JP7280610B2 - Diagnostic markers, methods for aiding diagnosis, methods for collecting data for diagnosis, methods for assessing susceptibility, and diagnostic kits for diseases associated with intracellular cholesterol levels - Google Patents

Description

Article 30, Paragraph 2 of the Patent Law applies https://www. abstractsonline. com/pp8/#! /7883/presentation/63319 (Released on September 21, 2019)

The present invention relates to diagnostic markers, methods for assisting diagnosis, methods for collecting data for diagnosis, methods for assessing morbidity, and diagnostic kits for diseases associated with intracellular cholesterol levels.

In 1906, Alois Alzheimer, a German psychiatrist, reported the first case of an Alzheimer's disease patient. More than 100 years have already passed, but no fundamental treatment has been established yet. However, in the last twenty-odd years, research on Alzheimer's disease has made great strides, and it is believed that the framework of the onset mechanism has almost been understood. That is, the constituent proteins of senile plaques and neurofibrillary tangles, which are the two major pathologies in Alzheimer's disease, were identified as amyloid beta protein (Aβ) and tau protein (τ), respectively, and causative genes that cause familial Alzheimer's disease. have been identified, and the analysis of the causative genes and proteins has progressed. As a result of the clarification of the pathological cascade that causes the molecular pathogenesis of Alzheimer's disease, it has become possible to develop treatment methods based on scientific evidence, and many attempts have been made to develop fundamental treatment methods that intervene in the onset mechanism. On the other hand, in 1993, it was revealed that the allele ε4 of apolipoprotein E (ApoE), which controls cholesterol metabolism through high-density lipoprotein (HDL) neoplasia, is a genetic risk factor. It has been reported that high cholesterol levels are correlated with the onset of Alzheimer's disease, and that statins, drugs that lower blood cholesterol levels, reduce the incidence of Alzheimer's disease. rice field. Since then, the amyloid-β precursor protein (APP), which is a substrate involved in Aβ production, and cleavage enzymes are all membrane proteins, so there has been interest in the relationship with other lipid metabolism that constitutes the membrane. It has been reported one after another in relation to the onset of the disease. How are events in the brain (molecular pathology of Alzheimer's disease) and events outside the brain (systemic circulation system, food intake, etc.) related, and how lipid metabolism in the brain is related to molecular pathogenesis of Alzheimer's disease? , there are still unexplained parts. Since most cases of Alzheimer's disease are sporadic cases, the significance of analysis of risk factors and related molecules/phenomena and their application is becoming increasingly important from the viewpoint of prevention/treatment development (Non-Patent Document 1). .

Makoto Michikawa, "Lipids and Alzheimer's Disease", Journal of the Japan Geriatrics Society, 2014, Vol. 51, No. 2, p. 109-116

About 60% of the cholesterol required by the human body is mainly synthesized in cells such as the liver, and the rest is taken in from food. Since blood cholesterol levels do not directly reflect intracellular cholesterol levels, they are not optimal biomarkers for diseases associated with intracellular cholesterol levels.

Accordingly, the present invention provides diagnostic markers, methods for assisting diagnosis, methods for collecting data for diagnosis, methods for assessing morbidity, and diagnostic kits for diseases associated with intracellular cholesterol levels. The task is to

[1] A diagnostic marker for diseases associated with intracellular cholesterol levels, comprising an exosome marker.
[2] The diagnostic marker according to [1], wherein the exosome marker is at least one selected from the group consisting of flotillin, CD63, HSP90 and exosomes.
[3] The diseases associated with intracellular cholesterol levels include Alzheimer's disease, diabetes, dyslipidemia, hypertension, hyperuricemia, obesity, impaired glucose tolerance, cerebrovascular disease, heart disease, cardiovascular disease and artery disease. The diagnostic marker of [1] or [2], which is at least one selected from the group consisting of sclerosis.
[4] A method of aiding diagnosis of diseases associated with intracellular cholesterol levels, comprising:
measuring the concentration of the diagnostic marker according to any one of [1] to [3] in a blood sample collected from a subject;
A method, wherein said concentration is compared to a reference value.
[5] A method of collecting data for the diagnosis of diseases associated with intracellular cholesterol levels, comprising:
A method of measuring the concentration of the diagnostic marker according to any one of [1] to [3] in a blood sample collected from a subject.
[6] A method for assessing the susceptibility to a disease associated with intracellular cholesterol levels, comprising:
measuring the concentration of the diagnostic marker according to [1] or [2] in a blood sample collected from a subject;
comparing said concentration to a reference concentration;
A method, wherein if said concentration is low compared to said reference concentration, said subject is likely to be suffering from a disease associated with said intracellular cholesterol level.
[7] The diseases associated with intracellular cholesterol levels include Alzheimer's disease, diabetes, dyslipidemia, hypertension, hyperuricemia, obesity, impaired glucose tolerance, cerebrovascular disease, heart disease and cardiovascular disease. The method of [6], which is at least one selected from the group.
[8] A diagnostic kit for diseases associated with intracellular cholesterol levels, comprising an antibody specific for the diagnostic marker of any one of [1] to [3].

INDUSTRIAL APPLICABILITY According to the present invention, diagnostic markers, methods for assisting diagnosis, methods for collecting data for diagnosis, methods for evaluating morbidity, and diagnostic kits for diseases associated with intracellular cholesterol levels are provided. can.

FIG. 1 is a diagram showing experimental results of Example 1. FIG. FIG. 2 is a diagram showing experimental results of Example 2. FIG. 3 is a diagram showing experimental results of Example 3. FIG. 4 is a diagram showing experimental results of Example 4. FIG. 5 is a diagram showing experimental results of Example 5. FIG. 6 is a diagram showing experimental results of Example 6. FIG. 7 is a diagram showing experimental results of Example 7. FIG. 8 is a diagram showing experimental results of Example 8. FIG. 9 is a diagram showing experimental results of Example 9. FIG. 10 is a diagram showing the experimental results (cerebrospinal fluid) of Example 10. FIG. 11 shows the experimental results (serum) of Example 10. FIG.

A numerical range represented using "-" shall include the numerical values on both sides of "-".
"Presymptomatic Alzheimer's disease" refers to a pathological condition in which accumulation of Aβ in the brain is observed but no symptoms of cognitive impairment are present.
Blood may be whole blood, plasma or serum, preferably plasma or serum, more preferably serum.
Diagnostic markers for diseases associated with intracellular cholesterol levels are sometimes simply referred to as "diagnostic markers."
Methods that aid in the diagnosis of diseases associated with intracellular cholesterol levels are sometimes simply referred to as "diagnostic methods."
Methods of collecting data for diagnosis of diseases associated with intracellular cholesterol levels are sometimes simply referred to as "methods of collecting data for diagnosis".
Methods for evaluating the morbidity of diseases associated with intracellular cholesterol levels are sometimes simply referred to as "methods for evaluating morbidity."
Diagnostic kits for diseases associated with intracellular cholesterol levels are sometimes simply referred to as "diagnostic kits."
A biomarker is "a characteristic that is objectively measured and evaluated as an indicator of a normal biological process, pathological process, or pharmacological response to therapeutic intervention" (Definition).
SD means Standard Deviation. The standard deviation provides information on how much each data is scattered around the mean value for normally distributed data. The range of 1 SD above and below the mean (abbreviated as ±1 SD) covers 68.26% of all data. ±2 SD includes 95.44%. ±3 SD includes 99.74%.

Embodiments for carrying out the present invention will be described below, but the present invention is not limited to the embodiments described later, and various modifications are possible as long as the gist of the present invention is not changed.

[Diagnostic marker]
The diagnostic markers of the present invention consist of exosome markers.

<Exosome marker>
The exosome marker is not particularly limited as long as it is a biomarker that can be used to quantify exosomes in a blood sample of a subject, but at least one selected from the group consisting of flotillin, CD63, HSP90 and exosomes is preferred.
By combining two or more selected from the group consisting of flotillin, CD63, HSP90 and exosomes, more accurate diagnosis becomes possible.

The diagnostic markers of the present invention are preferably contained in human bodily fluids. When the diagnostic marker of the present invention is contained in human bodily fluids, sample collection is less invasive and testing is simpler than biotests and the like. In addition, there is the advantage that examinations for diseases related to intracellular cholesterol levels can be performed at places other than medical institutions such as homes.
Specific examples of bodily fluids include bodily fluids such as blood, lymph, cerebrospinal fluid, and amniotic fluid. However, bodily fluids are not limited to these examples.
The bodily fluid may be a treated fluid obtained by pretreatment such as removing unnecessary components from the bodily fluid, or a culture solution obtained by culturing cells contained in the bodily fluid.
Among these, blood is preferable as the body fluid because it is easy to collect and can be used to measure biomarkers other than exosome markers. The blood may be whole blood, plasma or serum.

《Flotillin》
Flotilin is a type of endosome-associated protein, and is taken up by exosomes when exosomes are formed inside multivesicular endosomes (MVE) by collapse of the endosomal membrane. Flotilin is one of the common exosome markers.

The present inventors analyzed the intracellular cholesterol level of astrocyte cultured cells cultured from ApoE knockout mice (atherosclerosis disease model mice) and the amount of flotillin secreted into the culture medium. ~ (4) results were obtained.
(1) Astrocyte cultured cells cultured from ApoE knockout mice had lower flotillin concentrations in the culture supernatant than wild-type astrocyte cultured cells. That is, flotillin secretion was decreased.
(2) No decrease in flotillin secretion was observed in astrocyte cultured cells cultured from ApoE3 or ApoE4 knocked-in mice in ApoE knockout mice.
(3) When astrocyte cultured cells cultured from ApoE-deficient mice were treated with β-cyclodextrin, a cholesterol-removing agent, the decrease in flotillin secretion was restored.
(4) When cholesterol was added to the culture medium of astrocyte cultured cells cultured from ApoE3 or ApoE4 knocked-in mice in ApoE knockout mice, the concentration of flotillin in the culture supernatant was higher than that of the astrocyte cultured cells in (2). was declining. That is, flotillin secretion was decreased.
From these results, the present inventors found that when the intracellular cholesterol level is high, flotillin secretion from cells is reduced, that is, exosome secretion is reduced.

Therefore, a high intracellular cholesterol level leads to a decrease in flotillin secretion, and a low intracellular cholesterol level leads to an increase in flotillin secretion. was gotten.
This indicates that flotillin can be used as a marker for intracellular cholesterol levels and is useful as a diagnostic marker for diseases associated with intracellular cholesterol levels.

《CD63》
CD63 is a member of the tetraspanins, a family of transmembrane proteins expressed in the membrane of exosomes, and is one of the common exosome markers.

The present inventors analyzed the intracellular cholesterol level of astrocyte cultured cells cultured from ApoE knockout mice (atherosclerosis disease model mice) and the amount of CD63 secreted into the culture medium. ~ (4) results were obtained.
(1) The CD63 concentration in the culture supernatant of astrocyte cultured cells cultured from ApoE knockout mice was lower than that of wild-type astrocyte cultured cells. That is, CD63 secretion was decreased.
(2) No decrease in CD63 secretion was observed in astrocyte cultured cells cultured from ApoE3- or ApoE4-knocked-in ApoE knockout mice.
(3) When astrocyte cultured cells cultured from ApoE-deficient mice were treated with β-cyclodextrin, a cholesterol-removing agent, the decrease in CD63 secretion was restored.
(4) When cholesterol was added to the culture medium of astrocyte cultured cells cultured from ApoE3 or ApoE4 knocked-in mice in ApoE knockout mice, the CD63 concentration in the culture supernatant was higher than that of the astrocyte cultured cells in (2). was declining. That is, CD63 secretion was decreased.
From these results, the present inventors found that when intracellular cholesterol levels are high, CD63 secretion from cells is reduced, ie, exosome secretion is reduced.

Therefore, the finding that when the intracellular cholesterol level is high, the secretion of CD63 decreases, and when the intracellular cholesterol level is low, the secretion of CD63 increases, in other words, there is an inverse correlation between the intracellular cholesterol level and CD63 secretion. was gotten.
This indicates that CD63 can be used as a marker for intracellular cholesterol levels and is useful as a diagnostic marker for diseases associated with intracellular cholesterol levels.

《HSP90》
HSP90 is a protein with a molecular weight of about 90 kDa and is contained inside exosomes. HSP90 is one of the common exosome markers.

The present inventors analyzed the intracellular cholesterol level of astrocyte cultured cells cultured from ApoE knockout mice (atherosclerosis disease model mice) and the amount of HSP90 secreted into the culture medium. ~ (4) results were obtained.
(1) The HSP90 concentration in the culture supernatant of astrocyte cultured cells cultured from ApoE knockout mice was lower than that of wild-type astrocyte cultured cells. That is, HSP90 secretion was decreased.
(2) No decrease in HSP90 secretion was observed in astrocyte cultured cells cultured from ApoE3 or ApoE4 knocked-in ApoE knockout mice.
(3) When astrocyte cultured cells cultured from ApoE-deficient mice were treated with β-cyclodextrin, a cholesterol-removing agent, the decrease in HSP90 secretion was restored.
(4) When cholesterol was added to the culture medium of astrocyte cultured cells cultured from ApoE3 or ApoE4 knocked-in mice in ApoE knockout mice, the HSP90 concentration in the culture supernatant was higher than that of the astrocyte cultured cells in (2). was declining. That is, HSP90 secretion was decreased.
From these results, the present inventors found that when the intracellular cholesterol level is high, HSP90 secretion from cells is reduced, that is, exosome secretion is reduced.

Therefore, if the intracellular cholesterol level is high, the secretion of HSP90 is decreased, and if the intracellular cholesterol is low, the secretion of HSP90 is increased. was gotten.
This indicates that HSP90 can be used as a marker for intracellular cholesterol levels and is useful as a diagnostic marker for diseases associated with intracellular cholesterol levels.

《Exosome》
Flotilin described above, CD63 and HSP90 are all common exosome markers, the higher the intracellular cholesterol level, the lower the secretion of exosomes, and the lower the intracellular cholesterol, the higher the secretion of exosomes. Elevated, in other words, intracellular cholesterol levels were found to be inversely correlated with exosome secretion.
This shows that exosomes can be used as markers for intracellular cholesterol levels and are useful as diagnostic markers for diseases associated with intracellular cholesterol levels.

Furthermore, the present inventors directly quantified exosomes in the culture supernatant by ELISA or Western blotting, and found that the concentration of exosomes in the culture supernatant and the intracellular cholesterol level are inversely correlated. was taken.
This also shows that exosomes can be used as markers for intracellular cholesterol levels and are useful as diagnostic markers for diseases related to intracellular cholesterol levels.

<Disease associated with intracellular cholesterol level>
Diseases associated with intracellular cholesterol levels include, for example, Alzheimer's disease, diabetes, dyslipidemia, hypertension, hyperuricemia, obesity, impaired glucose tolerance, cerebrovascular disease, heart disease, cardiovascular disease and arteriosclerosis. and preferably at least one selected from the group consisting of these diseases.

If the concentration of the diagnostic marker of the present invention in a sample collected from a subject is lower than the reference value, it can be determined that the subject may have a disease associated with intracellular cholesterol levels.
The reference value is, for example, the average value ±1 SD of the concentration of the diagnostic marker of the present invention in specimens collected from healthy subjects.

Examples of diseases associated with the concentration of the diagnostic marker of the present invention being lower than the reference value include Alzheimer's disease, diabetes, dyslipidemia, hypertension, hyperuricemia, obesity, impaired glucose tolerance, and cerebrovascular disease. Diseases include heart disease, cardiovascular disease and arteriosclerosis.
Arteriosclerosis particularly includes atherosclerosis.
Dyslipidemia includes hypercholesterolemia and hyperlipidemia.

[Method for Aiding Diagnosis]
The method of the present invention for assisting diagnosis of diseases associated with intracellular cholesterol levels (sometimes simply referred to as "method for assisting diagnosis") is a method of detecting the presence of the diagnostic marker of the present invention in a blood sample collected from a subject. It is characterized by measuring the concentration and comparing the concentration with a reference value.

The method for assisting diagnosis of the present invention provides information for determining whether or not a subject is suffering from a disease associated with intracellular cholesterol levels. Diagnosis of a disease associated with intracellular cholesterol levels may be made based on such information.

In the method of aiding diagnosis of the present invention, the concentration of the diagnostic marker of the present invention is measured in a blood sample taken from a subject. The concentrations are then compared to a reference value.

A method for collecting a blood sample from a subject is not particularly limited. A method used in general inspection may be used. For example, blood can be collected by venipuncture.

Detection and quantification of the diagnostic marker of the present invention in a blood sample is preferably performed using an antibody specific for said diagnostic marker.
Detection and quantification of flotillin in blood specimens is preferably carried out using an anti-flotillin antibody.
Anti-CD63 antibodies are preferably used for the detection and quantification of CD63 in blood samples.
Detection and quantification of HSP90 in blood samples are preferably performed using anti-HSP90 antibodies.
Detection and quantification of flotillin, CD63 or HSP90 are preferably carried out using antigen-antibody reactions such as ELISA (Enzyme-Linked Immunosorbent Assay) and Western blotting, with ELISA being particularly preferred. ELISA may be a direct adsorption method, a sandwich method, or a competitive method.

Detection and quantification of exosomes, for example, capture exosomes with immobilized anti-CD9 antibody, detect and quantify with labeled anti-CD63 antibody, capture exosomes with immobilized anti-CD9 antibody, labeled A method of detecting and quantifying with an anti-CD9 antibody, capturing exosomes with a solid-phased anti-CD63 antibody, detecting and quantifying with a labeled anti-CD63 antibody, capturing exosomes with a solid-phased anti-HSP90 antibody, A method of detecting and quantifying with a labeled anti-HSP90 antibody, a method of capturing exosomes with a solid-phased anti-flotillin antibody, and detecting and quantifying with a labeled anti-flotillin antibody are preferred. Further, after isolating exosomes from blood specimens, FACS (Fluorescence-Activated Cell Sorting) may be used for detection and quantification. In addition, after isolating exosomes from blood samples, detection and quantification may be performed by Western blotting. Antibodies for detection and quantification of exosomes include anti-flotillin antibody, anti-CD63 antibody, anti-HSP90 antibody and the like.

The concentration of the diagnostic marker of the invention in the blood sample is then compared to the reference value.
The reference value is, for example, the average value ±1 SD of the concentration of the diagnostic marker of the present invention in specimens collected from healthy subjects.

Examples of diseases related to intracellular cholesterol levels when the concentration of the diagnostic marker of the present invention in a blood sample is lower than the reference value include Alzheimer's disease, diabetes, dyslipidemia, hypertension, and hyperuricemia. , obesity, glucose intolerance, cerebrovascular disease, heart disease, cardiovascular disease and arteriosclerosis. Arteriosclerosis specifically includes atherosclerosis. Dyslipidemia includes hypercholesterolemia and hyperlipidemia.

According to the diagnostic aid method of the present invention, information for diagnosing diseases associated with intracellular cholesterol levels with high accuracy can be obtained.
The information obtained by the diagnostic aid method of the present invention can be used for comparison with baseline values. As such, the information is useful in providing decision material for diagnosing diseases associated with intracellular cholesterol levels.

[How to collect data for diagnosis]
The method of collecting data for diagnosing a disease related to intracellular cholesterol levels of the present invention (sometimes simply referred to as "method of collecting data for diagnosis") is the , characterized by measuring the concentration of the diagnostic marker of the present invention.

The method for measuring the concentration of the diagnostic marker of the present invention can be the same as described in the above "Method for Aiding Diagnosis".

According to the method of collecting data for diagnosis of the present invention, information for diagnosing diseases associated with intracellular cholesterol levels with high accuracy can be obtained.
The information obtained by the method of collecting data for diagnosis of the present invention can be used for comparison with baseline values. As such, the information is useful in providing decision material for diagnosing diseases associated with intracellular cholesterol levels.
The specific contents of the reference value and the method for comparing the concentration of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

In Vitro Methods to Aid Diagnosis
In the in vitro method for aiding diagnosis of diseases associated with intracellular cholesterol levels of the present invention (sometimes simply referred to as "an in vitro method for aiding diagnosis"), a blood sample collected from a subject of the diagnostic marker of the present invention.
Methods for detection and quantification of diagnostic markers for diseases associated with intracellular cholesterol levels can be similar to those described in "Methods to Aid Diagnosis" above.

According to the in vitro method for aiding diagnosis of the present invention, information can be obtained for diagnosing diseases associated with intracellular cholesterol levels with high accuracy.
Information obtained by the in vitro methods to aid diagnosis of the present invention can be used for comparison with baseline values. As such, the information is useful in providing decision material for diagnosing diseases associated with intracellular cholesterol levels.
The specific contents of the reference value and the method for comparing the concentration of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

[Diagnostic method]
In the method for diagnosing diseases associated with intracellular cholesterol levels of the present invention (sometimes simply referred to as "diagnostic method"), the concentration of the diagnostic marker of the present invention in a blood sample collected from a subject is measured, The concentration is compared with a reference value.
The diagnostic method of the present invention is a method for diagnosing whether or not a subject has a disease associated with intracellular cholesterol levels.
Methods for detection and quantification of diagnostic markers for diseases associated with intracellular cholesterol levels can be similar to those described in "Methods to Aid Diagnosis" above.
The specific content of the reference value and the details and preferred aspects of comparison of the expression level of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

In the diagnostic method of the invention, the concentration of the diagnostic marker of the invention is compared to a reference value.

[Determination method]
In the method for determining a disease associated with intracellular cholesterol levels of the present invention (sometimes simply referred to as "determining method"), the concentration of the diagnostic marker of the present invention in a blood sample collected from a subject is measured, The concentration is compared with a reference value.
The determination method of the present invention is a method for determining the presence or absence of a disease related to intracellular cholesterol levels in a subject.
Methods for detection and quantification of diagnostic markers for diseases associated with intracellular cholesterol levels can be similar to those described in "Methods to Aid Diagnosis" above.
The specific contents of the reference value and the method for comparing the concentration of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

In the determination method of the present invention, the concentration of the diagnostic marker of the present invention is compared with a reference value.

[Test method]
In the test method for diseases related to intracellular cholesterol levels of the present invention (sometimes simply referred to as "test method"), the concentration of the diagnostic marker of the present invention in a blood sample collected from a subject is measured, The concentration is compared with a reference value.
The test method of the present invention is a method for testing the presence or absence of a disease related to intracellular cholesterol levels in a subject.
Methods for detection and quantification of diagnostic markers for diseases associated with intracellular cholesterol levels can be similar to those described in "Methods to Aid Diagnosis" above.
The specific contents of the reference value and the method for comparing the concentration of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

In the test method of the invention, the concentration of the diagnostic marker of the invention is compared to a reference value.

[Method for evaluating the likelihood of disease]
In one embodiment of the method of evaluating the morbidity of diseases associated with intracellular cholesterol levels of the present invention (simply "method of evaluating morbidity"), measuring the concentration of a diagnostic marker, comparing said concentration to a reference concentration, and if said concentration is low compared to said reference concentration, said subject is suffering from a disease associated with said intracellular cholesterol level evaluated as highly probable.

The method of evaluating the morbidity of the present invention is a method of evaluating the morbidity of a disease associated with intracellular cholesterol levels in a subject.
Methods for detection and quantification of diagnostic markers for diseases associated with intracellular cholesterol levels can be similar to those described in "Methods to Aid Diagnosis" above.
The specific contents of the reference value and the method for comparing the concentration of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

In the method of assessing susceptibility of the present invention, the concentration of the diagnostic marker of the present invention is compared to a reference value.

Diseases associated with intracellular cholesterol levels when the concentration of a diagnostic marker in a blood sample is lower than the reference value include, for example, Alzheimer's disease, diabetes, dyslipidemia, hypertension, hyperuricemia, obesity, Impaired glucose tolerance, cerebrovascular disease, heart disease, cardiovascular disease and arteriosclerosis. Arteriosclerosis specifically includes atherosclerosis. Dyslipidemia includes hypercholesterolemia and hyperlipidemia.

[Method of treatment]
In the method of treating diseases associated with intracellular cholesterol levels of the present invention (sometimes simply referred to as "therapeutic method"), the concentration of the diagnostic marker of the present invention in a blood sample collected from a subject is measured, The concentration is compared to a reference value, and if the concentration is lower than the reference value, the subject is administered a therapeutic agent for the disease.
The therapeutic methods of the present invention are directed to treating disorders associated with intracellular cholesterol levels in a subject.
Methods for detection and quantification of diagnostic markers for diseases associated with intracellular cholesterol levels can be similar to those described in "Methods to Aid Diagnosis" above.
The specific contents of the reference value and the method for comparing the concentration of the diagnostic marker with the reference value can be the same as those described in the above-mentioned "Method for Aiding Diagnosis".

In the therapeutic method of the present invention, a subject with a diagnostic marker of the present invention having a lower value than the reference value is administered medication.

Examples of therapeutic agents administered when the concentration of the diagnostic marker of the present invention is lower than the reference value and the intracellular cholesterol level is high include HMG-CoA inhibitors (statins). HMG-CoA inhibitors can lower intracellular cholesterol levels by inhibiting cholesterol synthesis within cells. An HMG-CoA inhibitor may be used in combination with ezetimibe, fibrate, or the like.

[Diagnostic kit]
The diagnostic kit for diseases associated with intracellular cholesterol levels of the present invention (the unit may be referred to as "diagnostic kit") is a kit used to test for the presence or absence of diseases associated with intracellular cholesterol levels. . The diagnostic kits of the invention contain antibodies specific for the diagnostic markers of the invention. Such a specific antibody is preferably at least one selected from the group consisting of anti-flotillin antibody, anti-CD63 antibody, anti-HSP90 antibody and exosome-specific antibody.

<Anti-flotillin antibody>
An anti-flotillin antibody is an antibody that has specific binding to flotillin. The type and origin of the anti-flotillin antibody are not particularly limited as long as it has a specific binding property to flotillin. Moreover, any of a polyclonal antibody, an oligoclonal antibody, and a monoclonal antibody may be used. Polyclonal antibodies or oligoclonal antibodies that can be used include IgG fractions derived from antiserum obtained by immunizing animals, as well as antigen-based affinity-purified antibodies. Anti-flotillin antibodies may be antibody fragments such as Fab, Fab', F(ab') ₂ , scFv and dsFv antibodies.

An anti-flotillin antibody can be prepared using an immunological technique, a phage display method, a ribosome display method, or the like. A polyclonal antibody can be prepared by an immunological technique as follows. An antigen (flotillin or part thereof) is prepared and used to immunize animals such as rabbits. Antigens are obtained by purifying biological samples. Recombinant antigens can also be used. Recombinant antigens can be produced, for example, by introducing a gene encoding flotillin (which may be part of the gene) into a suitable host cell using a vector, and expressing the gene in the resulting recombinant cells. It can be prepared by

An antigen conjugated with a carrier protein may be used to enhance the immunogenic effect. KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), OVA (ovalbumin) and the like are used as carrier proteins. A carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimidobenzoyloxysuccinimide) method, or the like can be used for binding the carrier protein. On the other hand, an antibody in which flotillin (or part thereof) is expressed as a fusion protein with GST (glutathione S-transferase), β-galactosidase, maltose-binding protein, histidine (His) tag, or the like can also be used. Such fusion proteins can be conveniently purified by general-purpose methods.

The immunization is repeated as necessary, and when the antibody titer is sufficiently increased, the animal is sampled and serum is obtained by centrifugation or the like. The resulting antiserum is affinity purified to obtain polyclonal antibodies.

On the other hand, monoclonal antibodies can be prepared by the following procedure. First, immunization is performed by the same procedure as above. Immunization is repeated as necessary, and antibody-producing cells are isolated from the immune cells when the antibody titer has sufficiently increased. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain hybridomas. Subsequently, after monoclonalization of this hybridoma, clones producing antibodies with high specificity to flotillin are selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the target antibody can also be obtained by growing the hybridoma to a desired number or more, then transplanting it into the peritoneal cavity of an animal (for example, a mouse), growing it in the ascites, and purifying the ascites. Affinity chromatography using protein G, protein A or the like is preferably used for purification of the culture fluid or ascites. Affinity chromatography in which an antigen (flotillin) is immobilized can also be used. Furthermore, a method such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation or centrifugation can also be used. These methods are used alone or in combination.

Various modifications can be made to the resulting antibody provided that it retains its specific binding to flotillin. Such modified antibodies may be used as flotillin-specific antibodies.

If a labeled antibody is used as the anti-flotillin antibody, it is possible to directly detect the amount of the conjugate using the amount of label as an indicator. Therefore, a simpler inspection method can be constructed. On the other hand, in addition to the need to prepare an anti-flotillin antibody to which a labeling substance is bound, there is a problem that the detection sensitivity is generally low. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound, or a method using a polymer to which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having specific binding property to the anti-flotillin antibody. For example, when an anti-HSP antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used. Labeled secondary antibodies that can be used against antibodies of various species such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Cosmo Bio Co., etc.), and an appropriate one can be selected and used.

Labeling substances include, for example, fluorescent dyes such as fluorescein, rhodamine, Texas Red and Oregon Green; enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase and β-D-galactosidase; chemiluminescence such as luminol and acridine dyes; compounds, radioactive isotopes such as ³² P, ³⁵ S, ¹³¹ I, ¹²⁵ I, and biotin.

In one aspect, flotillin-specific antibodies are immobilized for their intended use. The insoluble support used for solid phase formation is not particularly limited. For example, an insoluble support made of a water-insoluble substance such as a resin such as polystyrene resin, polycarbonate resin, silicone resin or nylon resin, or glass can be used. An antibody can be attached to an insoluble support by physical adsorption or chemisorption.

The diagnostic kit of the present invention may contain reagents other than the flotillin-specific antibody used for measuring flotillin in the blood of a subject. Such reagents include buffer solutions, part-locking reagents, substrates for enzymes, coloring reagents, and the like. In addition, instruments or devices such as containers, reaction devices, fluorescence readers, etc., used in measuring flotillin in the blood of a subject may be included. In addition, it is preferable to include flotillin in the kit as a standard sample.

<Anti-CD63 antibody>
The type and origin of the anti-CD63 antibody are not particularly limited as long as it has specific binding property to CD63. An anti-CD63 antibody can be prepared according to the method for preparing an anti-flotillin antibody.

<Anti-HSP90 antibody>
The type and origin of the anti-HSP90 antibody are not particularly limited as long as it has a specific binding property to HSP90. Anti-HSP90 antibodies can be prepared according to the method for preparing anti-HSP90 antibodies.

<Antibody specific to flotillin>
Anti-flotillin antibodies, anti-CD63 antibodies, anti-HSP90 antibodies, anti-CD9 antibodies, anti-CD81 antibodies, and the like can be used as flotillin-specific antibodies.

<When multiple antibodies are included>
The diagnostic kit of the present invention contains one or more of an anti-flotillin antibody, an anti-CD63 antibody, an anti-HSP90 antibody and an exosome-specific antibody.

Anti-flotillin antibody, anti-CD63 antibody, anti-HSP90 antibody and when containing two or more of the exosome-specific antibodies, each antibody may be housed in a separate container, housed in the same container may be

In the diagnostic kit of the present invention, reagents including antibodies may be stored in separate containers and mixed by the user at the time of use. It may be provided pre-mixed as a mix.

The diagnostic kit of the present invention is preferably further accompanied by an instruction manual (protocol).

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples described later, and various modifications are possible without departing from the gist of the present invention.

[Example 1]
<The concentration of flotillin in the conditioned medium of astrocytes cultured from ApoE knockout mice is decreased>
(1) Cell Culture Test Cerebral cortices were collected from wild-type C57BL/6J mice and ApoE-deficient C57BL/6J mice on the first day after birth, and then the meninges were removed and minced using a scalpel. The minced brain tissue was placed in DMEM medium (Dulbecco's Modified Eagle's Medium) containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37° C. for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, cultured in a serum-free medium for 72 hours, and the supernatant was collected.
Next, using the supernatant collected by the above procedure, the levels of flotillin and ApoE secreted into the culture medium were detected by Western blot analysis.
(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and goat anti-apolipoprotein E (ApoE) antibody (manufactured by Millipore, Cat No. .AB947) diluted 1,000-fold with TBS-T buffer was added to the membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.

The results shown in FIG. 1 were obtained.
Both flotillin and ApoE in conditioned media (CM; Conditioned Media) cultured for 72 hours were at lower concentrations in ApoE knockout cells (ApoE-KO) than in wild-type cells (WT) (Fig. 1A, B).
Also, flotillin in cell lysates did not differ between ApoE knockout cells (ApoE-KO) and wild-type cells (WT), whereas ApoE It was at a lower concentration than cells (WT) (Fig. 1C, D).
These findings indicate that exosome secretion is reduced in ApoE knockout cells.

[Example 2]
<Comparison of exosome secretion in conditioned medium of astrocytes cultured from wild-type mice, ApoE knockout mice, ApoE3 knock-in mice, ApoE4 knock-in mice>
(1) Cell culture test Cerebral cortex was collected from 1 day old wild-type C57BL/6J mice, ApoE-deficient C57BL/6J mice, ApoE3-knock-in mice, and ApoE4-knock-in mice, then the meninges were removed, and a scalpel was used. It was finely chopped using The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, cultured in serum-free medium for 24 hours, 48 hours, and 72 hours, and the supernatant was collected.
Next, using the supernatant collected by the above procedure, the levels of flotillin, ApoE and HSP90 secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and goat anti-apolipoprotein E (ApoE) antibody (manufactured by Millipore, Cat No. AB947) and a mouse anti-HSP90 monoclonal antibody (manufactured by BD Bioscience) diluted 1,000-fold with TBS-T buffer were added to the membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.

The results shown in FIG. 2 were obtained.
The concentrations of flotillin, HSP90, and ApoE in conditioned media (CM) cultured for 24 hours, 48 hours, or 72 hours were lower in ApoE knockout mice than in wild-type mice, ApoE3 knockin mice, and ApoE4 knockin mice. rice field. From this, it was found that although exosome secretion is reduced in ApoE knockout cells, it is restored to wild-type levels by knocking in ApoE3 or ApoE4.

[Example 3]
<Phosphorylated Akt and phosphorylated PI3 kinase are enhanced in astrocytes cultured from ApoE knockout mice>
(1) Cell culture test Cerebral cortex was collected from 1 day old wild-type C57BL/6J mice, ApoE-deficient C57BL/6J mice, ApoE3-knock-in mice, and ApoE4-knock-in mice, then the meninges were removed, and a scalpel was used. It was finely chopped using The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, cultured in a serum-free medium for 48 hours, and the supernatant was collected.
Next, using the supernatant collected by the above procedure, flotillin, phosphorylated PI3 kinase, PI3 kinase, phosphorylated Akt, and Akt levels secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-mouse phosphorylated PI3 kinase p85/p55 polyclonal antibody (manufactured by Cell Signaling Technology), rabbit anti-human PI3 kinase p85 monoclonal antibody (Cell Signaling Technology), rabbit anti-mouse phosphorylated Akt polyclonal antibody (Cell Signaling Technology), and rabbit anti-mouse Akt polyclonal antibody (Cell Signaling Technology) diluted 1,000-fold with TBS-T buffer. It was placed in a membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.

The results shown in FIG. 3 were obtained.
There was no significant difference in Akt levels between wild type, ApoE knockout, ApoE3 knockin and ApoE4 knockin, but P-Akt (phosphorylated Akt) levels were highest in ApoE knockout and lowest in wild type.
There was no significant difference in the amount of PI3K (PI3 kinase) among wild type, ApoE knockout, ApoE3 knockin and ApoE4 knockin, but the amount of P-PI3K (phosphorylated PI3 kinase) was highest in ApoE knockout and lower in wild type. was the least
α-tubulin was constantly expressed and used as a positive control.

[Example 4]
<High intracellular cholesterol levels in astrocytes cultured from ApoE knockout mice>
(1) Cell culture test Cerebral cortex was collected from 1 day old wild-type C57BL/6J mice, ApoE-deficient C57BL/6J mice, ApoE3-knock-in mice, and ApoE4-knock-in mice, then the meninges were removed, and a scalpel was used. It was finely chopped using The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, cultured in a serum-free medium for 48 hours, and the supernatant was collected.
Next, using the supernatant collected by the above procedure, the levels of flotillin, ApoE, HSP90 and α-tubulin secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and goat anti-apolipoprotein E (ApoE) antibody (manufactured by Millipore, Cat No. AB947), a mouse anti-HSP90 monoclonal antibody (manufactured by BD Bioscience), and a mouse anti-α-tubulin monoclonal antibody (Sigma ALdrich, USA) diluted 1,000-fold with TBS-T buffer. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.
(3) Cholesterol quantification In cholesterol quantification of cultured cells, the cholesterol in the cells was dried and then incubated with a hexane/isopropanol solution (3:2) for 30 minutes. It was quantified using a quantification kit (Kyowa Hakko, Wako Pure Chemical). After removing the solution, the plates were dried, the protein was quantified, and the amount of cholesterol per unit protein was quantified.

The results shown in FIG. 4 were obtained.
Flotilin, HSP90 and ApoE concentrations in the conditioned medium were lowest in ApoE knockout cells (Fig. 4A).
Also, the concentrations of flotillin and HSP90 in cell lysates were not significantly different between ApoE knockout cells and other cells, but the concentration of ApoE was lowest in ApoE knockout cells (Fig. 4B).
Intracellular cholesterol levels were significantly higher in ApoE knockout cells, and were approximately the same in wild-type, ApoE3 knockin and ApoE4 knockin cells.

[Example 5]
<Flotilin/HSP90 concentration in the culture medium is reduced by the addition of cholesterol>
(1) Cell culture test Cerebral cortex was collected from 1 day old wild-type C57BL/6J mice, ApoE-deficient C57BL/6J mice, ApoE3-knock-in mice, and ApoE4-knock-in mice, then the meninges were removed, and a scalpel was used. It was finely chopped using The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, and cholesterol (dissolved in ethanol) with different concentrations was added to each. The added contents were ethanol only and 0 μM, 5 μM and 10 μM cholesterol. The cultured cells treated with these concentrations were cultured in a serum-free medium for 48 hours, after which the supernatant was collected.
Next, using the supernatant collected by the above procedure, the levels of flotillin, ApoE and HSP90 secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and goat anti-apolipoprotein E (ApoE) antibody (manufactured by Millipore, Cat No. AB947) and a mouse anti-HSP90 monoclonal antibody (manufactured by BD Bioscience) diluted 1,000-fold with TBS-T buffer were added to the membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.
(3) Cholesterol quantification In cholesterol quantification of cultured cells, the cholesterol in the cells was dried and then incubated with a hexane/isopropanol solution (3:2) for 30 minutes. It was quantified using a quantification kit (Kyowa Hakko, Wako Pure Chemical). After removing the solution, the plates were dried, the protein was quantified, and the amount of cholesterol per unit protein was quantified.

The results shown in FIG. 5 were obtained.
In both wild-type and ApoE knockout cells, cholesterol addition decreased flotillin and HSP90 levels in conditioned media, but did not alter ApoE levels (FIGS. 5A, CE).
Cholesterol addition increased intracellular cholesterol levels in both wild-type and ApoE-knockout cells, but the levels were higher in ApoE-knockout cells than in wild-type cells (Fig. 5B).

[Example 6]
<Extraction of cholesterol from cells by β-cyclodextrin treatment increases flotillin/HSP90 secretion>
(1) Cell culture test Cerebral cortices were collected from wild-type C57BL/6J mice and ApoE-deficient C57BL/6J mice on the first day after birth, and then the meninges were removed and minced using a scalpel. The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, and β-cyclodextrin (dissolved in ethanol) with different concentrations was added to each of the astrocytes at concentrations of 0 mM, 5 mM and 10 mM in ethanol alone. β-Cyclodextrin has the effect of drawing cholesterol out of cells, lowering cellular cholesterol levels. The cultured cells treated with these concentrations were cultured in a serum-free medium for 48 hours, after which the supernatant was collected.
Next, using the supernatant collected by the above procedure, the levels of flotillin, ApoE and HSP90 secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and goat anti-apolipoprotein E (ApoE) antibody (manufactured by Millipore, Cat No. AB947) and a mouse anti-HSP90 monoclonal antibody (manufactured by BD Bioscience) diluted 1,000-fold with TBS-T buffer were added to the membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.
(3) Cholesterol quantification In cholesterol quantification of cultured cells, the cholesterol in the cells was dried and then incubated with a hexane/isopropanol solution (3:2) for 30 minutes. It was quantified using a quantification kit (Kyowa Hakko, Wako Pure Chemical). After removing the solution, the plates were dried, the protein was quantified, and the amount of cholesterol per unit protein was quantified.

The results shown in FIG. 6 were obtained.
Addition of β-cyclodextrin to the culture media increased flotillin and HSP90 concentrations in conditioned media of ApoE knockout cells to levels similar to those of wild-type cells, but did not change ApoE concentrations (Fig. 6A, C-E).
Addition of β-cyclodextrin reduced intracellular cholesterol levels in both wild-type and ApoE-knockout cells, but in ApoE-knockout cells, the addition of 2 mM reduced the levels to levels similar to those in wild-type cells (Fig. 6B). ).

[Example 7]
<When cholesterol is extracted from cells by β-cyclodextrin treatment, phosphorylated PI3 kinase and phosphorylated Akt decrease>
(1) Cell culture test Cerebral cortices were collected from wild-type C57BL/6J mice and ApoE-deficient C57BL/6J mice on the first day after birth, and then the meninges were removed and minced using a scalpel. The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, and β-cyclodextrin (dissolved in ethanol) with different concentrations was added to each of the astrocytes at concentrations of 0 mM, 5 mM and 10 mM in ethanol alone. β-Cyclodextrin has the effect of drawing cholesterol out of cells, lowering cellular cholesterol levels. The cultured cells treated with these concentrations were cultured in a serum-free medium for 48 hours, after which the supernatant was collected.
Next, using the supernatant collected by the above procedure, flotillin, phosphorylated PI3 kinase, PI3 kinase, phosphorylated Akt, and Akt levels secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and rabbit anti-mouse phosphorylated PI3 kinase p85/p55 polyclonal antibody (manufactured by Cell Signaling Technology), rabbit anti-human PI3 kinase p85 monoclonal antibody (Cell Signaling Technology), rabbit anti-mouse phosphorylated Akt polyclonal antibody (Cell Signaling Technology), and rabbit anti-mouse Akt polyclonal antibody (Cell Signaling Technology) diluted 1,000-fold with TBS-T buffer. It was placed in a membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.

The results shown in FIG. 7 were obtained.
Akt levels in cell lysates were not affected by β-cyclodextrin treatment, whereas P-Akt (phosphorylated Akt) levels were decreased by β-cyclodextrin treatment (FIGS. 7A-C).
PI3K (PI3 kinase) levels in cell lysates were not affected by β-cyclodextrin treatment, but P-PI3K (phosphorylated PI3 kinase) levels were reduced by β-cyclodextrin treatment in ApoE knockout cells ( 7A, D, E).

[Example 8]
A PI3 Kinase Inhibitor (LY294002) Ameliorated Flotilin/HSP90 Decrease in ApoE Knockouts
(1) Cell culture test Cerebral cortices were collected from wild-type C57BL/6J mice and ApoE-deficient C57BL/6J mice on the first day after birth, and then the meninges were removed and minced using a scalpel. The minced brain tissue was placed in a DMEM medium containing trypsin (0.25%) and DNase I (0.1 mg/mL) and incubated at 37°C for 15 minutes to crush the tissue. Then, after centrifugation at 800 rpm for 5 minutes, the supernatant was aspirated, a DMEM solution containing 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin was added, and the cells were seeded and cultured in culture flasks (37° C., 5% CO ₂ ). . As soon as the cells became confluent, they were shaken for 1 hour at 37° C. and 200 rpm using a constant temperature shaker to remove microglia with weak adhesion.
Then, 1×10 ⁶ astrocytes were seeded on a 60 mm dish, and different concentrations of PI3 kinase inhibitor (LY294002) (dissolved in DMSO) were added to each plate at concentrations of 2, 10, and 20 μM. The cultured cells treated with these concentrations were cultured in a serum-free medium for 48 hours, after which the supernatant was collected.
Next, using the supernatant collected by the above procedure, the levels of flotillin and HSP90 secreted into the culture medium were detected by Western blot analysis.

(2) Western blot analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed three times with washing buffer (TBS-T) for 10 minutes, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and mouse anti-HSP90 monoclonal antibody (manufactured by BD Bioscience) were added to TBS-T buffer. A 1,000-fold diluted antibody solution was added to the membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.

The results shown in FIG. 8 were obtained.
Addition of a PI3 kinase inhibitor (LY294002) increased the concentrations of flotillin and HSP90 in the culture medium of ApoE knockout cells to levels comparable to those of wild-type cells (FIGS. 8A-C).

[Example 9]
<Correlation between flotillin concentration and CD63/9 using human serum samples (MCI (+), MCI (-))>
For the collection of human samples, sera from patients in the MCI(−) (without mild cognitive impairment) and MCI(+) (with mild cognitive impairment) groups were collected. These patients were diagnosed by cognitive function tests, MRI tests, amyloid PET tests, and the like.
A 100-fold dilution of each serum in PBS was used and the contained flotillin levels were quantified by Western blot analysis. First, a solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was placed on a 10% polyacrylamide gel and electrophoresed to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed with washing buffer (TBS-T) for 10 minutes three times, and an antibody solution obtained by diluting rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) 1,000 times with TBS-T buffer was applied to the membrane. and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.
In serum exosome level quantification, the serum was diluted 20-fold and analyzed using a CD9/CD63 exosome quantification kit (Cosmo Bio Co., Ltd.) according to the protocol.

The results shown in FIG. 9 were obtained.
Between the MCI (-) (no mild cognitive impairment) group and the MCI (+) (with mild cognitive impairment) group, a significant difference in serum exosome concentration was observed (FIG. 9A-C).

[Example 10]
(1) Analysis of the effect of a high-fat diet (high-cholesterol diet) on mice Prepare 20 3-month-old wild-type C57BL/6J mice, and 11 mice that are fed a normal diet and fed a high-fat diet. Nine mice were used. After breeding under these conditions for 4 months, cerebrospinal fluid, serum and brain samples were collected. At the time of recovery, three types of anesthetics were medetomidine (Domitor (registered trademark), manufactured by Nihon Zenyaku Kogyo Co., Ltd.) at 0.3 mg/kg, midazolam (Dormicum (registered trademark), manufactured by Astellas Pharma Inc.) at 4.0 mg/kg, and butorphanol. (Betorfal (registered trademark), manufactured by Meiji Seika Pharma) A mixture was prepared so that it could be administered at a rate of 5.0 mg/kg, and was dissolved in pure water and used according to the body weight of the mouse.
As for the administration method, 0.01 mL per g body weight was administered into the peritoneal cavity of mice.
Under anesthesia, the occipital region of the mouse was opened, and cerebrospinal fluid was collected and stored frozen (-80°C).
4° C. PBS was injected from the heart to perfuse the whole body (including the brain).

(2) Western Blot Analysis A solution obtained by adding 2.5 μL of 4×SDS sample buffer to 10 μL of the supernatant of each sample was added to a 10% polyacrylamide gel and subjected to electrophoresis to separate proteins. The separated proteins were transferred to a PVDF (polyvinylidene difluoride) membrane and then blocked (5% skimmed milk/TBS-Tween 20, TBS-T) at room temperature for 1 hour. Thereafter, the membrane was washed three times with washing buffer (TBS-T) for 10 minutes, and rabbit anti-flotillin-1 antibody (manufactured by Sigma-Aldrich) and mouse anti-HSP90 monoclonal antibody (manufactured by BD Bioscience) were added to TBS-T buffer. A 1,000-fold diluted antibody solution was added to the membrane and reacted at 4° C. for 16 hours. After washing the membrane with washing buffer for 10 minutes three times, HRP (horseradish peroxidase)-labeled goat secondary antibody (diluted 5,000 times with 2.5% skimmed milk/TBS-Tween 20) was added and reacted at room temperature for 1 hour. After exposure, chemiluminescence was detected with an Amersham Imager-600 (GE Healthcare Life Sciences) instrument.

As shown in FIG. 10, the levels of flotillin and HSP90 in the cerebrospinal fluid of mice fed a high-fat diet were significantly reduced compared to those of mice fed a normal diet.
Also, as shown in FIG. 11, serum flotillin and HSP90 levels in the serum of mice fed a high-fat diet were also significantly lower than those of mice fed a normal diet.

According to the diagnostic marker, the method for assisting diagnosis, the method for collecting data for diagnosis, the method for evaluating the possibility of morbidity, and the diagnostic kit of the present invention, intracellular cholesterol levels that cannot be directly measured can be estimated. , useful for the diagnosis of diseases associated with intracellular cholesterol levels.

Claims (6)

A diagnostic marker for mild cognitive impairment consisting of an exosome marker (excluding flotillin) . The diagnostic marker according to claim 1, wherein the exosome marker is at least one selected from the group consisting of CD63 , CD9, and HSP90 . A method for assisting diagnosis of mild cognitive impairment , comprising:
measuring the concentration of the diagnostic marker according to claim 1 or 2 in a blood sample collected from a subject;
A method, wherein said concentration is compared to a reference value. A method of collecting data for diagnosis of mild cognitive impairment , comprising:
A method of measuring the concentration of the diagnostic marker according to claim 1 or 2 in a blood sample collected from a subject. A method for assessing the susceptibility of mild cognitive impairment , comprising:
measuring the concentration of the diagnostic marker according to claim 1 or 2 in a blood sample collected from a subject;
comparing said concentration to a reference concentration;
A method wherein, if said concentration is low compared to said reference concentration, it indicates that said subject is likely suffering from mild cognitive impairment . A diagnostic kit for mild cognitive impairment comprising an antibody specific for the diagnostic marker according to claim 1 or 2 .

Images