a labeled spleen cell sample. A labeled red blood cell sample was prepared in the same manner.

(2) Measurement The fluorescence intensities of the spleen cell sample and the red blood cell sample labeled with the eight labeling agents of the present invention prepared as described above were determined as follows.
Each was measured with a flow cytometer (FACScan, manufactured by Becton Dickinson Immunocytometry System).

(3) Analysis The results obtained by the flow cytometer are shown in FIG. 4 as a fluorescence pattern consisting of a histogram with the type of sugar chain on the vertical axis and the relative fluorescence intensity on the horizontal axis. However, the fluorescence pattern of spleen cells is shown in FIG. 4 (A), and the fluorescence pattern of red blood cells is shown in FIG. 4 (B). It can be seen that the fluorescence patterns of spleen cells and red blood cells are clearly different.
In addition, the relative fluorescence intensity in FIG. 4 represents a relative value with respect to the fluorescence intensity obtained from the cells treated with the fluorescent beads not adsorbing the sugar chain polymer. That is, these values reflect the specific effect obtained by coating the fluorescent beads having no cell specificity with the sugar chain polymer.

Example 3 FIG. 5 shows a fluorescence pattern obtained by applying the cytodiagnosis system of the present invention to healthy human peripheral blood mononuclear cells (PBMC) in the same manner as in Example 2. On the other hand, PBMC cells are collected from a patient with human acute myeloid leukemia (AML), and fluorescence patterns (AML1, AML2) obtained by applying the cytodiagnosis system of the present invention in the same manner.
Is also shown in FIG. In the fluorescent pattern of AML1,
Although the overall fluorescence intensity is higher than the fluorescence pattern obtained from a healthy subject, the affinity for PV-Man and PV-GlcNAc is particularly increased. Further, in the fluorescence pattern of AML2, the affinity for PV-LA and PV-G is increased as compared with healthy subjects.

The PBMC cells whose fluorescence patterns are shown in FIG. 5 show the difference between healthy subjects and those who have developed AML.
Even those affected by ML showed different fluorescence patterns depending on the degree of the difference. From these facts, by analyzing the fluorescence pattern obtained by applying the cell diagnostic system of the present invention, useful information to know the presence or absence of a disease in the cell and the degree of the disease can be obtained.

Example 4 As another embodiment of the labeling agent of the present invention, a labeling agent was prepared by directly binding a fluorescent molecule to PV-LA. (1) Synthesis of PV-LA As in Example 1, first, the monomer Np-vinylbenzyl- [o-β-D-galactopyranosyl- (1 → 4)-
D-Gluconamide was synthesized, and the monomer was homopolymerized to synthesize PV-LA.

(2) Fixation of fluorescent dye 500 mg of PV-LA (corresponding to 10 × 10 ⁻³ mol of monomer units) was dissolved in 5 ml of dry dimethyl sulfoxide to which 3 drops of pyridine had been added. 50 mg of FITC (manufactured by Wako Pure Chemical Industries) was added to the solution, and 1
After adding 5 mg of dibutyltin dilaurate, 90 ° C.
For 2 hours. The obtained reaction mixture was recrystallized several times in ethanol and filtered to obtain the labeling agent of this example.

The amount of FITC immobilized on the labeling agent of the present example thus synthesized was measured using a fluorescence spectrophotometer (RF-5).
00, manufactured by Shimadzu Corporation. As a result, P
It was found that lactose was fixed at a ratio of 1 molecule per 40 molecules in the V-LA polymer.

In order to confirm the affinity between this PV-LA and the asialosaccharide receptor present on the surface of the hepatocyte, PV-LA and PV-MA not immobilized with a fluorescent substance, asialofetwin, fetuin, and Rat hepatocytes pretreated with LDL were labeled with the labeling agent of this example, and the fluorescence intensity of the labeled hepatocytes was measured using a flow cytometer (FA).
CScan, manufactured by Becton Dickinson Immunocytometry System).

Here, asialofetuin is a natural ligand of asialoglycoprotein receptor, and fetuin is a compound in which sialic acid is bound to asialofetuin. In this pretreatment, each of the above substances and BSA
PBS containing 0.1% by weight and 0.1% by weight of NaN ₃
The hepatocytes were suspended therein and performed at 4 ° C. for 40 minutes. The labeling treatment was performed in the same manner as in Example 2 using a PV-LA labeling agent solution having a concentration of 100 μg / ml. The results are expressed as relative intensity to the fluorescence intensity of hepatocytes labeled without pretreatment, and are shown in Table 1.

[0075]

[Table 1]

In the case of pretreatment with PV-LA and asialofetuin, since the asialoglycoprotein receptor on the surface of the hepatocyte is blocked by PV-LA and asialofetuin, the label that binds in the subsequent labeling treatment is used. The amount of the agent is reduced, and the relative fluorescence intensity is significantly reduced. On the other hand, in hepatocytes pretreated with PV-MA and fetuin, little change in fluorescence intensity is seen. That is, PV-
LA was found to have an affinity for the asialoglycoprotein receptor over that of the natural ligand asialofetuin.

(Example 6) Measurement was performed using a hepatoma cell labeled with the labeling agent of Example 5 with a flow cytometer. The labeling method was the same as in Example 2. The types of cancer cells used were human-derived Hep G2 and Chang.
Liver. Table 2 shows the results.

[0078]

[Table 2]

From the above results, liver cancer cells labeled with the labeling agent of the present invention containing PV-LA emit fluorescence with different intensities depending on the type of the cancer. That is, the labeling agent of the present invention reflects the pathological state of the cell in detail, and by using the diagnostic system of the present invention, for example, only by flow cytometry measurement of hepatocytes, the presence or absence of carcinogenesis, It turns out that the type can be identified.

[0080]

The labeling agent of the present invention utilizes a specific interaction between a sugar chain and a cell and can be synthesized without complicated operations such as a labeling agent using a conventional monoclonal antibody. Inexpensive and easily available without cost. In particular, when a sugar chain polymer in which a sugar chain is bonded to a styrene-based polymer is used, the adsorptivity to ordinary fluorescent beads is good, and it is stable and easy to maintain quality compared to conventional labeling agents. It is. In addition, in the cell diagnostic system of the present invention using the above-described labeling agent, it is not necessary to prepare a special antibody for each cell. Can be. Therefore, it is possible to easily identify the phenotype of the cell and grasp the disease state.

[Brief description of the drawings]

FIG. 1 is a schematic view showing the structure of the labeling agent of the present invention.

FIG. 2 is a diagram showing an example of a fluorescence pattern obtained by the cell diagnostic system of the present invention.

FIG. 3 is a histogram showing the degree of interaction between a sugar chain polymer serving as a labeling agent of the present invention and four types of lectins labeled with fluorescence.

FIG. 4 is a diagram showing a fluorescence pattern obtained by applying the cytodiagnosis system of the present invention to hepatocytes (A) and red blood cells (B).

FIG. 5: PB of a healthy person and a person having a disease (AML)
It is a figure which shows together the fluorescence pattern obtained by applying the cytodiagnosis system of this invention to MC cell.

[Explanation of symbols]

 1 ... labeling agent, 2 ... fluorescent substance, 3 ... sugar chain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 33/566 G01N 33/566 (56) References JP-A-7-35754 (JP, A) , A) WO 95/4751 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/53 C08B 37/00 C12Q 1/02 G01N 33/543 597 G01N 33/566

Claims (7)

(57) [Claims] 1. The method according to claim 1, wherein the amount of the sugar chain polymer to which the fluorescent substance is fixed is reduced.
A cell diagnosis comprising a labeling means for mixing two kinds of cells with a cell to fluorescently label the cell, a measuring means for measuring the fluorescence intensity of the labeled cell, and an analyzing means for obtaining a fluorescence pattern from the measured fluorescence intensity system. Wherein said measuring means, cytology system according to claim 1, characterized in that from the flow cytometry. 3. Furthermore, cytology system according to claim 1, characterized in that it comprises a storage unit for storing the fluorescent pattern. 4. The apparatus according to claim 3, further comprising logical operation means for comparing the fluorescence pattern stored in said storage means with a fluorescence pattern obtained from the fluorescence intensity measured by said measurement means. Cell diagnostic system. 5. The method according to claim 1, wherein the amount of the sugar chain polymer to which the fluorescent substance is fixed is reduced.
2. The composition according to claim 1, wherein the two kinds are contained .
Labeling agent for cytodiagnosis system . 6. The labeling agent according to claim 5 , wherein the fluorescent substance is a fluorescent bead. 7. The sugar chain polymer is poly (N-p-vinylbenzyl- [O-β-D-galactopyranosyl- (1 → 4) -D-gluconamide]), poly (N- p-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4) -D-gluconamide]), poly (Np-vinylbenzyl- [O-β-D-mannopyranosyl- (1 → 4)) -D-mannanamide]), poly (Np-vinylbenzyl- [O-α-D-galactopyranosyl- (1 → 6) -D-gluconamide]), poly (Np-vinylbenzyl-) [O-6-carboxymethyl-β-D-galactopyranosyl- (1 → 4) -OD-6-carboxymethyl-gluconamide]), poly (3-O-4′-vinylbenzyl-D) -Glucose), poly (Np-vinylbenzyl- [O-2-acetamido-2]
-Deoxy-β-D-glucopyranosyl- (1 → 4) -OD-
2-acetamido-2-deoxy-β-D-glucopyranosyl
-(1 → 4) -OD-2-acetamido-2-deoxy-β-
D-gluconamide]) and / or poly (Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-
Glucopyranosyl- (1 → 4) -OD-2-acetamide-
2-deoxy-β-D-gluconamide]), poly (Np-vinylbenzyl-D-gluconamide), and poly (Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 is selected from → 3)-D-gluconic amide]
Labeling agent according to claim 5, wherein a.