JP4746276B2 - Method for analyzing membrane function of biological structure having membrane structure - Google Patents

Description

  The present invention relates to a method for analyzing the membrane function of a biological structure by measuring the fluctuation strength of a marker molecule in the membrane structure of the biological structure (for example, a cell or an organelle). The present invention also provides a method for evaluating the properties of a biological structure by analyzing the membrane function of the biological structure, and analyzing the membrane function of the biological structure in the presence and absence of the test substance. The present invention relates to a method for evaluating the influence of a test substance on a living body. Especially this invention is useful when evaluating the toxicity which the test substance gives to the anatomy.

  In order to evaluate the effect of a compound on a living body, in recent years, a shift is being made from administration experiments using animals to methods using cultured cells. When a compound acts on a cell, first, its involvement in the cell membrane occurs as the first step, where an interaction with a substance that forms a membrane structure, that is, (sugar) lipid or protein occurs. Next, it is expected that the environment inside and outside the membrane will change due to the changed membrane structure, thereby affecting the cell signal system. When an adverse effect is exerted, the function of the cell itself is eventually lost, leading to cell death. Thus, the time required for the influence of the compound to appear (until cell death) varies depending on the type of cell or compound. However, the first step, the relationship between the compound and the membrane, is a very short-term phenomenon. By accurately monitoring these, the effects of the compound on the cells (cell death) can be predicted early. It is considered possible.

  As a method for measuring the toxicity of a compound to a membrane, a method using liposome has been conventionally used. For example, there is a method in which a pH-sensitive dye (acridine orange) is encapsulated in a liposome, and the fluorescence of the dye that flows out when an external compound breaks the liposome membrane is measured (Patent Document 1 (Special Tables 2002-517726, 20). page)). In addition, as a method using liposomes, an immunoassay method has been reported in which the fluorescence of dye released from liposomes is measured by the formation of an antigen-antibody complex on the surface of the liposome membrane and the action of complement acting on it. (Patent Document 2 (Japanese Patent Laid-Open No. 7-199103, page 5)).

  On the other hand, in fluorescence correlation spectroscopy, the molecular diffusion rate, the number of molecules at one molecule level, and intermolecular interactions are required from these information by measuring fluctuations of fluorescent molecules in a minute laser focal region. . According to this method, a phenomenon in which liposome-encapsulated rhodamine flows out of the membrane by a compound (Melittin) has been measured (Non-patent Document 1 (Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy., Pramanik A, Thyberg). P, Rigler R., Chem Phys Lipids 2000 Jan; 104 (1): 35-47)). In addition, regarding the interaction evaluation of receptors and ligands on biological structures such as cells using fluorescence correlation spectroscopy, the invention of Rigler et al. Has been reported as a prior art (Patent Document 3 (Patent Document 3). 11-502608, p. 115).

  Although the conventional method using liposomes has the advantage of simplifying the experimental system, the liposome system is based on the fact that the physiological functions of actual biological structures (cells, etc.) are maintained by their complex structures. It is considered to be a very special model for anatomy. Furthermore, a certain amount of know-how and man-hours are required to create a uniform liposome. Therefore, an evaluation system using an actual biological structure as it is for an experiment is required, and a system in which measurement is performed under the condition that the physiological function is maintained normally is required.

  Further, in the case of a cytotoxicity test using a conventional biological structure, there is a drawback that a sufficient amount of cells are required and it takes time to evaluate the influence of the test substance on the cells.

Special Table 2002-517726, page 20

JP 7-1991033 A, page 5

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy., Pramanik A, Thyberg P, Rigler R., Chem Phys Lipids 2000 Jan; 104 (1): 35-47

No. 11-502608, page 115

  In view of the above circumstances, an object of the present invention is to provide a method for analyzing a membrane function of a living body structure in a short time using a minute amount of living body structure (cell or the like) that maintains a physiological function. In particular, an object of the present invention is to provide a method for evaluating the influence (for example, toxicity) of a test substance on a living body in a short time using a very small amount of biological structure (cells, etc.) that maintains physiological functions. is there.

  The inventors have solved the above-mentioned problem, that is, a method for analyzing the membrane function of a biological structure in a short time by measuring the fluctuation strength of the marker molecule on the membrane of the biological structure maintaining physiological functions. developed.

That is, the present invention provides the following means.
(1) A method for analyzing a membrane function of a biological structure having a membrane structure,
Fixing a biological structure having a membrane structure to a measurement container under conditions in which the biological structure can maintain physiological functions;
Binding marker molecules to the membrane structure of the biological structure;
Measuring the intensity fluctuation of the marker molecule bound to the membrane structure.
(2) A method for evaluating the properties of the biological structure by analyzing the membrane function of the biological structure having a membrane structure,
Fixing a biological structure having a membrane structure to a measurement container under conditions in which the biological structure can maintain physiological functions;
Binding marker molecules to the membrane structure of the biological structure;
Measuring intensity fluctuations of the marker molecules bound to the membrane structure;
Associating the measurement result obtained by the step with the property of the anatomy based on a predetermined reference value of intensity fluctuation.
(3) A method for evaluating the influence of a test substance on a living body by analyzing the membrane function of a biological structure having a membrane structure,
Fixing a biological structure having a membrane structure to a measurement container under conditions in which the biological structure can maintain physiological functions;
Binding marker molecules to the membrane structure of the biological structure;
Reacting a biological structure having the marker molecule with a test substance;
Measuring intensity fluctuations of the marker molecules bound to the membrane structure;
A method comprising a step of comparing a measurement result obtained by the above step with a measurement result in the absence of a test substance.

(4) The evaluation method according to (3), wherein the intensity fluctuation is measured when the test substance and the marker molecule are suspected to have a competitive reaction with the biological structure. From the result, (i) the diffusion rate of the marker molecule and (ii) the ratio of the marker molecule in a state bound to the membrane structure and the marker molecule in a state released from the membrane structure are calculated.
(5) The method according to any one of (1) to (4), wherein the anatomical structure is an animal or plant-derived cell or organelle.
(6) The method according to any one of (1) to (4), wherein the biological structure is a blood cell or a nerve cell.
(7) The method according to any one of (1) to (6), wherein the marker molecule is a fluorescent marker molecule, and fluorescence intensity fluctuation of the fluorescent marker molecule is detected by fluorescence correlation spectroscopy or fluorescence. A method characterized by being performed by a polarization method.
(8) An evaluation article in which a biological structure having a membrane structure and having a marker molecule bound to the membrane structure is held under conditions that allow the biological structure to maintain physiological functions, The ratio [x / y] between the intensity fluctuation [x] of the marker molecule that freely moves in the vicinity and the intensity fluctuation [y] of the marker molecule that is bound to the membrane structure of the living body structure and the free movement is suppressed, An evaluation article characterized by being 0.2 or less.

  Since the method of the present invention uses a biological structure that maintains its physiological function, it can be analyzed and evaluated under conditions close to those of a living body. In addition, since the method of the present invention measures the intensity fluctuation of the marker molecule bound to the membrane structure, it can be measured by using a very small amount of biological structure and can be measured in a short time (several tens of seconds). Is possible.

  Hereinafter, the present invention will be described in detail, but the following description is for explaining the present invention and is not intended to limit the present invention.

[1] Method for Analyzing Membrane Function of Biological Structure Having Membrane Structure According to one aspect, the method of the present invention for analyzing the membrane function of a biological structure having a membrane structure,
(1) fixing a biological structure having a membrane structure to a measurement container under a condition in which the biological structure can maintain a physiological function;
(2) binding a fluorescent marker molecule to the membrane structure of the biological structure;
(3) measuring a fluorescence intensity fluctuation of the fluorescent marker molecule bound to the membrane structure.

  In the present invention, the biological structure refers to any structure existing in a living body having a membrane structure, and more specifically refers to a cell, an organelle, a nucleus, and a mitochondria. Hereinafter, the case where a cell is used as a living body structure will be described as an example. The biological structure is not limited to a natural substance obtained by extraction or excision from the living body, but may be a substance synthesized or cultured so as to have the same function in the living body. Further, even a naturally derived biological structure may be subjected to chemical or physical treatment so as to meet the purpose of analysis.

  In the step (1), the cells are fixed to the measurement container by adhering to the bottom of the measurement container and culturing the cells at room temperature in the cell culture solution in the measurement container. For floating cells such as blood cells, the cells are fixed to the bottom of the glass via an adhesion factor such as lectin (see FIG. 1). As the cell culture solution, a solution having a physiological condition in which cells can grow (eg, PBS) can be used, and a pH adjusted to around 7.4 can be used. In particular, during the FCS measurement, the red phenol dye contained in the cell culture medium that is usually used may interfere with the measurement. Therefore, a medium from which the dye is removed is preferably used instead. As a medium not containing such a dye, a commercially available medium called Opti-MEM (trademark) may be used.

  In step (2), a fluorescent marker molecule is bound to the membrane structure of the cell. In the present invention, a fluorescent marker molecule is generally a marker molecule having a hydrophobic site, but a marker molecule having a long hydrophobic portion is particularly preferable. For example, Octadecyl Rhodamine B Chrolide (hereinafter referred to as R18) can be used. In addition, it may be a general dye having no hydrophobic site, or a dye labeled with a binding probe having a hydrophobic site. Examples of such binding probes include Phosphatidylcholin or DPH (diphenylhexatoriene). ). By adding such a fluorescent marker molecule to several nM of a cell-containing solution (cell concentration 30 to 50% by weight) and culturing for 10 to 60 minutes, R18 is bound to the biological membrane. The molecular structure of R18 is shown in FIG. Steps (1) and (2) may be performed first.

  In step (3), the fluorescence intensity fluctuation of the fluorescent marker molecule bonded to the film structure is measured. For example, FCS measurement is performed for about 10 seconds by placing the laser focus of the fluorescence correlation spectrometer near the biological membrane to which the fluorescent marker molecule (R18) is bound. If sufficient fluorescence intensity and the associated autocorrelation function are obtained, the “diffusion rate” is calculated. Further, since R18 that is not bonded in the vicinity of the film is freely diffusing, the “ratio” between the film-bound R18 and R18 that is not bonded to the film is calculated by this autocorrelation function.

[2] Method for evaluating properties of biological structure having membrane structure The method of the present invention for evaluating the properties of biological structure having a membrane structure, according to one embodiment,
(1) fixing a biological structure having a membrane structure to a measurement container under a condition in which the biological structure can maintain a physiological function;
(2) binding a fluorescent marker molecule to the membrane structure of the biological structure;
(3) measuring the fluorescence intensity fluctuation of the fluorescent marker molecule bound to the membrane structure;
(4) including a step of associating the measurement result obtained by the step with the property of the biological structure based on a predetermined reference value of the fluorescence intensity fluctuation.

  For the above steps (1) to (3), refer to the description of steps (1) to (3) in “Method for analyzing membrane function of biological structure having membrane structure”. In step (4), the measurement result obtained in step (3) is associated with the properties of the living body structure based on a predetermined reference value of the fluorescence intensity fluctuation. For example, based on the measurement result (reference value) of the fluorescence intensity fluctuation of the fluorescent marker molecule of the normal cell structure (reference sample), the measurement result of the fluorescence intensity fluctuation of the fluorescent marker molecule of the cell structure (test object) And associated with the properties of the anatomy (presence of aging or disease). Alternatively, using a cell structure having various aging (or disease) levels in addition to a normal cell structure as a reference sample, the fluorescence intensity fluctuations of these fluorescent marker molecules are measured, and a plurality of reference values are set. Thus, the measurement result of the fluorescence intensity fluctuation of the fluorescent marker molecule of the cell structure (subject to be examined) can be associated with the degree of aging or disease (see Example 2 described later).

  Thus, the property of a biological structure can be evaluated by performing the dynamic analysis of the fluorescent molecule in the biological membrane using the evaluation method of the present invention. In addition, the present invention uses a biological structure evaluated as being in a state of membrane modification due to a disease, administers a therapeutic agent thereto, and measures the fluorescence intensity fluctuation using the evaluation method of the present invention. It is also possible to monitor the effects of therapeutic agents.

[3] Method for evaluating the influence of a test substance on a living body The method of the present invention for evaluating the influence of a test substance on a living body, according to one embodiment,
(1) fixing a biological structure having a membrane structure to a measurement container under a condition in which the biological structure can maintain a physiological function;
(2) binding a fluorescent marker molecule to the membrane structure of the biological structure;
(3) reacting the biological structure having the fluorescent marker molecule with a test substance;
(4) measuring the fluorescence intensity fluctuation of the fluorescent marker molecule bound to the membrane structure;
(5) including a step of comparing the measurement result obtained by the above step with the measurement result in the absence of the test substance.

This method can be carried out in the same manner as the above method. Therefore, only steps different from the above method will be described below.
In the step (3), the biological structure having the fluorescent marker molecule is reacted with the test substance. The reaction here is, for example, culturing cells (cell concentration 30 to 50% by weight) as a biological structure and a test substance of 10 to 80 nM for 10 to 60 minutes under the same conditions as in the above step (1). Can be done.

  In step (5), the measurement result of the fluorescence intensity fluctuation measured in the presence of the test substance is compared with the measurement result of the fluorescence intensity fluctuation measured in the absence of the test substance. By this comparison, it is possible to detect a case where the test substance affects the film structure and increases or decreases the viscosity of the film.

  For example, if it is suspected that the analyte and the fluorescent marker molecule on the membrane have a competitive reaction with the biological structure, the “diffusion rate” of the fluorescent marker molecule can be determined from the measurement result of the fluorescence intensity fluctuation. In addition to the calculation, the “ratio” between the fluorescent marker molecule bound to the membrane structure and the fluorescent marker molecule released from the membrane structure may be calculated from the measurement result of the fluorescence intensity fluctuation. That is, “diffusion rate” and “ratio” may be compared with “diffusion rate” and “ratio” data obtained in the absence of the test substance, respectively (see Example 3 and Table 2 below). . As shown in Example 3, when the test substance is a substance that competes with the fluorescent marker molecule, the “diffusion rate” of the fluorescent marker molecule is slowed by the addition of the test substance, and the fluorescent marker bound to the membrane structure It is observed that the “ratio” of molecules decreases and the “ratio” of fluorescent marker molecules released from the membrane structure increases.

  Also, by using the evaluation method of the present invention, the change in the influence of the test substance on the living body structure is evaluated over time by measuring the fluorescence intensity fluctuation over time after the test substance is given to the cell. be able to.

[4] Method for Measuring Fluorescence Intensity Fluctuation In the present invention, the fluctuation in fluorescence intensity emitted by the fluorescent marker molecule can be measured using fluorescence correlation spectroscopy (FCS) or fluorescence polarization.

  In fluorescence correlation spectroscopy, the excitation laser beam is narrowed down by an objective lens with a high NA (1.2), and the fluorescence marker molecules enter and exit in a very small area (sub-femtoliter) in the sample on the stage, that is, the fluctuation of fluorescence intensity is high. This is a method of detecting by a sensitivity detector. An autocorrelation function is obtained from the obtained data, and the average number of molecules passing through the minute region, the average diffusion rate of the molecules, etc. are obtained (FIG. 3). The autocorrelation function is expressed by the following equation.

  Thus, fluorescence correlation spectroscopy can be measured at the single molecule level due to its high sensitivity, and many examples of in vitro or in vivo measurement have been reported so far. Furthermore, the combination of FCS and a microscope provides non-invasiveness of FCS and high spatial resolution using a microscope. In particular, cell (in vivo) measurements can capture the movement of proteins, DNA, etc. in cells. . An example of the apparatus is shown in FIG. In FIG. 4, the graph shown in the Digital Correlator shows the fluorescence fluctuation and autocorrelation function measured in order from the top.

  Fluorescence polarization, on the other hand, is a method of measuring fluorescence intensity fluctuations due to the rotational Brownian motion inherent to each molecule. Based on the phenomenon of depolarization, the speed of rotational motion of molecules that generate fluorescence signals is fast (fluorescence intensity This is a method of detecting a change in the polarization state under such a condition that the polarized light is canceled when the fluctuation is large) and the polarized light is stored without being canceled when the rotational speed is reduced.

[5] Evaluation article According to one aspect, the present invention provides an evaluation article having the following characteristics. The evaluation article of the present invention has a membrane structure having a membrane structure in which a fluorescent marker molecule is bound to the membrane structure under conditions (in solution) under which the organism structure can maintain physiological functions. Intensity fluctuation [x] of a fluorescent marker molecule that freely moves in the vicinity of a living body structure and an intensity fluctuation of a fluorescent marker molecule that is bound to the membrane structure of the living body structure and has free movement suppressed [ The ratio [x / y] to y] is 0.2 or less. Here, “intensity fluctuation [x]” and “intensity fluctuation [y]” are values that reflect the total number of fluorescent marker molecules, respectively. The evaluation article of the present invention can be used for evaluating the properties of a biological structure having a membrane structure, or for evaluating the influence of a test substance on a living body.

  In each of the methods and articles of the present invention described above, it is preferable that the biological structure having a membrane structure and the marker molecule are selected so as to have the following relationship in the characteristics of molecular motion in the liquid. That is, as described in the evaluation article [5], the ratio [x / y] is preferably 0.2 or less. By washing the evaluation article with a buffer solution or the like in which the article is suspended, the number of “unbound marker molecules that freely move in the vicinity of the biological structure” can be reduced.

  In general, the rate at which marker molecules bound to biological structures are detached during the measurement time is considered to be about 5 to 30% (about 20% for erythrocytes and about 5% for nerve cells), and is synthesized like a liposome. If it is a living body structure, there may be 20 to 50% detachment. In the present invention, focusing on the rate at which the fluorescent molecules are detached with time, the ratio of [x / y] is set to 0.2 or less in consideration of this rate, and the marker is not bound to the film structure that may cause measurement noise. The amount of molecules is reduced in advance. In other words, even if there is a marker molecule that is not bound to the biological structure from the beginning, if the ratio of [x / y] is 0.2 or less, the above-mentioned ratio of the marker molecule is detached. Even measurement is possible.

  When measuring only the binding part (for example, membrane structure) of a marker molecule in a biological structure, the unbound marker molecule does not cause measurement noise, but in this case too, excess light of excitation light from the unbound marker molecule In order to reduce absorption, the ratio [x / y] is preferably 0.2 or less.

  On the other hand, when unbound marker molecules are present in detectable amounts, the marker molecules behave in such a way that the marker molecules bound to the membrane structure leave or the unbound marker molecules bind to the membrane structure. Can also be monitored continuously. When the unbound marker molecule is positively used in this way, the [x / y] ratio is preferably 0.05 to 0.2. In any case, when the ratio of [x / y] is 0.2 or less, the measurement value of the intensity fluctuation of the marker molecule bound to the biological structure and the measurement value of the intensity fluctuation of the unbound marker molecule are effective. Can be distinguished.

  FCS measurement: All measurements were performed on a commercial instrument, ConfoCor2 (Carl Zeiss). Excitation was performed with a helium-neon laser (543 nm), and fluorescence was detected with a detector (APD) through a band-pass filter BP560-615 nm.

[Example 1] Measurement of human erythrocyte membrane denatured by physicochemical change (1) Erythrocyte membrane change when changing buffer salt concentration to make hypotonic solution Normally, normal erythrocytes are isotonic solution buffer In (NaCl concentration: 140 mM), the center shows a concave shape, and the shape can be flexibly deformed so that it can pass through even a thin blood vessel. However, when the extracellular buffer is replaced with a hypotonic solution (NaCl concentration: 70 mM), water enters the cell from the outside due to the osmotic pressure, and the cell shape becomes spherical. Moreover, in this case, the deformability is greatly reduced as compared with the normal case.

Since the cell membrane is expected to change its viscosity due to such environmental changes, the fluctuation strength on the membrane of the fluorescent marker (R18) bound to the cell membrane was measured by FCS.
Method:
Human erythrocytes are collected in EDTA / Tris buffer, centrifuged / washed, diluted to an appropriate amount in 10 mM Tris / HCl buffer (10 mM Glucose, 0.1% BSA, 140 mM, 70 mM or 50 mM NaCl, pH = 7.4), and lectin. Red blood cells were allowed to stand in the coated FCS measurement chamber. Thereafter, 10 nM R18 was added to erythrocytes and incubated for 30 minutes, and then FCS measurement was performed.

result:
When the diffusion of R18 on the erythrocyte membrane in hypotonic or isotonic solutions was compared, it showed a slow diffusion rate in a slightly hypotonic solution (70 mM), but the difference was not significant. However, in ghost cells from which hemoglobin in erythrocytes flowed out at a salt concentration of 50 mM, a significant delay in the diffusion rate was observed. From this, it seems that the shape of erythrocytes greatly changes due to the change in extracellular salt concentration, but changes such as membrane viscosity at the micro level do not occur much. However, it was suggested that the membrane with a large change such as intracellular hemoglobin efflux is greatly damaged (FIGS. 5-1 and 5-2).

(2) Measurement of erythrocyte membranes that have been altered by cholesterol-removing drugs Cholesterol is one of the most important constituents of cell membranes. FCS measurement was performed on a membrane from which cholesterol of cells (erythrocytes) was removed by about 10 to 20% using methyl β cyclodextrin (MβC), a drug that specifically removes cholesterol from the membrane.

Method:
Example 1 (1) After preparing red blood cells in the same manner, treat with MβC1-5mM in the measurement chamber, incubate for 30 minutes at 37 ° C, wash the cells with buffer, add R18, and measure FCS. went.

result:
As the state of red blood cells changed with MβC treatment, the diffusion rate tended to decrease. In particular, diffusion on ghosted erythrocytes was about 4 times slower than normal. This suggests that cholesterol is an important factor in the smooth diffusion of cell membrane lipids.

[Example 2] Measurement of membrane changes due to aging of cells or diseases (1) Measurement of changes in membrane function due to aging of erythrocytes and cultured cells In the case of erythrocytes, after collecting blood from a living body, the buffer salt concentration is increased or decreased in a test tube. Pseudo-senescent erythrocytes are prepared by applying osmotic stress by alternation.
In the case of normal cultured cells (not cancerous), proliferation is finally stopped by repeated passage. Cell aging is the process of slowing growth, and cell membrane changes at this stage are followed. The method of the present invention is effective for evaluating the functional deterioration of cells aging in normal living bodies against such experimentally manipulated senescent cells.

  Guess: Cell aging causes changes in its membrane function, especially when the membrane viscosity increases and becomes harder, resulting in cell damage and cell death. In FCS measurement, it is considered possible to find micro changes in the cell membrane as this initial stage.

(2) Measurement of changes in the viscosity of erythrocyte membranes in diseases, especially diabetic patients Since erythrocytes in diabetics are known to be highly viscous and adversely affect intravascular movements Measure whether it is denatured. Thereby, there is a possibility that the difference in the viscosity of the erythrocyte membrane depending on the disease type and medical history can be known, and a method for monitoring the therapeutic effect may be provided.

Method:
Normal human erythrocytes and diabetic patient erythrocytes were collected, and each erythrocyte was placed in a measurement chamber by the preparation method shown in Example 1, and the R18 marker was bound to the cell membrane to perform FCS measurement.
result:
It was found that the diffusion rate of R18 in patient erythrocytes was significantly (1.2 times) slower than normal erythrocytes. This suggests that the patient erythrocyte membrane is less flexible. This FCS evaluation method is suitable for the purpose of examining the effects of new antidiabetic drugs at the cellular level.

[Example 3] Toxicity measurement of amyloid β peptide on erythrocytes and nerve cultured cells Amyloid β peptide, which is said to be a causative factor of Alzheimer's disease, affects the cell from the outside through the cell membrane, and finally causes nerve cells to die. By measuring the toxicity of amyloid β peptide to the membrane, it is possible to know the disease mechanism, and this method is also effective as a monitor for various therapeutic agents.

Method:
The cells used in the experiment were (1) erythrocytes and (2) cultured neurons. The erythrocytes were prepared according to Example 1, and the cultured neurons were mouse brain neurons (Neuro2a) cultured in medium (D-MEM) and 10% FBS. After binding R18 to the cell membrane as a molecular marker, various concentrations of amyloid β peptide (number of amino acids 1-28 or 1-40) were added to the cells, and the addition rate of R18 on the membrane and the membrane binding ratio were not added as controls. Changes were measured by FCS.

The added amyloid β peptide has a rhodamine dye (Rh) at the N-terminus, and the amino acid sequence is shown below. Since the 28-amino acid peptide lacks the 29-40 amino acid site, which is the transmembrane site, the binding to the cell membrane is expected to be weak.
Rh-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (1-40) (Ab40; SEQ ID NO: 1)
Rh-DAEFRHDSGYEVHHQKLVFFAEDVGSNK (1-28) (Ab28; SEQ ID NO: 2)

result:
When amyloid β peptide (1-40) was added to erythrocytes, the diffusion rate of R18 on the membrane was delayed (approximately 1.3 times), and the decrease in the component ratio of membrane-bound R18 (20%) was dependent on the peptide concentration. Was seen.
On the other hand, on cultured neurons, the addition of amyloid β peptide with 40 amino acids significantly delayed the R18 diffusion rate (1.6 times), but the effect of amino acid 28 peptide was not observed.

Furthermore, the ratio of membrane-bound R18 also decreased (5%), but to a lesser extent than the decrease in red blood cells (20%). Contrary to the decrease in the membrane binding ratio of R18, the proportion of free R18 molecules released from the membrane increased. This is considered to be observed as a free molecule in the vicinity of the membrane as a result of the markedly different cell membrane environment due to peptide membrane binding.
From this, it is possible to quantify the phenomenon in which bound R18 is released by adding a test substance such as a peptide to the membrane. Moreover, the degree seems to correlate with the degree of toxicity of the test substance to the membrane.

The figure which shows typically the cultured cell and red blood cell which were adhere | attached on the measurement cover glass on a stage. The figure which shows the molecular structural formula of R18. The figure which shows the FCS measurement principle. (A) shows a measurement region by laser light, (b) shows an autocorrelation function equation, and (c) shows molecular information obtained therefrom. The figure which shows a fluorescence correlation spectroscopy apparatus. The figure which shows the FCS measurement result in a R18 binding erythrocyte membrane. (A) shows the fluorescence intensity profile on the z axis in an isotonic solution (50 mM NaCl), and (b) shows the fluorescence fluctuation and autocorrelation function in the isotonic solution. The figure which shows the FCS measurement result in a R18 binding erythrocyte membrane. (C) shows the fluorescence intensity profile on the z axis in a hypotonic solution (50 mM NaCl), and (d) shows the fluorescence fluctuation and autocorrelation function in the hypotonic solution.

Claims (4)

In order to examine cell aging of a subject, a method of measuring fluctuations of a fluorescent marker bound to a subject-derived cell that is a biological structure having a membrane structure,
Fixing the cell to the measurement container under conditions that allow the cell to maintain physiological function;
Binding a fluorescent marker molecule to the membrane structure of the cell;
Measuring fluorescence intensity fluctuations of the fluorescent marker molecules bound to the membrane structure;
Comparing the measurement result obtained by the above step with a reference value of fluorescence intensity fluctuation determined in advance using cells derived from a normal subject . A method for measuring fluctuations of a fluorescent marker bound to a red blood cell membrane derived from a subject that is a biological structure having a membrane structure in order to examine aging or diabetes of the subject ,
Fixing red blood cells to a measurement container under conditions that allow the red blood cells to maintain physiological function;
Binding a fluorescent marker molecule to the membrane structure of the red blood cells;
Measuring fluorescence intensity fluctuations of the fluorescent marker molecules bound to the membrane structure;
And a step of comparing the measurement result obtained by the above step with a reference value of fluorescence intensity fluctuation determined in advance using red blood cells derived from normal subjects .   The method according to claim 1 or 2, wherein the fluorescence intensity fluctuation of the fluorescent marker molecule is detected by fluorescence correlation spectroscopy or fluorescence polarization.   The fluorescent marker molecule-binding biological structure obtained by the above-described step of binding the fluorescent marker molecule to the membrane structure of cells or erythrocytes is a fluorescence intensity fluctuation [x] of the fluorescent marker molecule that freely moves in the vicinity of the biological structure; The ratio [x / y] to the fluorescence intensity fluctuation [y] of the fluorescent marker molecule, which is bound to the membrane structure of the biological structure and whose free movement is suppressed, is 0.2 or less, The method according to 1 or 2.

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