CN117535393A

CN117535393A - Intracranial calcification drug target and application thereof

Info

Publication number: CN117535393A
Application number: CN202210945346.3A
Authority: CN
Inventors: 刘静宇; 徐旋
Original assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Current assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Priority date: 2022-08-08
Filing date: 2022-08-08
Publication date: 2024-02-09

Abstract

The invention provides a pharmaceutical target for intracranial calcification and application thereof, in particular to a group of markers for detecting intracranial calcification and evaluating the effect of intracranial calcification treatment, wherein the markers are selected from the group consisting of: osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), myosin heavy chain 15 (MYH 15), aspartyl Glucosaminidase (AGA), avian sarcoma virus 17 putative transforming gene (Jun), β -Actin (ACTB), phospholipase C (PLCG 2), mitogen activated protein kinase 14 (MAP 3K 14), integrin β2 (ITGB 2), death-related protein kinase (DAP), maestro heat-like repeat family member 7 (MROH 7), cytochrome p450 family member (CYP 2J 13), chemokine CXC motif ligand 10 (CXCL 10), C-type lectin domain family member 7A (CLEC 7A), alpha-2 subunit of type IV collagen (COL 4 A2), or a combination thereof.

Description

Intracranial calcification drug target and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a medicine target for intracranial calcification and application thereof.

Background

Intracranial calcification is a type of neurodegenerative disease characterized by the occurrence of calcification in the brain as the main pathological feature. Clinically, it is now found that the incidence of intracranial calcification is very high, increasing with age, by neuroimaging [ mainly by Computed Tomography (CT) techniques ], with incidence of about 1% in young people and up to 20% in elderly people.

With the exacerbation of intracranial calcification, a variety of extrapyramidal clinical symptoms are often induced in patients ranging from occasional migraine to severe movement disorders (e.g., dystonia, ataxia, parkinsonism-like symptoms, etc.) and neuropsychiatric disorders (e.g., memory decline, affective disorders, confusion, dementia, etc.), and possibly for a lifetime of the patient.

To date, in the progress of treatment research of intracranial calcification, no effective treatment method is still found at home and abroad, and the method has a great relation with the reasons of slow research of pathogenesis of intracranial calcification, no known intracranial calcification drug targets and the like. Therefore, the pathogenesis of intracranial calcification and drug target screening studies are urgent.

Disclosure of Invention

The present invention aims to study the pathogenesis of intracranial calcification and the corresponding drug targets, in particular the present invention provides a group of drug targets which can be used for detecting intracranial calcification diseases, the targets being selected from: osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), myosin heavy chain 15 (MYH 15), aspartyl Glucosaminidase (AGA), avian sarcoma virus 17 putative transforming gene (Jun), β -Actin (ACTB), phospholipase C (PLCG 2), mitogen activated protein kinase 14 (MAP 3K 14), integrin β2 (ITGB 2), death-related protein kinase (DAP), maestro heat-like repeat family member 7 (MROH 7), cytochrome p450 family member (CYP 2J 13), chemokine CXC motif ligand 10 (CXCL 10), C-type lectin domain family member 7A (CLEC 7A), alpha-2 subunit of type IV collagen (COL 4 A2), or a combination thereof.

The present invention provides in a first aspect the use of a marker or detection reagent thereof for the preparation of a diagnostic reagent or kit for (a) detecting an intracranial calcification disease, and/or (b) assessing the effect of a treatment for an intracranial calcification disease;

wherein the marker is selected from the group consisting of:

(Z1) an Osteopontin (OPN) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z2) Osteocalcin (OCN) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z3) alkaline phosphatase (ALP) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z4) an Osteoprotegerin (OPG) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z5) myosin heavy chain 15 (MYH 15) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z6) aspartyl aminoglucosidase (AGA) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z7) avian sarcoma virus 17 putative transgene (Jun) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z8) beta-Actin (ACTB) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z9) a phospholipase C (PLCG 2) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z10) mitogen-activated protein kinase 14 (MAP 3K 14) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z11) integrin beta 2 (ITGB 2) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z12) death-related protein kinase (DAP) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z13) Maestro heat-like repeat family member 7 (MROH 7) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z14) a cytochrome p450 family member (CYP 2J 13) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z15) chemokine CXC motif ligand 10 (CXCL 10) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z16) c-type lectin domain family 7 member a (CLEC 7A) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z17) alpha-2 subunit of collagen type IV (COL 4 A2) gene, or mRNA thereof, or cDNA thereof, or protein thereof; and/or

(Z18) (Z1) to (Z17).

In another preferred embodiment, the detection reagent is selected from the group consisting of: primers, probes, antibodies, nucleic acid aptamers, sequencing libraries, nucleic acid chips (e.g., DNA chips), protein chips, or combinations thereof.

In another preferred example, the intracranial calcification disease comprises pathological intracranial calcification caused by genetic factors, various congenital syndromes, viral infections, trauma, toxins, physiological injuries, hypoparathyroidism, abnormal calcium-phosphorus metabolism, and physiological intracranial calcification caused by age aging.

In another preferred embodiment, the detection reagent comprises:

(a) A specific antibody, specific binding molecule for one or more of markers Z1-Z17; and/or

(b) Primers or primer pairs, probes or chips (e.g., nucleic acid chips or protein chips) that specifically amplify mRNA or cDNA of one or more of the markers Z1-Z17.

In another preferred embodiment, the diagnosis includes early diagnosis, auxiliary diagnosis.

In another preferred embodiment, the markers Z1-Z17 are derived from a mammal.

In another preferred embodiment, the mammal includes a human and a non-human mammal, preferably a primate (e.g., human).

In another preferred embodiment, the markers Z1-Z17 are of human origin.

In another preferred embodiment, the markers Z1-Z17 are derived from a patient diagnosed with or suspected of having an intracranial calcification disease.

In another preferred embodiment, the detection is for an ex vivo sample.

In another preferred embodiment, the ex vivo sample comprises: tissue samples, cerebrospinal fluid and blood.

In another preferred embodiment, the sample is a sample isolated from brain tissue.

In another preferred embodiment, the detection is the detection of the expression level of one or more of the markers Z1-Z17 in brain tissue.

In another preferred embodiment, the detection reagent is coupled to or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically binding to polynucleotides (mRNA or cDNA) of one or more of the markers Z1-Z17.

In another preferred embodiment, the protein chip comprises a substrate and specific antibodies spotted on the substrate, wherein the specific antibodies comprise specific antibodies against one or more of the markers Z1-Z17.

In another preferred example, when the expression level of the marker gene is restored to the level of normal population, it is suggested that the prevention and treatment effect of the intracranial calcification disease is better or the intracranial calcification disease is substantially cured or the deterioration of the intracranial calcification disease is suppressed.

In another preferred embodiment, the reagents are PCR primers.

In another preferred embodiment, the primers are primers for amplifying markers Z1-Z17.

In another preferred embodiment, the primer comprises a primer pair as shown in SEQ ID No. 3 and SEQ ID No. 4.

In another preferred embodiment, the primer comprises a primer pair as shown in SEQ ID No. 5 and SEQ ID No. 6.

In another preferred embodiment, the primer comprises a primer pair as shown in SEQ ID NO.7 and SEQ ID NO. 8.

In another preferred embodiment, the marker is used as a standard (or quality control) in a diagnostic kit.

In another preferred embodiment, the diagnostic reagent or kit is used for detecting a cerebrospinal fluid sample.

In a second aspect, the invention provides a kit comprising a detection reagent for detecting a marker selected from the group consisting of:

(Z18) (Z1) to (Z17).

In another preferred embodiment, the kit contains one or more of the markers Z1-Z17 as a control or quality control.

In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used for (a) detecting an intracranial calcification disease, and/or (b) assessing the effect of a treatment for an intracranial calcification disease.

In another preferred embodiment, the reagents are PCR primers.

In another preferred embodiment, the marker concentration comprises protein concentration, mRNA concentration, or a combination thereof.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the kit is used for (a) detecting an intracranial calcification disease, and/or (b) assessing the effect of a treatment for an intracranial calcification disease.

In another preferred embodiment, the label or description refers to the following:

when the expression level of each marker gene (or a combination thereof) or the expression level of each marker protein (or a combination thereof) or the concentration of each marker is restored to the level in the normal control population, the prevention and treatment effect of the intracranial calcification disease is suggested to be better or the intracranial calcification disease is basically cured or the deterioration of the intracranial calcification disease is restrained.

The third aspect of the present invention provides a detection method, comprising the steps of:

(a) Providing a detection sample, wherein the detection sample is a sample separated from brain tissue;

(b) Detecting the expression level of one or more genes of the markers Z1-Z17 or the expression amount of one or more proteins of the markers Z1-Z17 or the concentration of one or more of the markers Z1-Z17 in the detection sample, and marking as E1; and

(c) The expression level of the gene or genes of the markers Z1-Z17 or the expression level of the protein or proteins of the markers Z1-Z17 or the concentration E1 of the protein or proteins of the markers Z1-Z17 is compared with a control reference value E0.

In another preferred embodiment, the sample is from a test object.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, if the expression level of one or more genes of markers Z1-Z4, Z7-Z17 or the expression level of one or more proteins of markers Z1-Z4, Z7-Z17 or the concentration E1 of one or more of markers Z1-Z4, Z7-Z17 is higher than the control reference value E0, the subject has a greater chance of suffering from an intracranial calcification disease than the general population (normal control population).

In another preferred embodiment, if the expression level of one or more genes of markers Z5-Z6 or the expression level of one or more proteins of markers Z5-Z6 or the concentration E1 of one or more of markers Z5-Z6 is lower than the control reference value E0, the subject has a greater chance of suffering from an intracranial calcification disease than the general population (normal control population).

In another preferred embodiment, said control reference value E0 is the expression level or amount or concentration of one or more of the markers Z1-Z17 in the same sample of said normal control population or in the same sample of a population of brain disease patients other than intracranial calcification disease or in the same sample of the test subject itself prior to receiving a medication for intracranial calcification disease.

In another preferred example, when the expression level of one or more genes of the markers Z1 to Z17 or the expression amount of one or more proteins of the markers Z1 to Z17 or the concentration E1 of one or more of the markers Z1 to Z17 is restored to the control reference value E0, it is suggested that the prevention effect of the intracranial calcification disease is better or the intracranial calcification disease is substantially cured or the deterioration of the intracranial calcification disease is suppressed.

In another preferred embodiment, the level of expression of one or more RNA of markers Z1-Z17 in the sample is detected by RT-PCR, in situ hybridization or transcriptome sequencing, and the level of expression of one or more protein of markers Z1-Z17 in the sample is detected by immunoblotting or immunohistochemistry.

In another preferred embodiment, the concentration of one or more of the markers Z1-Z17 is detected using an Elisa kit, western immunoblotting, proteomics, etc.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a fourth aspect, the invention provides a method of diagnosing an intracranial calcification disease, comprising the steps of:

a) Providing a test sample from a subject, the test sample comprising brain tissue;

b) Detecting the expression level of one or more genes or the expression level or the concentration of proteins of markers Z1-Z17 in brain tissues in the test sample, and marking as E1; and

c) Comparing the expression level of the gene or the expression amount or concentration E1 of the protein of one or more of the markers Z1 to Z17 with a control reference value E0,

wherein the expression level of one or more genes of markers Z1-Z4, Z7-Z17 or the expression level of one or more proteins of markers Z1-Z4, Z7-Z17 or the concentration E1 of one or more of markers Z1-Z4, Z7-Z17 in the sample is higher than a control reference value E0, and the subject has a greater chance of suffering from an intracranial calcification disease than in the general population (normal control population); or (b)

If the expression level of one or more genes of markers Z5-Z6 or the expression level of one or more proteins of markers Z5-Z6 or the concentration E1 of one or more of markers Z5-Z6 is lower than the control reference value E0, the subject has a greater chance of suffering from an intracranial calcification disease than the general population (normal control population).

In another preferred embodiment, the diagnosis comprises an early diagnosis, an auxiliary diagnosis, or a combination thereof.

In another preferred embodiment, the subject includes human and non-human mammals.

In another preferred embodiment, the general population comprises a healthy population or a population not suffering from intracranial calcification disease.

In another preferred embodiment, the subject is a patient with intracranial calcification disease or a suspected patient.

In another preferred embodiment, the quantitative PCR is used to detect the expression level of one or more genes of markers Z1-Z17, and data processing is performed, wherein the data processing determines whether the object to be tested is a patient with intracranial calcification disease according to the expression level of one or more genes of markers Z1-Z17 of the object to be tested.

In a fifth aspect, the present invention provides a method of evaluating the effect of treatment of intracranial calcification disease, comprising the steps of:

(a) Providing a test sample, wherein the test sample is a brain tissue sample, cerebrospinal fluid or blood isolated from a subject, wherein the subject is a patient during or after treatment for an intracranial calcification disease;

(b) Detecting the expression level of one or more genes of markers Z1-Z17 or the expression level of one or more proteins of Z1-Z17 or the concentration of one or more markers Z1-Z17 in brain tissue, cerebrospinal fluid or blood in the detection sample, and recording as E1; and

(c) Comparing the expression level of the one or more genes of the markers Z1-Z17 or the expression amount of the one or more proteins of the markers Z1-Z17 or the concentration E1 of the one or more of the markers Z1-Z17 with a control reference value E0, wherein if the expression level of the one or more genes of the markers Z1-Z17 or the expression amount of the one or more proteins of the markers Z1-Z17 or the concentration E1 of the one or more of the markers Z1-Z17 is restored to the control reference value E0, a better prevention effect of the intracranial calcification disease or a substantial cure of the intracranial calcification disease or inhibition of the deterioration of the intracranial calcification disease is suggested.

In another preferred embodiment, the method further comprises: comparing the expression level or the expression amount or the concentration E1 with E2, wherein E2 is the expression level of one or more genes of the markers Z1-Z17 or the expression amount of one or more proteins of the markers Z1-Z17 or the concentration of one or more of the markers Z1-Z17 in the same sample of the test subject before receiving the intracranial calcification disease drug treatment.

In a sixth aspect, the present invention provides the use of a marker for diagnosing and/or evaluating the efficacy of a treatment for intracranial calcification disease, wherein said marker is selected from the group consisting of:

(Z1) an Osteopontin (OPN) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z2) Osteocalcin (OCN) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z3) alkaline phosphatase (ALP) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z4) an Osteoprotegerin (OPG) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z5) myosin heavy chain 15 (MYH 15) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z6) aspartyl aminoglucosidase (AGA) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z7) avian sarcoma virus 17 putative transgene (Jun) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z8) beta-Actin (ACTB) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z9) a phospholipase C (PLCG 2) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z10) mitogen-activated protein kinase 14 (MAP 3K 14) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z11) integrin beta 2 (ITGB 2) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z12) death-related protein kinase (DAP) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z13) Maestro heat-like repeat family member 7 (MROH 7) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z14) a cytochrome p450 family member (CYP 2J 13) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z15) chemokine CXC motif ligand 10 (CXCL 10) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z16) c-type lectin domain family 7 member a (CLEC 7A) gene, or mRNA thereof, or cDNA thereof, or protein thereof;

(Z18) (Z1) to (Z17).

In a seventh aspect, the present invention provides a system for diagnosing an intracranial calcification disease, the system comprising:

(a) The characteristic receiving module is used for receiving brain tissue samples, cerebrospinal fluid or blood characteristic data; the characteristic data includes: expression level or amount or concentration of a marker in a brain tissue sample, cerebrospinal fluid or blood, said marker being selected from the group consisting of:

(Z18) (Z1) to (Z17) combinations;

(b) The data processing module is used for comparing the expression level or the expression quantity or the concentration of the marker in the brain tissue sample, the cerebrospinal fluid or the blood with a standard value so as to obtain a judgment result of the intracranial calcification disease; and

(c) And the output module is used for receiving and outputting the judging result.

In another preferred embodiment, the subject is a human.

In another preferred embodiment, the standard value is the expression level or amount or concentration of one or more of the markers Z1-Z17 in the same brain tissue sample of the normal control population, the same brain tissue sample of the brain disease patient population of cerebral spinal fluid or blood or non-intracranial calcification disease.

In another preferred embodiment, a higher level or amount or concentration of expression of a marker in the brain tissue sample, cerebrospinal fluid or blood than a standard value indicates that the subject has an intracranial calcification disease.

In another preferred embodiment, the characteristic receiving module comprises a sample collector and a characteristic signal input end.

In another preferred embodiment, the data processing module includes a processor and a memory, wherein the memory stores threshold information of intracranial calcification disease.

In another preferred embodiment, the output module comprises any terminal, preferably a display, a printer, a tablet (PAD), a smart phone.

In another preferred embodiment, the modules are connected by wire or wirelessly.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the mRNA expression level of calcification-related genes. As shown in FIG. 1, the expression levels of Spp1 (A), mgp (B) and Col4a2 (C) at the mRNA level were significantly increased in the mouse thalamus of the intracranial calcification model (Slc 20a2-Ki homozygote) compared with the wild-type (WT) thalamus. The number of WT and Slc20a2-Ki homozygous mice was 5, respectively; error bars are expressed in mean ± s.e.m; * Represents p value less than 0.01; the p-value is calculated from the rank sum test.

FIG. 2 is an examination of the bone morphogenetic hormone (OPN) in brain tissue of a mouse homozygous for Slc20a 2-Ki. As shown in FIG. 2, (A) for immunofluorescence detection of OPN, nuclei were stained with DAPI. And (B) staining calcification by HE staining method. (A '-B') is an enlarged view of the broken line box in the (A-B) drawing. (A) and (B) are serial sections. Observing the relationship of OPN to intracranial calcification, it was found that there was a significant accumulation of OPN at the location of the intracranial calcified nodules, indicating that OPN is associated with the formation of intracranial calcification and is one of the organic components of intracranial calcification. The number of the Slc20a2-Ki homozygous mice in (A-B) was 3; the lengths of the scales in (A-B) and (A '-B') were 250 μm and 25 μm, respectively.

FIG. 3 shows the experimental groups WT+H ₂ Group O (WT mice given sterile water), ki+H ₂ Concentration of inorganic phosphorus (Pi) in cerebrospinal fluid (CSF) of mice in group O (Slc 20a2-Ki homozygous mice given sterile water), and Ki+LJY-001 (Slc 20a2-Ki homozygous mice given LJY-001 drug). As shown in FIG. 3, is WT+H ₂ O、Ki+H ₂ Pi concentrations in CSF of the O, ki +LJY-001 groups of mice were seen to be identical to Ki+H ₂ Group O is compared, pi concentration in the CSF of Ki+LJY-001 group mice is unchanged, which indicates that the traditional Chinese medicine of the invention does not inhibit calcification development by changing the Pi concentration in the brain of the mice; wt+h ₂ O,n＝12；Ki+H ₂ O, n=12; ki+ljy-001, n=12; ns, no significant difference; * P <0.001 is the ANOVA test result.

FIG. 4 shows the experimental groups WT+H ₂ Group O, ki+H ₂ Concentration of OPN in CSF from mice in group O, ki+LJY-001. As shown in FIG. 4, WT+H ₂ O、Ki+H ₂ OPN concentration in CSF of O, ki +LJY-001 mice group, which was seen to be similar to Ki+H ₂ Group O control, OPN concentration in CSF was significantly down-regulated and restored to wt+h in ki+ljy-001 mice ₂ Group O level, the process of inhibiting calcification by the traditional Chinese medicine of the invention relates to reducing OPN level in the brain of mice; wt+h ₂ O,n＝12；Ki+H ₂ O，n＝12；Ki+LJY-001, n=12; ns, no significant difference; * P<0.001 is the ANOVA test result.

FIG. 5 shows alkaline phosphatase (ALP) examination in brain tissue of Slc20a2-Ki homozygous mice. As shown in fig. 5, the relationship of ALP to intracranial calcification was observed, and a large amount of ALP accumulation was found inside the calcified nodule, indicating that ALP was related to formation of intracranial calcification and was one of the organic components of the calcified nodule. ALP staining, black positive color, and hematoxylin staining of nuclei. The position indicated by the black arrow is a calcification site; the experimental number of the Slc20a2-Ki homozygous mice is 3; the length of the scale bar is 25 μm.

FIG. 6 shows an Osteoprotegerin (OPG) assay in brain tissue of a Slc20a2-Ki homozygous mouse. As shown in fig. 5, the relationship of OPG to intracranial calcification was observed, and it was found that there was a large amount of OPG accumulation inside the calcified nodules, indicating that OPG was associated with formation of intracranial calcification and was one of the organic components of the calcified nodules. Immunohistochemical staining of OPG, positive color brown, nuclei stained with hematoxylin. The position indicated by the black arrow is a calcification site; the experimental number of the Slc20a2-Ki homozygous mice is 3; the length of the scale bar is 25 μm.

FIG. 7 is a thermal graph analysis of gene recovery to WT levels after LJY-001 extract solution administration. As shown in FIG. 7, it can be seen that the reaction with Ki+H ₂ Compared with group O, the expression level of the gene in thalamus of mice in the Ki+LJY-001 group is obviously changed and restored to WT+H ₂ The total of 16 genes at the O group level are Gm12689, gm36560, myh15, aga, jun, col a2, actb, plcg2, map3k14, itgb2, dap, mroh7, cyp2j13, gm13453, cxcl10 and Clec7a, respectively, which indicate that the traditional Chinese medicine of the invention is involved in recovering the expression level of related abnormal expression genes in thalamus in the process of inhibiting calcification occurrence and development. In the first round of experiments, WT+H ₂ Group O mice 5, ki+H ₂ Group O mice 5, ki+LJY-001 group 5; in the second round of experiments, WT+H ₂ Group O mice 4, ki+H ₂ Group O mice 4, ki+LJY-001 group 4; in the third experiment, WT+H ₂ Group O mice 4, WT+LJY-001 group 4, ki+H ₂ Group O mice 4, ki+LJY-001 group 4.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found a group of drug targets that can be used for detecting intracranial calcification diseases, on the basis of which the present invention has been completed.

Specifically, the present study found that the expression level of Osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), and Osteoprotegerin (OPG) in cerebrospinal fluid, and the like, and myosin heavy chain 15 (MYH 15), aspartyl Glucosaminidase (AGA), avian sarcoma virus 17 putative transgene (Jun), β -Actin (ACTB), phospholipase C (PLCG 2), mitogen activated protein kinase 14 (MAP 3K 14), integrin β2 (ITGB 2), death-related protein kinase (DAP), maestro heat-like repeat family member 7 (MROH 7), cytochrome p450 family member (CYP 2J 13), chemokine CXC motif ligand 10 (CXCL 10), C-type lectin domain family 7 member a (CLEC 7A), and α -2 subunit of type IV collagen (COL 4 A2) can be used as targets for detecting intracranial calcification. In the examples of the present invention, it was found that the expression amounts of desmin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP) and Osteoprotegerin (OPG) were significantly increased in the cerebrospinal fluid of intracranial calcified mice, and that the expression amounts of myosin heavy chain 15 (MYH 15), aspartyl Glucosaminidase (AGA), avian sarcoma virus 17 putative transforming gene (Jun), β -Actin (ACTB), phospholipase C (PLCG 2), mitogen activated protein kinase 14 (MAP 3K 14), integrin β2 (ITGB 2), death-related protein kinase (DAP), maestro heat-like repeat family member 7 (MROH 7), cytochrome p450 family member (CYP 2J 13), chemokine CXC motif ligand 10 (CXCL 10), C-type lectin domain family 7 member a (CLEC 7A), and α -2 subunit of type IV collagen (COL 4 A2) were significantly changed in the thalamus of intracranial calcified mice. The set of targets or combinations thereof may thus be used for the preparation of a diagnostic reagent or diagnostic kit for detecting intracranial calcification diseases and/or for assessing the efficacy of a treatment for intracranial calcification diseases.

Terminology

As used herein, "drug targets of the invention," "a set of drug targets that can be used to detect intracranial calcification disease," and the like are used interchangeably and refer to a substance in cerebrospinal fluid selected from the group consisting of:

(Z1) Osteopontin (OPN), or mRNA thereof, or cDNA thereof;

(Z2) Osteocalcin (OCN), or mRNA or cDNA thereof,

(Z3) alkaline phosphatase (ALP), or mRNA or cDNA thereof;

(Z4) Osteoprotegerin (OPG), or mRNA thereof, or cDNA thereof;

(Z5) myosin heavy chain 15 (MYH 15), or mRNA thereof, or cDNA thereof;

(Z6) Aspartyl Glucosaminidase (AGA), or mRNA thereof, or cDNA thereof;

(Z7) avian sarcoma virus 17 putative transgene (Jun), or mRNA thereof, or cDNA thereof;

(Z8) β -Actin (ACTB), or mRNA thereof, or cDNA thereof;

(Z9) phospholipase C (PLCG 2), or mRNA thereof, or cDNA thereof;

(Z10) mitogen-activated protein kinase 14 (MAP 3K 14), or mRNA thereof, or cDNA thereof;

(Z11) integrin β2 (ITGB 2), or mRNA thereof, or cDNA thereof;

(Z12) death-related protein kinase (DAP), or mRNA thereof, or cDNA thereof;

(Z13) Maestro heat-like repeat family member 7 (MROH 7), or mRNA thereof, or cDNA thereof;

(Z14) a cytochrome p450 family member (CYP 2J 13), or mRNA thereof, or cDNA thereof;

(Z15) chemokine CXC motif ligand 10 (CXCL 10), or mRNA thereof, or cDNA thereof;

(Z16) c-type lectin domain family 7 member a (CLEC 7A), or mRNA thereof, or cDNA thereof;

(Z17) alpha-2 subunit of type IV collagen (COL 4 A2), or mRNA thereof, or cDNA thereof; (Z18) (Z1) to (Z17).

(Z18) (Z1) to (Z17).

As used herein, "LJY-001 drug solution" is a combination of traditional Chinese medicines that was studied by the present inventors to be effective in treating intracranial calcification, the formulation of which is disclosed in patent application CN 202011504122.6.

Intracranial calcification

In 1850 Delacour first described intracranial calcification, and subsequently Bamberger et al reported an intracranial calcification in female patients with mental retardation and seizures, pathologically confirmed the presence of intracranial calcification.

Intracranial calcification can be classified into physiological calcification and pathological calcification, and can be regarded as intracranial physiological calcification if the calcification is limited to pallidum and does not involve other parts in the brain, and the patient still does not show neurological symptoms after 40 years old and has no abnormal calcium metabolism disease; pathological calcification may be caused by a number of factors, such as genetic factors, age-related aging, autoimmunity, TORCH syndrome, viral infection, trauma, toxins, physiological injury, hypoparathyroidism, etc., and various congenital syndromes may also be accompanied by cerebral calcification, such as Airdi-Goutene syndrome, cockayne syndrome, kenny-Caffey syndrome type I, krabbe disease, etc.

Although there are many factors inducing intracranial calcification, various intracranial calcifications have common characteristics, the pathological features are similar, bilateral symmetry is often adopted, the calcification is better developed on pallidum, in addition, dentate nuclei, thalamus, cerebellar cortex, other parts of the brain and the like can be related by calcification, and the calcification is gradually developed in the brain; the substance component for intracranial calcification comprises inorganic matters and organic matters, wherein the main component of the inorganic matters is hydroxyapatite.

Idiopathic basal ganglia calcification

Idiopathic basal ganglia calcification (Idiopathic basal gangl ia calcification, IBGC) is an intracranial calcification caused by genetic factors with a incidence of 0.21-2%, which stabilizes the inherited intracranial calcification phenotype, making it a reliable research object for pathogenesis of intracranial calcification diseases, drug targets and prevention and treatment studies thereof.

IBGC is mainly characterized by symmetric calcification of basal ganglia and other parts of the brain, commonly known as Fahr disease. Patients often develop between 30-60 years of age, and are overwhelmed by calcification often accompanied by parkinsonism, ataxia, dystonia, dementia, memory loss, confusion, affective disorders, epilepsy, and migraine. The common calcified parts of IBGC diseases include caudate nucleus, dentate nucleus, white matter, thalamus, cortex, midbrain, brain bridge and the like, and calcium salt particles are widely distributed on the blood vessel wall and the periphery thereof by researching the pathological process.

However, serum biochemical indexes such as blood phosphorus, blood calcium, parathyroid hormone (PTH), alkaline phosphatase, 1,25 (OH) 2D, fibroblast growth factor 23 (FGF 23) and the like of IBGC patients are normal. The disease is first reported by Delacour in 1850, the pathogenic mechanism of the disease is unknown before 2012, we start from families, clone and report the pathogenic gene SLC20A2 of the first IBGC for the first time through genetic analysis, and functional analysis finds that the gene mutation influences the transportation of inorganic phosphorus by cells, so that inorganic phosphorus (Pi) in extracellular matrix is accumulated to cause the disease. It has been found in the current study that about 50% of IBGC patients are caused by SLC20A2 gene mutation. Therefore, a mouse with the Slc20a2 gene mutation (p.S602W) is constructed, and the mouse is identified to have obvious intracranial calcification phenotype, so that the mouse is a good intracranial calcification model mouse; meanwhile, we find a traditional Chinese medicine composition LJY-001 with obvious inhibition effect on intracranial calcification, and the researches lay a foundation for screening a medicine target of the intracranial calcification.

Osteopontin (OPN)

Osteopontin (OPN), which is a secreted phosphoprotein that is a member of the N-linked glycoprotein (SIBLING) family of small integrin-binding ligands of cellular matrix proteins, is encoded by the Spp1 gene, also known as bone-regulating factor, and is involved in a variety of biological activities. OPN plays a role in bone metabolism and homeostasis. OPN is not only an important factor for neuronal mediation and endocrine regulation of bone mass, but also is involved in biological activities such as proliferation, migration, adhesion, etc. of various bone-related cells, including bone marrow mesenchymal stem cells, hematopoietic stem cells, osteoclasts, osteoblasts. OPN is closely related to the development of many bone related diseases, such as osteoporosis, rheumatoid arthritis, osteosarcoma, etc.

Osteocalcin (OCN)

Osteocalcin (OCN) is encoded by Mgp gene, also called Osteocalcin, and is an important component of bone extracellular matrix, and is non-collagenous protein with the most abundant content in bone. OCNs can bind to calcium ions and contain three highly conserved glutamyl groups (Glu), which can be carboxylated by vitamin k-dependent gamma-glutamyl carboxylase (GGCX), resulting in conformational changes that stabilize the alpha-helical portion of the protein while also imparting greater affinity to calcium ions and hydroxyapatite. Therefore, OCN plays a critical role in skeletal development, being a marker for bone formation and remodeling.

Alkaline phosphatase (ALP)

Alkaline phosphatase (alkaline phosphatase, ALP) is encoded by the ALPL gene and is a membrane-bound glycoprotein that hydrolyzes various monophosphate esters under high pH conditions. ALP is a rich enzyme existing in animal bones and widely distributed in human liver, bones, intestines, kidneys, placenta and other tissues, can rapidly hydrolyze hexose phosphate into phosphoric acid, plays an important role in human bone mineralization, and many cases of low phosphatase prove that the low phosphatase is a rare metabolic genetic disease caused by ALPL gene mutation.

Bone protecting element (OPG)

Osteoprotegerin (OPG), encoded by the TNFRSF11B gene, is a member of the TNF receptor superfamily. OPG is an important regulator of bone metabolism and plays a role in the vascular system. Studies have shown that OPG is an important arterial calcification inhibitor, and under certain pathological conditions, such as diabetes, chronic kidney disease and other metabolic diseases, endothelial cells release OPG, protecting it from survival.

Myosin heavy chain 15 (MYH 15)

Myosin heavy chain 15 is encoded by the MYH15 gene and is a slow-contracting myosin. It was cloned for the first time in 2002, and related functional studies are very few at present. Recent studies have found that genetic alterations in myosin heavy chain 15 are a risk factor for amyotrophic lateral sclerosis, and MYH15 may regulate expansion G ₄ C ₂ Toxicity of dipeptides produced by the replicates.

Aspartyl Glucosaminidase (AGA)

Aspartyl aminoglucosidase (AGA) is encoded by the AGA gene and is a key enzyme for the catabolism of n-oligosaccharides in glycoproteins. Mutation of the AGA gene results in accumulation of AGA substrate in vivo, causing an autosomal recessive lysosomal storage disease, aspartate glucosamine diabetes (AGU).

Fowl sarcoma virus 17 postulates to transform gene (Jun)

Avian sarcoma virus 17 assumes that the transgene (JUN) is encoded by the JUN gene and is an oncogene. Jun deficiency in mice results in embryonic death in mid and late gestation, manifested by impaired liver production, altered fetal liver erythropoiesis and systemic edema. JUN can prevent apoptosis by antagonizing p53 activity in early stages of tumor progression.

beta-Actin (ACTB)

beta-Actin (ACTB) is encoded by the ACTB gene and is essential for many cytoplasmic functions, such as regulation of cell shape, migration, nuclear function, gene expression, cell division, proliferation, etc.

Phospholipase C (PLCG 2)

Phospholipase C (PLCG 2) is encoded by the PLCG2 gene and is an essential enzyme for agonist-induced calcium entry into cells. PLCG catalyzes the hydrolysis of phospholipids to form water-soluble phosphorylated derivatives of diacylglycerol and lipidyl, and is specific for inosine phosphate.

Mitogen-activated protein kinase 14 (MAP 3K 14)

Mitogen-activated protein kinase 14 (MAP 3K 14) is encoded by MAP3K14 gene, also known as nuclear factor- κB (NF- κB) induced kinase, and can activate NF- κB, and participate in regulating and controlling cell proliferation and apoptosis genes and expression of genes responsive to inflammation and immune response.

Integrin beta 2 (ITGB 2)

Integrin beta 2 (ITGB 2) is encoded by the ITGB2 gene, also known as CD18, a leukocyte adhesion molecule, which belongs to the cell membrane glycoprotein. Beta 2-integrins are primarily involved in wound healing, which is achieved with the aid of cellular signals and microvascular responses, including pathogen or cell debris removal, etc.; beta 2-integrins can also mediate leukocyte adhesion and crawling in key processes for treatment of infections.

Death-related protein kinase (DAP)

Death-related protein kinase (DAP), encoded by the DAP gene, is a positive mediator of gamma-Interferon (IFNG) -induced apoptosis. Inactivation of DAP expression may reduce the sensitivity of IFNG-induced apoptosis in HeLa cells.

Maestro heat-like repeat family member 7 (MROH 7)

Maestro heat-like repeat family member 7 (MROH 7) is encoded by the MROH7 gene, MROH7 and the downstream gene tetra-tripeptide repeat domain protein 4 (TTC 4) act as apoptosis inhibitors in vascular endothelial cells lacking serum and FGF-2.

Cytochrome p450 family members (CYP 2J 13)

Cytochrome P450 family members (CYP 2J 13) are encoded by CYP2J13, and no exact protein function study is available to predict heme binding activity, isomerase activity and monooxygenase activity, involved in cyclooxygenase P450 pathway, linoleic acid metabolic process and heterologous metabolic process.

Chemokine CXC motif ligand 10 (CXCL 10)

Chemokine CXC motif ligand 10 (CXCL 10) is encoded by the CXCL10 gene, also known as IP10, and IP10 inhibits bone marrow colony formation as a member of the alpha chemokine family, has antitumor activity in vivo, is a chemotactic agent for human monocytes and T cells, and promotes adhesion of T cells to endothelial cells. IP10 is also a potent inhibitor of angiogenesis in vivo, likely to be involved in the regulation of angiogenesis during inflammation and tumorigenesis.

c-type lectin domain family 7 member a (CLEC 7A)

The c-type lectin domain family 7 member a (CLEC 7A) is encoded by CLEC7A gene, a pattern recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils), recognizes a variety of beta-1, 3-linked and beta-1, 6-linked glucans in fungal and plant cell walls, and triggers direct cellular antibacterial activity, including phagocytosis and reactive oxygen species production.

Alpha-2 subunit of type IV Collagen (Collagen alpha-2 (IV), col4a 2)

The alpha-2 subunit of type IV Collagen (Collagen alpha-2 (IV)) is encoded by the Col4a2 gene and is a component of type IV Collagen. Type IV collagen is associated with laminin, entactin, and heparin sulfate proteoglycans, forming a sheet-like basement membrane that separates epithelial cells from connective tissue, one of the marker proteins of vascular basement membrane. The research shows that the IV type collagen is accumulated in a great amount in kidney stones, and is one of organic components of kidney stone calcification matrixes.

Primer(s)

A primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, and is covalently linked to a reactant. Primers are typically two oligonucleotide sequences that are synthesized, one complementary to one strand of the DNA template at one end of the target region and the other complementary to the other strand of the DNA template at the other end of the target region.

In the present invention, in order to improve the sensitivity of the detection system, the corresponding gene fragment in the detection system is amplified in advance, and thus a primer corresponding to the sequence in which the mutation is located is designed.

In a preferred embodiment, for the cDNA of glyceraldehyde phosphate dehydrogenase (Gapdh), the optimal primer pair for detecting glyceraldehyde phosphate dehydrogenase (Gapdh) is designed as SEQ ID No. 1 and 2.

In another preferred embodiment, the optimal primer pair for detecting Spp1 (OPN) is designed for the cDNA of Spp1 (OPN) as SEQ ID Nos. 3 and 4.

In another preferred example, the optimal primer pair for Mgp (OCN) is designed for cDNA of Mgp (OCN) as SEQ ID Nos. 5 and 6.

In another preferred embodiment, the optimal primer pair for Col4a2 is designed for the cDNA of Col4a2 as SEQ ID Nos. 7 and 8.

Probe with a probe tip

As used herein, a "probe" refers to a nucleic acid sequence (DNA or RNA) with a detectable label and a known sequence that is complementary to a gene of interest (referred to herein). The gene probe is combined with the target gene through molecular hybridization to generate hybridization signals, and the target gene can be displayed from a vast genome. According to the hybridization principle, the nucleic acid sequence as a probe must have at least the following two conditions: (1) if the double strand is used, the double strand must be denatured; (2) should be provided with a label that is easily detectable. The nucleic acid probe may include the entire gene or may be only a part of the gene; either DNA itself or RNA transcribed from it. In the present invention, the probe also refers to a modified primer, wherein both ends or the middle of the modified primer are provided with chemical modification groups, and the chemical modification has special functions including but not limited to: signaling, enhancing the attachment to the reactants, etc.

Kit for detecting a substance in a sample

Based on the significantly higher expression level of the drug target of the present invention in a cerebrospinal fluid sample of a mouse model of intracranial calcification disease, the present invention provides a kit comprising a marker selected from the group consisting of:

(Z1) Osteopontin (OPN), or mRNA thereof, or cDNA thereof;

(Z2) Osteocalcin (OCN), or mRNA or cDNA thereof,

(Z3) alkaline phosphatase (ALP), or mRNA or cDNA thereof;

(Z4) Osteoprotegerin (OPG), or mRNA thereof, or cDNA thereof;

(Z5) myosin heavy chain 15 (MYH 15), or mRNA thereof, or cDNA thereof;

(Z6) Aspartyl Glucosaminidase (AGA), or mRNA thereof, or cDNA thereof;

(Z8) β -Actin (ACTB), or mRNA thereof, or cDNA thereof;

(Z9) phospholipase C (PLCG 2), or mRNA thereof, or cDNA thereof;

(Z11) integrin β2 (ITGB 2), or mRNA thereof, or cDNA thereof;

(Z12) death-related protein kinase (DAP), or mRNA thereof, or cDNA thereof;

(Z18) (Z1) to (Z17).

The main advantages of the invention include:

(a) The present invention discovers for the first time the expression levels of Osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP) and Osteoprotegerin (OPG) in the cerebrospinal fluid of mice, the expression levels of myosin heavy chain 15 (MYH 15), aspartyl Glucosaminidase (AGA), avian sarcoma virus 17 putative transgene (Jun), β -Actin (ACTB), phospholipase C (PLCG 2), mitogen activated protein kinase 14 (MAP 3K 14), integrin β2 (ITGB 2), death-related protein kinase (DAP), maestro coat-lipeat repeat family member 7 (MROH 7), cytochrome p450 family member (CYP 2J 13), chemokine CXC motif ligand 10 (CXCL 10), C-type lectin domain family member a (CLEC 7A), and alpha-2 subunit of collagen IV (COL 4 A2) in relation to the calcification of intracranial diseases.

(b) The present inventors found that, after having intracranial calcification disease, osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP) and Osteoprotegerin (OPG) expression were increased, and that in thalamus of intracranial calcified mice, the expression level of myosin heavy chain 15 (MYH 15), aspartyl Glucosaminidase (AGA), avian sarcoma virus 17 putative transgene (Jun), β -Actin (ACTB), phospholipase C (PLCG 2), mitogen-activated protein kinase 14 (MAP 3K 14), integrin β2 (ITGB 2), death-related protein kinase (DAP), maestro heat-like repeat family member 7 (MROH 7), cytochrome p450 family member (CYP 2J 13), chemokine CXC motif ligand 10 (CXCL 10), C lectin domain family 7 member a (CLEC 7A), and α -2 subunit of type IV collagen (COL 4 A2) were significantly increased. Therefore, the set of targets can be used for preparing a diagnostic reagent or a diagnostic kit for detecting intracranial calcification diseases and/or evaluating the therapeutic effect of the intracranial calcification diseases.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

The experimental model to be used in this example was established as follows:

model mice for intracranial calcification disease (slc 20a2-p.S602W Ki mice)

Prior to the present invention, we constructed model mice for intracranial calcification disease (slc 20a2-p.S602W Ki mice). The mouse slc20a2 Gene [ Ensembl Gene ID (ENSMUSG 00000037656)]The nucleotide CC at 1805-1806 of cDNA (Transcript: slc20a2-201 (ENSMUST 00000067786)) was mutated to GG to thereby mutate the 602 th codon TCC to TGG, and the encoded serine to tryptophan (p.S602W), to obtain Wild Type (WT) and heterozygous type (slc 20a 2) ^+/S602W ) And pure sum type (slc 20a 2) ^S602W/S602W ) And (3) a mouse.

By phenotypic analysis we determined that homozygous mice have a very similar phenotype to human intracranial calcification disease, and that homozygous mice have brain with calcified phenotypes and other symptoms that develop progressively with age, such as abnormal motor balance, anxiety behavior, learning and memory, and depressive behavior. The mechanical study found that the Pi concentration of homozygous mouse CSF was significantly increased compared to that of wild-type mouse CSF, indicating that the homozygous mutation of Slc20a2 disrupted normal Pi homeostasis in the mouse brain, consistent with that of Slc20a2 mutation causing local Pi homeostasis imbalance in the brain of human IBGC patients. Therefore, we used the homozygous mice described above as intracranial calcification model mice.

The various detection methods used in the examples are listed below:

1. proteomics

The iTRAQ technology is a new proteomic quantitative research technology recently developed, and can be obtained: typically 500 to 600 proteins, and differences in protein expression between different samples. The iTRAQ kit comprises eight amine active agents in the same amount, and can label the peptide fragment of protein hydrolysis, so that the peptide fragment can be accurately identified and quantified by adopting a tandem mass spectrometry method. The method comprises the following specific steps:

(1) Distributing 3 male wild mice and intracranial calcification model mice, taking fresh brain tissue, and extracting protein;

(2) Respectively carrying out protease hydrolysis on 6 parts of protein;

(3) Marking the enzymolysis fragments by adopting different marks and then mixing;

(4) Performing primary mass spectrometry using a combination of liquid chromatography and mass spectrometry;

(5) The same tagged peptide fragment of the same protein, of 6 different sources, appears as a peak on the primary mass spectrum.

(6) Performing secondary mass spectrometry on the peptide fragment added with the label, wherein the balance group is detached from the reporter group;

(7) After secondary mass spectrometry, the reporter group generates 6 reporter ion signals in the low mass region of the secondary mass spectrometry, and the intensities of the reporter ion signals respectively represent the same peptide segment of 6 marked samples;

(8) The peak area ratio of the reported ions is the ratio of the same peptide of the same protein between different samples;

(9) And carrying out biological information analysis, qualitative and quantitative research on data obtained by the mass spectrometer.

2. Real-time quantitative PCR experiments

The primers for the above genes were analyzed and designed by http:// asia. Ensembl. Org/index. Html search for the gene sequence information of glyceraldehyde phosphate dehydrogenase (Gapdh), osteomodulin (Spp 1), osteocalcin (Mgp) and collagen type IV α -2 subunit (Col 4a 2), using GeneTool 2.0 software and Oligo v7.56 software, respectively, and the primer design principle mainly includes: the primer spans two exons, and the size of the fluorescent quantitative product fragment is between 100 and 200 bp. Primer sequence information is shown in Table A:

table A sequence in this example

Fresh brain tissue of a mouse is extracted, thalamus is separated, and the brain tissue is put into a homogenizer which is filled with 1mL of TRIzol lysate in advance, and is ground uniformly, and the brain tissue is left to stand for 5-15min at room temperature, so that the tissue is completely lysed. The homogenate was transferred to a 2mL EP tube, 200. Mu.L of chloroform was added, mixed by vortexing, allowed to stand for 2min, and centrifuged at 12000rpm at 4℃for 15min. The supernatant solution containing RNA was taken into a fresh EP tube, 0.5mL of isopropanol was added to precipitate RNA, and the mixture was allowed to stand at room temperature for 10min and centrifuged at 12000rpm for 5-10min at 4 ℃. Removing supernatant, adding 0.5mL 75% ethanol, shaking up and down for several times, and centrifuging at 12000rpm at 4deg.C for 5-10min. Removing the supernatant, drying RNA at room temperature for 5-15min, adding proper amount of DEPC water for dissolving, and measuring concentration. And sucking a proper volume of RNA solution according to the concentration to carry out agarose gel electrophoresis, and detecting whether the extracted RNA has degradation. According to the RNA concentration, after calculating the volume of the required RNA template, the reverse transcription kit instruction of the first strand cDNA synthesis is used to prepare 10 mu L system, and the first strand cDNA is synthesized as a DNA template in the subsequent real-time quantitative PCR experiment. According to the fluorescent quantitative PCR kit Green Master Mix, vazyme Biotech) protocol prepares 10. Mu.L of reaction system: the forward and reverse primers were 0.3. Mu.L, 2 XMix 5. Mu. L, DEPC water 4.05. Mu.L and cDNA template 0.35. Mu.L, respectively. 3 parts of sample reaction system are prepared for each gene of each mouse, and 9.4 mu L (the tube wall is stained with liquid and 10 mu L of reaction liquid cannot be accurately sucked) is added into a 96-well plate. Carrying out real-time quantitative PCR reaction on the prepared reaction system on a StepOneGlus fluorescent quantitative PCR instrument (Life TechnologiesTM), wherein the reaction conditions comprise the preparation stage: pre-denaturation at 95 ℃ for 5min; and (3) a circulation stage: denaturation at 95℃for 10sec, extension at 60℃for 30sec,40 cycles; melting curve stage: 95℃for 15sec, 60℃for 1min, and 95℃for 15sec. After completion of the reaction, data from real-time quantitative PCR was analyzed using StepOne Software v 2.3.

3. Immunohistochemical staining

(1) Production of paraffin sections

After the target mice were sacrificed under anesthesia, fresh brain tissue was rapidly isolated, and the brain tissue was placed in 4% paraformaldehyde and fixed overnight (over 12 h). And taking out the fixed brain tissue, flushing the brain tissue for a plurality of times by using clear water, and trimming the brain tissue according to the required slice shape. The brain tissue is sequentially put into 75% alcohol (3-4 h), 85% alcohol (1.5-2.5 h), 90% alcohol (1-2 h), 95% alcohol (1 h), absolute ethyl alcohol I (0.5 h) and absolute ethyl alcohol II (0.5 h) for dehydration, and then the brain tissue is respectively subjected to transparency with alcohol benzene (absolute ethyl alcohol: xylene=1:1), xylene I and xylene II for 7-10min, and the transparency of the central part of the brain tissue is taken as a standard. The brain tissue was waxed 3 times with a paraffin solution at 70 c for 1h each time, followed by embedding. Finally, slicing is carried out, and slices with the thickness of 4-12 mu m are cut according to the requirement.

(2) Hematoxylin-eosin (HE) staining

Paraffin sections are dewaxed with xylene and gradient alcohol, and the sections are sequentially put into xylene I for 7-10min, xylene II for 7-10min, absolute ethyl alcohol for 5-10min, 95% alcohol for 5min, 85% alcohol for 5min, 70% alcohol for 5min and distilled water for 2min. The frozen sections are not required to be dewaxed, and can be subjected to subsequent staining after being taken out of a refrigerator and placed at 37 ℃ for 30min.

Placing the slices in hematoxylin dye solution, staining cell nuclei for 3min, taking out, washing with running water for 1min, differentiating with 1% hydrochloric acid alcohol for 5-10sec, washing with running water for 1min, returning to blue with 1% ammonia water for 30sec, and washing with running water for 1min. Placing the slices in eosin dye solution, dyeing cytoplasm for 1min, taking out, sequentially placing into absolute ethyl alcohol I5 min, absolute ethyl alcohol II 5min, dimethylbenzene I5 min and dimethylbenzene II 5min for dehydration, taking out the slices, airing, and sealing the slices with neutral resin. Finally, the pictures were observed and taken by microscopy.

(3) Immunofluorescent staining

The sections were taken and treated prior to staining of paraffin sections according to the HE staining procedure in experimental method (2). After distilled water washing, the tissue slice is placed in EDTA antigen retrieval liquid (pH=8.0), antigen retrieval is carried out by heating in a microwave oven, medium and low fire is carried out for 10min, after natural cooling at room temperature, PBS buffer is used for washing for 3 times, and each time is carried out for 5min. Tissue sections were blocked with 3% BSA solution for 30min. After the sealing is completed, the sealing liquid is gently thrown away, the prepared primary antibody solution is dripped on the surface of the tissue slice, and the tissue slice is placed in a wet box and incubated overnight at 4 ℃ for about 12 hours. After the primary antibody solution is gently thrown off the next day, the primary antibody solution is washed for 3 times by PBS buffer solution for 5min each time, and is gently dried after washing, the prepared secondary antibody solution is dripped, and the primary antibody solution is incubated for 50min. Then washing with PBS buffer solution for 3 times, each time for 5min, slightly spin-drying after washing, dripping DAPI dye solution, counterstaining the cell nuclei, and keeping away from light for 5-10min at room temperature. Washing with PBS buffer solution for 5min for 3 times, slightly drying, and sealing with anti-fluorescence quenching sealing tablet. Finally, observation under a fluorescence microscope and photographing were performed.

4. Inorganic phosphorus (Pi) concentration detection

The malachite green method detects Pi concentration in mouse CSF. The reaction principle is as follows: malachite green dye reacts with Pi's phosphomolybdate in molybdic acid solution, producing phosphomolybdate complex under acidic conditions to color, and the solution turns from brown to green.

The required reagents: KH (KH) ₂ PO ₄ (Pi standard), 4N hydrochloric acid (i.e., 4 mmol/l hydrochloric acid), ammonium molybdate tetrahydrate, malachite green; preparing a solution: a solution of 4.2% ammonium molybdate in 4N hydrochloric acid, 0.045% malachite green (as prepared); the specific experimental steps are as follows:

1 microliter of cerebrospinal fluid is diluted to 50 microliters, 75 microliter of malachite green solution and 25 microliter of ammonium molybdate solution are sequentially added, mixed and then added into a 96-well plate, an enzyme-labeled instrument detects the light absorption value of the solution at 650 nanometers, and the concentration of inorganic phosphorus in the solution is obtained according to a standard curve. Each sample was averaged 3 times.

5. OPN concentration detection

The OPN concentration was measured by an Elisa kit (Mouse Osteopontin ELISA Kit, bosterbio, USA) as follows: first, the sample and the standard were added and reacted at 37℃for 90 minutes. The biotin-labeled antibody was then added thereto and reacted at 37℃for 60 minutes. The 1 x wash buffer was washed 3 times. Then, avidin-peroxidase complex was added thereto and reacted at 37℃for 30 minutes. The 1X wash buffer was washed 5 times. Then TMB color development solution is added for reaction for 25-30 minutes at 37 ℃. Finally, adding TMB stopping solution, and detecting absorbance by using an enzyme-labeled instrument 450 nm.

6. Transcriptome sequencing (RNA-seq)

Transcriptomes are the collection of all RNAs transcribed by a particular tissue or cell at a certain developmental stage or functional state, including predominantly mRNA and non-coding RNAs. Transcriptome studies enable studies of gene function and gene structure from an overall level, revealing molecular mechanisms in specific biological processes and disease occurrence processes. Transcriptome sequencing (RNA-seq) refers to cDNA sequencing using second generation high throughput sequencing techniques to comprehensively and rapidly obtain almost all transcripts of a particular organ or tissue of a species in a particular state. The RNA-seq technique is specifically as follows:

(1) Total RNA was extracted from each thalamus sample separately and the RNA integrity and content was accurately determined.

(2) 1 microgram of total RNA was taken as the starting RNA for library construction, mRNA with poly A tail was enriched by magnetic beads, and the resulting mRNA was randomly disrupted with divalent cations. The first strand and the second strand of cDNA are synthesized respectively by using fragmented mRNA as a template and random oligonucleotides as primers. The purified double-stranded cDNA is subjected to end repair, adenylate tail addition and sequencing joint connection, cDNA with the size of 370-420 bases is screened out, and Polymerase Chain Reaction (PCR) amplification and PCR product purification are carried out, so that a library is finally obtained.

(3) And (3) detecting the quality of the library, and sequencing different libraries according to the effective concentration and the requirement of the target off-machine data volume after the library is qualified. The basic principle of sequencing is sequencing while synthesizing, adding four fluorescent labeled deoxynucleotide mixtures, DNA polymerase and adaptor primer into a sequencing flow cell for amplification, and when each sequencing cluster extends complementary strand, each fluorescent labeled deoxynucleotide mixture is added to release corresponding fluorescence, and a sequencer captures a fluorescence signal and converts the optical signal into a sequencing peak through computer software, so that the sequence information of the fragment to be detected is obtained.

(4) And carrying out data visualization analysis on the sequencing result, and presenting the result in the form of heat map analysis.

Example 1 establishment of OPN as a drug target for the prevention and treatment of intracranial calcification disease

1.1. Screening intracranial calcification targets by proteomics

In the invention, 5 mice of the initial 3 month old Wild Type (WT) and the initial intracranial calcification model (Slc 20a2-Ki homozygote) are taken to extract fresh brain tissues, and the brain tissues of the mice are subjected to proteomic analysis by isotope relative labeling and absolute quantification (iTRAQ), so that a plurality of protein expression levels possibly related to calcification are found to be up-regulated: protein expression amounts of Osteopontin (OPN), osteocalcin (OCN), and alpha-2 subunit of type IV collagen are shown in Table 1.

Table 1 3 month old WT and Slc20a2-Ki homozygous mice brain tissue proteomics Experimental differential protein analysis

1.2 Verification of intracranial calcification targets at mRNA level

And (3) verifying the expression of proteins such as OPN, OCN, IV type collagen and the like at the mRNA level through a real-time quantitative PCR experiment. 5 WT and Slc20a2-Ki homozygous mice at 3 months of age were taken, respectively, fresh brain tissue of each mouse was extracted, thalamus was isolated, RNA was rapidly extracted, and a subsequent real-time quantitative PCR experiment was performed, taking into consideration that the initial calcified region of the Slc20a2-Ki homozygous mice was thalamus. In the real-time quantitative PCR experiment, gapdh was used as an internal reference gene to examine the expression of OPN (Spp 1), OCN (Mgp) and type IV collagen alpha-2 subunit (Col 4a 2) genes, respectively. The results showed that the expression levels of Spp1, mgp and Col4a2 at the mRNA level were significantly increased in the Slc20a2-Ki homozygous thalamus compared to the WT thalamus (fig. 1A-C), which is consistent with the trend of change in proteomics.

Further, the role of OPN in the formation of intracranial calcification was explored, 3 mice homozygous for Slc20a2-Ki, 6 months old, were obtained, and after all brains were obtained, serial sections with a thickness of 5 μm were prepared. Taking two consecutive sections, one of which is immunofluorescent stained with an antibody to OPN; the other piece was HE stained to color intracranial calcification to dark purple. Observing the relationship of OPN to intracranial calcification, it was found that there was a significant accumulation of OPN at the location of the intracranial calcified nodules, indicating that OPN was associated with formation of intracranial calcification and was one of the organic components of intracranial calcification (fig. 2A-B, A '-B').

Since OPN is also a secreted protein, we speculate that OPN may be an early biomarker of intracranial calcification formation in mouse cerebrospinal fluid (CSF) as a potential intracranial calcification drug target.

1.3 in vivo experiments verify that OPN is a target for intracranial calcification diseases

Based on the above study, in this example, WT+H was designed by taking WT and Slc20a2-Ki homozygous mice as subjects and taking sterilized water as drinking water for WT mice ₂ Group O Ki+H with sterilized water as drinking water for Slc20a2-Ki homozygous mice ₂ Group O, ki+LJY-001 group with LJY-001 liquid medicine as drinking water of Slc20a2-Ki homozygous mice, starting experiment from 21 days after birth of mice, extracting mouse CSF when the age of each group of mice reaches 90 days, and checking OPN concentration in CSF by the detection method; meanwhile, the detection of Pi content in the CSF of the Slc20a2-Ki homozygous mice was known to be significantly increased compared to WT mice.

As a result, it was found that with WT+H ₂ Ki+H compared to group O mice ₂ The concentrations of Pi and OPN were significantly elevated in CSF in group O mice (fig. 3-4); and Ki+H ₂ The Pi concentration was not improved in the ki+ljy-001 group compared to group O mice (fig. 3), whereas the OPN concentration was significantly down-regulated and restored to wild type mouse levels (fig. 4). It is demonstrated that the mechanism of inhibiting calcification of the LJY-001 on mice with intracranial calcification model is not through regulating the Pi steady state of cerebrospinal fluid, but regulating the OPN expression of brain of calcified mice, so that the mice can be restored to the level of wild mice. Therefore, OPN concentration in CSF can be used as a drug target for preventing and treating intracranial calcification.

Example 2 establishment of OCN as a drug target for the prevention and treatment of intracranial calcification disease

We found that in example 1, the up-regulated OCN expressed in the brain of the mice homozygous for Slc20a2-Ki was also a secreted protein by proteomic (Table 1) and real-time quantitative PCR (FIG. 1B) assays, and the mice were tested in the same manner as in example 1, and the concentrations of OCN in the CSF were examined.

As a result, it was found that with WT+H ₂ Ki+H compared to group O mice ₂ The trend of the OCN concentration of mice in group O and Ki+LJY-001 was comparable to that of OPN concentration in example 1. Therefore, the concentration of OCN in CSF can be used as a drug target for preventing and treating intracranial calcification.

EXAMPLE 3 establishment of ALP as a drug target for the prevention and treatment of intracranial calcification disease

From the results of the study in example 1, it was found that OPN, OCN and type IV collagen associated with calcification formation are all bone formation-related proteins, based on which we speculate that formation of intracranial calcification is associated with the appearance of an osteogenic environment in the brain. We therefore examined the expression of the osteoblastic marker protein alkaline phosphatase (ALP) in the brain and ALP staining found that there was a large accumulation of ALP inside calcified nodules in the brain of the Slc20a2-Ki homozygous mice, indicating that ALP was associated with formation of intracranial calcification and was one of the organic components of calcified nodules (fig. 5A-C). ALP was also present in the CSF in an enriched amount, and a mouse experiment was performed in the same manner as in example 1, and the mouse CSF was extracted to examine the concentration of ALP in the CSF. As a result, it was found that with WT+H ₂ Ki+H compared to group O mice ₂ ALP concentration change trends of mice in group O and Ki+LJY-001 were comparable to that of OPN concentration in example 1. The ALP concentration in CSF can therefore be used as a drug target for the prevention and treatment of intracranial calcification.

Example 4 establishment of OPG as a drug target for the prevention and treatment of intracranial calcification disease

From the results of the study in example 1, it was found that OPN, OCN and type IV collagen associated with calcification formation are all bone formation-related proteins, based on which we speculate that formation of intracranial calcification is associated with the appearance of an osteogenic environment in the brain. We therefore examined the expression of Osteoprotegerin (OPG) in the brain, which plays an important role in vascular calcification formation, and immunohistochemical staining found that there was a large accumulation of OPG inside calcified nodules in the brain of the Slc20a2-Ki homozygous mice, indicating that OPG was involved in intracranial calcification formation and was one of the organic components of calcified nodules (fig. 6A-C). OPG was also present in the CSF in an enriched amount as a secreted protein, and the concentration of OPG in the CSF was examined by taking out the mouse CSF by the procedure of example 1.

As a result, it was found that with WT+H ₂ Ki+H compared to group O mice ₂ The trend of OPG concentration change in mice of group O and Ki+LJY-001 was comparable to that of OPN concentration change in example 1. Therefore, the concentration of OPG in CSF can be used as a drug target for preventing and treating intracranial calcification.

EXAMPLE 5 transcriptome sequencing (RNA-seq) 13 intracranial calcification targets were screened

In this example, WT and Slc20a2-Ki homozygous mice were used as subjects, and administration was performed by gastric lavage, and WT+H was designed for administration to WT mice using sterilized water, respectively ₂ Group O, WT+H administered as WT mice with LJY-001 extract solution ₂ Group O, ki+H administered in sterilized water as Slc20a2-Ki homozygous mice ₂ Group O, ki+LJY-001 group administered in mice homozygous for Slc20a2-Ki with LJY-001 extract solution. The LJY-001 extract solution is prepared by concentrating LJY-001 liquid medicine to obtain extract, dissolving the extract in sterile water before experiments to obtain extract solution, wherein each 0.3 ml of extract solution contains 10 mg of extract, and the extract solution is used for the gastric lavage administration of the drug group in the above mice experiments, and each mouse is subjected to gastric lavage by 0.3 ml of extract solution every day. Experiments were performed from 9 weeks of age for 4 weeks, and when the age reached 13 weeks (age at which intracranial calcification initially occurred), fresh brain tissue was extracted from each group of mice, thalamus (intracranial calcification initially formed site) was isolated, and after liquid nitrogen quick freezing, the mice were stored at-80 ℃ in an ultra-low temperature refrigerator. The thalamus tissue was then subjected to transcriptome sequencing (RNA-seq) to analyze the effect of the traditional Chinese medicine composition LJY-001 of the present invention on the mouse thalamus gene expression. The experiment is carried out for 3 rounds to ensure the true reliability of the differentially expressed genes obtained by sequencing.

Visual analysis of 3 rounds of RNA-seq data, thermal mapping analysis found a correlation with Ki+H ₂ Compared with group O, the expression level of the gene in thalamus of mice in the Ki+LJY-001 group is obviously changed and restored to WT+H ₂ The results of the above results confirm that the Chinese medicinal composition of the present invention can make the expression level abnormal gene Gm12689, gm36560, myh15, aga, jun, col4a2, actb, plcg2,Map3k14, itgb2, dap, mroh7, cyp2j13, gm13453, cxcl10, clec7a are restored to normal levels, thereby achieving the effect of inhibiting intracranial calcification in mice; among them, gm12689, gm36560, gm13453 are DNA sequences with unknown functions, and are genes that have not been confirmed. Therefore, the expression levels of 13 genes such as Myh15, aga, jun, col a2, actb, plcg2, map3k14, itgb2, dap, mroh7, cyp2j13, cxcl10, clec7a and the like in the thalamus can be used as drug targets for preventing and treating intracranial calcification.

Discussion:

prior to the present invention, we have also found a Chinese medicinal composition LJY-001 which has an obvious inhibitory effect on intracranial calcification. Taking brain of the intracranial calcification model mouse to prepare paraffin sections after taking the intracranial calcification model mouse for 27 weeks from the 4 th week after birth, and finding that intracranial calcification is obviously inhibited after the administration of the intracranial calcification model mouse; starting from 21.5 weeks of age, after continuous administration for 8.5 weeks, paraffin sections were prepared from brains of intracranial calcification model mice, and it was found that intracranial calcification was also significantly inhibited after administration of the intracranial calcification model mice. After taking LJY-001, the patient with intracranial calcification also has been found to significantly reduce the deposition of calcium in the brain and alleviate clinical symptoms (see Chinese patent application No. CN 202011504122.6).

The invention designs a sterilized water supply group and an LJY-001 supply group of wild type and intracranial calcification model mice. Starting from the 4 th week after birth, after continuous administration for 10 weeks, the cerebrospinal fluid of the mice was extracted, and the levels of OPN, OCN, ALP and OPG were measured using the Elisa kit. The results showed that the content of OPN, OCN, ALP and OPG etc. in the cerebrospinal fluid of mice with intracranial calcification model given sterilized water was significantly increased compared to wild type mice given sterilized water, whereas the content of OPN, OCN, ALP and OPG etc. in the cerebrospinal fluid of mice with intracranial calcification model given LJY-001 drug was significantly decreased without significant difference compared to wild type mice. In addition, the invention also designs a gastric lavage sterilization water supply group and an LJY-001 extract solution supply group of wild type and intracranial calcification model mice, and 3 rounds of repeated experiments are successively completed to ensure the reliability of experimental data. Experiments were performed starting at 9 weeks of age from mice, after 4 weeks, fresh thalamus were extracted and RNA-seq analysis was performed. The results showed that 13 genes such as Myh15, aga, jun, col a2, actb, plcg2, map3k14, itgb2, dap, mroh7, cyp2j13, cxcl10, clec7a were abnormally expressed in the thalamus of the mice in the intracranial calcification model of gavage sterilized water, and that the expression levels of 13 genes such as Myh15, aga, jun, col a2, actb, plcg2, map3k14, itgb2, dap, mroh7, cyp2j13, cxcl10, clec7a were restored to the levels of the mice in the wild type group of gavage sterilized water in the thalamus of the mice in the intracranial calcification model of gavage lJY-001 extract solution.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. Use of a marker or a detection reagent thereof for preparing a diagnostic reagent or kit for (a) detecting an intracranial calcification disease, and/or (b) assessing the effect of a treatment for an intracranial calcification disease;

wherein the marker is selected from the group consisting of:

(Z18) (Z1) to (Z17).

2. A kit comprising a detection reagent for detecting a marker selected from the group consisting of:

(Z18) (Z1) to (Z17).

3. A method of detection comprising the steps of:

4. The method of claim 3, wherein the subject has a greater chance of suffering from an intracranial calcification disease than the general population (normal control population) if the expression level of the one or more genes of markers Z1-Z4, Z7-Z17 or the expression level of the one or more proteins of markers Z1-Z4, Z7-Z17 or the concentration E1 of the one or more markers Z1-Z4, Z7-Z17 is higher than the control reference value E0.

5. The method of claim 3, wherein the subject has a greater chance of suffering from an intracranial calcification disease than the general population (normal control population) if the expression level of the one or more genes of markers Z5-Z6 or the expression level of the one or more proteins of markers Z5-Z6 or the concentration E1 of the one or more markers Z5-Z6 is lower than the control reference value E0.

6. A method of diagnosing an intracranial calcification disease comprising the steps of:

7. A method of evaluating the effect of treatment of intracranial calcification disease, comprising the steps of:

8. Use of a marker for diagnosing and/or evaluating the efficacy of a treatment for an intracranial calcification disease, wherein the marker is selected from the group consisting of:

(Z18) (Z1) to (Z17).

9. A system for diagnosing an intracranial calcification disease, the system comprising:

(Z18) (Z1) to (Z17) combinations;

10. The system of claim 9, wherein the standard value is the expression level or amount or concentration of one or more of the markers Z1-Z17 in the same brain tissue sample of the normal control population, in the cerebrospinal fluid or blood, or in the same brain tissue sample of a population of brain disease patients other than intracranial calcification disease.