WO2020248771A1 - 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法 - Google Patents

基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法 Download PDF

Info

Publication number
WO2020248771A1
WO2020248771A1 PCT/CN2020/090794 CN2020090794W WO2020248771A1 WO 2020248771 A1 WO2020248771 A1 WO 2020248771A1 CN 2020090794 W CN2020090794 W CN 2020090794W WO 2020248771 A1 WO2020248771 A1 WO 2020248771A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
hsa
diabetes
medicament
sequence fragment
Prior art date
Application number
PCT/CN2020/090794
Other languages
English (en)
French (fr)
Other versions
WO2020248771A9 (zh
Inventor
汪道文
陈琛
李华萍
王峰
殷中伟
Original Assignee
华中科技大学同济医学院附属同济医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201910500477.9A external-priority patent/CN110295227B/zh
Priority claimed from CN201910500630.8A external-priority patent/CN110327363A/zh
Application filed by 华中科技大学同济医学院附属同济医院 filed Critical 华中科技大学同济医学院附属同济医院
Priority to EP20822198.6A priority Critical patent/EP3984541A4/en
Priority to US17/904,597 priority patent/US20230313182A1/en
Publication of WO2020248771A1 publication Critical patent/WO2020248771A1/zh
Publication of WO2020248771A9 publication Critical patent/WO2020248771A9/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention belongs to the field of diagnosis, prevention and treatment of molecular medicine and metabolic diseases, and specifically relates to a preparation method of an hsa-miR-320a-based diabetes early warning and/or diagnosis kit, a drug for preventing and treating diabetes and a screening method thereof and Preparation.
  • microRNA is a newly discovered type of endogenous small molecule non-coding single-stranded RNA with a length of approximately 22 nucleotides derived from the endogenous hairpin structure transcript.
  • the untranslated region (3'untranslated region, 3'UTR) specifically binds to promote the degradation of target mRNA, or inhibit the translation of target mRNA, reduce the level of encoded protein, so as to realize the regulation of gene expression after transcription.
  • miRNA is closely related to many diseases of the body, including neurodegenerative diseases, heart disease, kidney disease and tumors. Recent studies have found that miRNA plays an important role in the regulation of glucose and lipid metabolism and insulin resistance.
  • Diabetes diabetes (diabetic mellitus, DM) is a major disease that seriously threatens human life and health.
  • the current global prevalence rate is 4.4%-17.9%, which brings a heavy burden to patients and society.
  • epidemiological statistics the number of diabetes patients worldwide reached 370 million in 2011, 80% of which were in developing countries. In that year, a total of 4.6 million people died of diabetes worldwide, and the global medical expenditure on diabetes was as high as US$465 billion.
  • the prevalence of diabetes has increased significantly in the past 30 years.
  • the latest epidemiological survey and research warned that the prevalence of diabetes in adults in my country was 11.6% (about 110 million), and the prevalence of prediabetes was as high as 50.1% (about 490 million).
  • insulin resistance is a disorder of glucose and lipid metabolism caused by the interaction of a variety of genetic and environmental factors, including defects in the insulin signaling pathway, abnormal expression of insulin targets, cross-action with other hormone systems, and other metabolism Channel imbalance, etc.
  • its specific pathophysiological mechanism and molecular signal network are far from being fully understood and elucidated.
  • drug treatment strategies for diabetes are mostly based on correcting glucose and lipid metabolism disorders, including promoting insulin secretion (sulfonylureas, glinides, DPP-4 inhibitors) and drugs that lower blood sugar through other mechanisms (biguanides, TZDs, ⁇ -Glycosidase inhibitors). Even if patients receive treatments including insulin injections, only 40% of patients have good blood sugar control. Moreover, the metabolic disorders caused by diabetes cannot be effectively corrected, so it is urgent to develop new treatments. On the other hand, the etiology and risk factors of diabetes have not been fully elucidated, so it is necessary to find new risk factors and adopt more effective risk assessment methods.
  • miRNA As an important endogenous regulatory factor, miRNA is widely involved in the regulation of signal transduction pathways in animals and plants. Although the regulatory mechanism of miRNA has not yet been fully elucidated, existing research data shows that miRNA plays an important role in the regulation of glucose and lipid metabolism, not only as a new diagnostic marker for diabetes, but also in regulating the occurrence and development of diabetes insulin resistance and its complications . A series of studies have found that compared with the control population, the circulating miRNA expression profile of diabetic patients has significant differences. miRNA-375 is specifically expressed in pancreatic ⁇ -cells, and can participate in the regulation of insulin secretion through PI3K/PDK1/PKB and other pathways, while miR-375 knockout mice present a phenotype of glucose and lipid metabolism disorder.
  • let-7b in the whole body or pancreatic islets will lead to impaired glucose tolerance and decreased insulin secretion.
  • miR-503 expression is increased in endothelial cells, and it participates in the regulation of diabetic vascular function through Cdc25A and CCNE1.
  • RNA interference technology has entered clinical trials for many products in just a few years since its appearance, and achieved great success.
  • the applicant of the present invention found that miR-320a has a predictive role in atherosclerotic diseases in the preliminary research work, and researched and determined the antisense sequence anti-miR-320a of miR-320a Therapeutic role in atherosclerotic diseases.
  • the relationship between miR-320a and diabetes has not been discovered in the art, and the prior art lacks effective products or drugs in molecular diagnosis and molecular therapy of diabetes.
  • the present invention further determines the role of miR-320a in diabetic disease risk prediction based on original basic work; and the role of miR-320a in the regulation of glucose metabolism and the development of diabetes and anti- The therapeutic effect of miR-320a in diabetes.
  • the present invention provides a new medical use of endogenous non-coding small RNA, and more specifically relates to microRNA-320a (hsa-miR-320a) and its antisense nucleotide sequence hsa-anti-miR-320a. Use in risk assessment, prevention and treatment of diabetes.
  • the present invention also relates to a method for constructing and preparing a recombinant adeno-associated virus recombinant (rAAV-miR-320a/rAAV-anti-miR-320a) capable of regulating human miR-320a, and more specifically to hsa-miR-320a and anti-miR-320a
  • a recombinant adeno-associated virus recombinant rAAV-miR-320a/rAAV-anti-miR-320a
  • One aspect of the present invention provides a biomarker for diagnosing and/or early warning of diabetes, which is characterized by comprising: sequence fragments including hsa-miR-320a; the hsa-miR-320a is as SEQ ID No Shown in .1.
  • the biomarkers include: the hsa-miR-320a.
  • the biomarker is the hsa-miR-320a.
  • the second aspect of the present invention provides a kit for diagnosing and/or early warning of diabetes, which is characterized in that it comprises a reagent for quantitatively detecting the biomarker.
  • the kit includes reagents for quantitative detection of hsa-miR-320a.
  • the reagent for quantitative detection of hsa-miR-320a includes a specific primer pair of the hsa-miR-320a.
  • the specific primer pair is a commercially available primer product miRQ0000510-1-1.
  • the primer product miRQ0000510-1-1 was purchased from Guangzhou Ruibo Biological Company.
  • the reagents for quantitative detection of hsa-miR-320a also include reverse transcription reagents; and/or reverse transcription PCR reagents;
  • the reverse transcription reagents include: RT Primer Mix, 2 ⁇ TS reaction buffer, RNase free H 2 O, TS enzyme;
  • the reverse transcription PCR reagent includes: 2 ⁇ SYBR Green Mix, RNase free H 2 O.
  • the quantitative detection refers to fluorescent quantitative PCR detection
  • the kit includes: miR-320a reverse transcription primer, miR-320a real-time PCR primer F, miR-320a real-time PCR primer R, SYBR Green I, TS reaction enzyme, TS reaction buffer, DEPC ddH 2 O.
  • the third aspect of the present invention provides the application of the biomarker in the preparation of a diagnostic reagent for diabetes.
  • the fourth aspect of the present invention provides a medicine for preventing and treating diabetes, which is characterized in that the medicinal ingredient of the medicine takes hsa-miR-320a as the drug target, and is combined, and/or captured, and/ Or degrade and/or down-regulate the expression of the hsa-miR-320a to have the efficacy of preventing and treating diabetes; the hsa-miR-320a is shown in SEQ ID NO.1.
  • the medicinal ingredients of the drug include substances that can degrade and/or down-regulate the expression of the hsa-miR-320a; preferably, substances that can down-regulate the expression of the hsa-miR-320a.
  • the medicinal component of the drug comprises a sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a;
  • the antisense complement refers to a sequence fragment of 15-25 bases in length in which the full length or partial sequence of the anti-hsa-miR-320a and the hsa-miR-320a are reverse complementary.
  • the medicinal component of the drug is a recombinant plasmid that can express a sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a.
  • the active ingredient of the drug is that the active ingredient of the drug is a recombinant adeno-associated virus plasmid pAAV-D(+)-anti-miR-320a, and the expressed sequence fragments anti-hsa-miR-320a and hsa-miR -320a antisense complement.
  • the pAAV-D(+)-anti-miR-320a is inserted into the adenovirus expression vector pAAV-D(+) by inserting the primer pair shown in SEQ ID NO. 4 and SEQ ID NO. ).
  • the medicine also includes pharmaceutically acceptable excipients, and/or reagents for buffering, culturing, and/or expanding the recombinant adeno-associated virus plasmid pAAV-D(+)-anti-miR-320a.
  • the fifth aspect of the present invention provides a method for screening drugs for preventing and treating diabetes, which is characterized by detecting whether the candidate substance can bind, capture, degrade, and/or down-regulate the expression of hsa-miR-320a;
  • a substance capable of reducing the expression level of hsa-miR-320a is screened out.
  • the sixth aspect of the present invention provides a method for preparing a medicine for preventing and treating diabetes, which is characterized in that it comprises: degrading, and/or down-regulating the expression of the hsa-miR-320a substance;
  • sequence fragment anti-hsa-miR-320a that is complementary to the antisense of hsa-miR-320a;
  • the recombinant adeno-associated virus plasmid expressing the antisense complementary sequence fragment anti-hsa-miR-320a with hsa-miR-320a is used as the active ingredient of anti-heart failure drugs.
  • the preparation method also includes: inserting a primer pair capable of expressing a sequence fragment anti-hsa-miR-320a complementary to hsa-miR-320a antisense into an expression vector, thereby preparing a stable expression and antisense hsa-miR-320a Recombinant plasmid with complementary sequence fragment anti-hsa-miR-320a.
  • the primer pair that can express the sequence fragment anti-hsa-miR-320a that is complementary to the antisense hsa-miR-320a is shown in SEQ ID NO. 4 and SEQ ID NO. 5; the expression vector is an adeno-associated virus expression Vector pAAV-D(+).
  • the inventors have discovered through a large number of experiments that the expression of miR-320a in the peripheral blood of diabetic patients is significantly increased. Furthermore, the present inventors designed and synthesized the sequences expressing hsa-miR-320a and the antisense complementary anti-hsa-miR-320a respectively based on the base sequence of hsa-miR-320a, and successfully inserted them into the eukaryotic expression vector pAAV- D(+) constitutes the recombinant plasmids pAAV-D(+)-miR-320a and pAAV-D(+)-anti-miR-320a.
  • the real-time PCR method was used to determine the titer.
  • the two recombinant adeno-associated viruses (rAAV-miR-320a and rAAV-anti-miR-320a) of the same serotype prepared by packaging were injected into db/db diabetic mice through the tail vein, and it was found that the db/db was small.
  • the blood glucose of mice is significantly regulated, and the expression of anti-hsa-miR-320a mediated by recombinant adeno-associated virus can significantly improve the elevated blood glucose of db/db mice and improve cardiac function.
  • the hsa-miR-320a further aggravated the increase in blood sugar in db/db mice and worsened the heart function. These results further support the therapeutic effect of anti-hsa-miR-320a on diabetes and its complications.
  • the first objective of the present invention is to provide the expression of miR-320a in the peripheral blood of diabetic patients.
  • the results show that the expression of miR-320a in the peripheral blood of diabetic patients is significantly increased, which means that miR-320a can be used as a kind of Biomarkers for early diagnosis and prediction of diabetes.
  • the second objective of the present invention is based on the first objective.
  • the inventors confirmed through experiments that the antisense nucleotide anti-miR-320a of miR-320a has a protective effect on diabetes, and that anti-miR-320a can become A medicine to treat diabetes and its complications.
  • the hsa-miR-320a discovered in the present invention can be used as an effective biomarker for diagnosing diabetes risk and diagnosing diabetes.
  • a kit based on hsa-miR-320a is a molecule used in the field of diabetes diagnosis. High-efficiency and accurate products of biological means.
  • the hsa-miR-320a-based diabetes prevention and treatment drug of the present invention provides an effective and safe drug for the treatment of diabetes in the field of molecular biology, and provides a new choice for the treatment of diabetes for patients and medical personnel.
  • Figure 1 shows the correlation between the blood glucose level of diabetic patients and the expression level of hsa-miR-320a in the peripheral blood;
  • A Real-time PCR method detects the expression of hsa-miR-320a in the peripheral blood of diabetic patients; U6 is used as the internal reference, so The comparison method is 2 - ⁇ CT method;
  • B Diabetes patients' fasting blood glucose value;
  • C Peripheral blood hsa-miR-320a expression and fasting blood glucose correlation analysis;
  • DM diabetic patients;
  • Non-DM normal control population.
  • Figure 2 shows the plasmid structure of pAAV-D(+)-miR-320a and pAAV-D(+)-anti-miR-320a.
  • Figure 3 is a fluorescence microscope observation of GFP (40X, green shows cells successfully transfected) after the purified virus is transfected into cells.
  • the transfection efficiency can reach more than 90%.
  • Figure 4 shows the effects of different microRNA treatments on fasting blood glucose in db/db mice.
  • Fasting blood glucose testing shows that rAAV-miR-320a and rAAV-anti-miR-320a can significantly regulate glucose metabolism in db/db mice (the former promotes blood glucose rise High, the latter makes blood sugar lower).
  • Figure 5 shows the effects of different microRNA treatments on the cardiac ejection fraction of db/db mice.
  • Cardiac ultrasound testing shows that rAAV-anti-miR-320a can significantly improve the cardiac ejection fraction of db/db mice; while rAAV-miR-320a It significantly reduces the cardiac ejection fraction of db/db mice.
  • mice and db/db diabetic mice were from Nanjing Model Animal Center; 293T cells were purchased from the Typical Species Collection of Wuhan University.
  • biomarker for diagnosis and/or early warning of diabetes.
  • the biomarkers have the following common features: the biomarkers include: sequence fragments including hsa-miR-320a; the hsa-miR-320a is as SEQ ID No Shown in .1.
  • the biomarker includes: the hsa-miR-320a.
  • the biomarker is the hsa-miR-320a.
  • the second group of embodiments, the diabetes diagnosis/early warning/risk assessment kit of the present invention is the diabetes diagnosis/early warning/risk assessment kit of the present invention.
  • kits for diagnosing and/or early warning of diabetes All the examples in this group have the following common feature: the kit includes reagents for quantitatively detecting any of the biomarkers described in the first group of examples.
  • the kit includes reagents for quantitative detection of hsa-miR-320a.
  • the reagent for quantitative detection of hsa-miR-320a includes a specific primer pair of hsa-miR-320a.
  • the specific primer pair is a commercially available primer product miRQ0000510-1-1.
  • the reagent for quantitative detection of hsa-miR-320a further includes a reverse transcription reagent; and/or a reverse transcription PCR reagent;
  • the reverse transcription reagents include: RT Primer Mix, 2 ⁇ TS reaction buffer, RNase free H 2 O, TS enzyme;
  • the reverse transcription PCR reagent includes: 2 ⁇ SYBR Green Mix, RNase free H 2 O.
  • the quantitative detection refers to fluorescent quantitative PCR detection
  • the kit includes: miR-320a reverse transcription primer, miR-320a real-time PCR primer F, miR-320a real-time PCR primer R, SYBR Green I, TS reaction enzyme, TS reaction buffer, DEPC ddH 2 O.
  • This group of examples provides the application of any one of the biomarkers described in the first group of examples in the preparation of diagnostic reagents for diabetes.
  • the fourth group of examples, the diabetes prevention and treatment drugs of the present invention is the diabetes prevention and treatment drugs of the present invention.
  • This group of embodiments provides a medicine for preventing and treating diabetes. All the examples in this group have the following common feature: the medicinal ingredients of the drug are hsa-miR-320a as the drug target, and are expressed by binding, and/or capturing, and/or degradation, and/or down-regulation.
  • the hsa-miR-320a has the efficacy of preventing and treating diabetes; the hsa-miR-320a is shown in SEQ ID NO.1.
  • the medicinal ingredient of the drug includes a substance that can degrade and/or down-regulate the expression of the hsa-miR-320a; preferably, a substance that can down-regulate the expression of the hsa-miR-320a.
  • the medicinal ingredient of the drug comprises a sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a;
  • the antisense complement refers to the length of the anti-hsa-miR-320a and the full-length or partial sequence of the hsa-miR-320a is 15-25 bases in reverse complementarity. Sequence fragments.
  • the medicinal ingredient of the drug is a recombinant plasmid that can express a sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a.
  • the medicinal ingredient of the drug is that the medicinal ingredient of the drug is a recombinant adeno-associated virus plasmid pAAV-D(+)-anti-miR-320a, and the expressed sequence fragment anti-hsa -miR-320a is antisense complementary to hsa-miR-320a.
  • the pAAV-D(+)-anti-miR-320a is inserted into the adenovirus expression vector pAAV by inserting a primer pair shown in SEQ ID NO. 4 and SEQ ID NO. 5 -D(+) built in.
  • the drug further includes pharmaceutically acceptable excipients, and/or used for buffering, culturing, and/or expanding the recombinant adeno-associated virus plasmid pAAV-D(+)-anti- miR-320a reagent.
  • This group of examples provides a method for screening drugs for preventing and treating diabetes.
  • the common features of all the embodiments in this group are as follows: the screening method has the following features: detecting whether the candidate substance can bind, capture, degrade, and/or down-regulate the expression of hsa-miR-320a;
  • substances that can reduce the expression level of hsa-miR-320a are screened out.
  • the sixth group of examples the preparation method of the diabetes medicine of the present invention
  • the preparation method includes: degrading and/or down-regulating the substance that expresses the hsa-miR-320a; antisense to hsa-miR-320a A complementary sequence fragment anti-hsa-miR-320a; a recombinant plasmid expressing a sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a; and/or, an expression complementary to hsa-miR-320a antisense
  • the recombinant adeno-associated virus plasmid of the sequence fragment anti-hsa-miR-320a is used as the active ingredient of anti-heart failure drugs.
  • the preparation method further includes: inserting a primer pair capable of expressing a sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a into an expression vector, thereby preparing a stable expression and hsa-miR-320a antisense complementary sequence fragment anti-hsa-miR-320a recombinant plasmid.
  • the primer pair that can express the sequence fragment anti-hsa-miR-320a that is antisense complementary to hsa-miR-320a is shown in SEQ ID NO. 4 and SEQ ID NO. 5;
  • the expression vector is an adeno-associated virus expression vector pAAV-D(+).
  • RNA quality 1. Collect peripheral blood of 200 diabetic patients and 200 normal controls. After sampling, centrifuge at 3,500 rpm for 6 min at room temperature, and take the upper layer of plasma and store it in a refrigerator at -80°C. Add 1ml TRIZOL LS (Invitrogen) to every 0.25ml peripheral blood plasma, extract RNA, and process the sample with RNasey Mini Kit (Qiagen). use ND-1000 detects RNA quality.
  • TRIZOL LS Invitrogen
  • the detection kit includes a set of specific reverse transcription primers for amplifying miR-320a and a set of real-time PCR primer pairs; specific reverse transcription primers and real-time for amplifying control RNA (U6)
  • a set of PCR primer pairs and related reagents, the components and contents are as follows (100 times), stored at -20 degrees:
  • the currently commonly used methods for diagnosing diabetes in the field are fasting blood glucose testing and OGTT experiments. 100 blood samples of patients with diabetes previously diagnosed by this method are collected, and then the biomarkers provided in any one of the first group embodiments of the present invention are used. Diabetes early warning/diagnostic kit provided by any one of the second group of embodiments and/or the second group of embodiments performs molecular detection, and the diagnostic criteria for the detection result is: the expression level of miR-320a is higher than? ? (There is currently no large-scale population data) The diagnosis is diabetes; the final result is that there is a total? ? The expression level of miR-320a in the blood samples of patients with diabetes is higher than? ? What is the accuracy of the diabetes early warning/diagnostic kit of the present invention? ? % (There is currently no large-scale population data)
  • Diabetes early warning/diagnostic kit provided by any one of the second group of embodiments and/or the second group of embodiments performs molecular detection, and the diagnostic criteria of the detection result is: the expression level of miR-320a is higher than 50 nmol/L to diagnose diabetes; The final result found that the expression level of miR-320a in blood samples of 83 diabetic patients was higher than 50 nmol/L, indicating that the accuracy of the diabetes early warning/diagnostic kit of the present invention was 83%.
  • the constructed plasmids were identified by double enzyme digestion and sequencing, and the eukaryotic expression plasmids pAAV-D(+)-miR-320a and pAAV-D(+)-anti-miR-320a were obtained.
  • the structure is shown in Figure 3. .
  • 293T cells human embryonic kidney epithelial cells grow to 90%, 1-2hr before calcium and phosphorus transfection, change each petri dish to 12-15ml of fresh medium (containing serum), and add calcium chloride ( CaCl 2 ), then add the plasmid to form a Ca-DNA mixture, mix well, slowly add 2XHEBS BUFFER dropwise to the Ca-DNA mixture to form a Ca-DNA-P mixture, add 2XHEBS while shaking the centrifuge tube, fully Mix well to form calcium and phosphorus particles. After 8-12hr, change to 18-20ml serum-free medium. After 72hr, aspirate the medium and wash with PBS 3 times. Add 1ml Tris+NaCl (pH 8.5) to each petri dish, scrape the cells with a spatula, Collect in a clean centrifuge tube and freeze at -80°C.
  • Tris+NaCl pH 8.5
  • the cells frozen at -80°C were taken out, thawed and thawed at 37°C, repeated freezing and thawing 4 times, centrifuged at 8,000g for 15min, put the supernatant into a clean centrifuge tube, and discard the cell pellet.
  • the instrument used is an ultrasound instrument equipped with a 30MHz high frequency probe. After anesthetizing the mouse with isoflurane, the mouse was placed supine on the detection platform, and two-dimensional images of the left ventricle were collected along the horizontal short-axis and long-axis views of the left ventricular papillary muscles of the left ventricle next to the sternum, and at the same time under the guidance of the two-dimensional images. Acquire M-mode ultrasound images of more than 5 consecutive cardiac cycles, and use software analysis results based on the collected images to obtain cardiac hemodynamic indicators for cardiac ultrasound detection.
  • Heart rate (HR) , Left Ventricular Internal Dimension (Diastole, LVIDd), Left Ventricular Internal Dimension (systole, LVIDs), Left Ventricular Posterior Wall, diastole, LVPWd) , Left Ventricular Posterior Wall (systole, LVPWs), Interventricular Septal Thickness (Diastole, IVSd), Interventricular Septal Thickness (systole, IVSs), Ejection Blood fraction (Ejection Fraction, EF) and Fractional Shortening (FS), etc.
  • the present invention uses the diabetes prevention and treatment drugs of the present invention to treat hundreds of diabetic mice.
  • the symptoms of each diabetic mouse before treatment include: high blood sugar, diabetic complications, for example, impaired cardiac contractility, etc. .
  • the diabetes treatment is specifically: using the diabetes prevention and treatment drugs provided in any embodiment of the fourth group of the present invention, and/or, the diabetes prevention and treatment drugs screened by the screening method provided in any embodiment of the fifth group, and/or,
  • the diabetes prevention and treatment drugs prepared by the preparation method provided in any embodiment of the sixth group were injected once through the tail vein at the age of 12 weeks in db/db mice, and the virus titer was 1 ⁇ 10 11 PFU/mouse.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Diabetes (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Emergency Medicine (AREA)
  • Endocrinology (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明"基于hsa-miR-320a的防治糖尿病的药物及其筛选方法和制备方法"，属于分子医药及代谢性疾病的诊断、预防和治疗领域。所述药物的药效成分以hsa-miR-320a为药物靶点，且通过结合、和/或捕获、和/或降解、和/或下调表达所述hsa-miR-320a起到所述防治糖尿病的药效；所述hsa-miR-320a如SEQ ID NO.1所示。同时本发明还提供了所述药物的筛选方法和制备方法。采用本发明的药物可显著降低糖尿病患者的血糖浓度，有效缓解受试动物的糖尿病并发症症状，使受试动物长时间内保持健康稳定的血糖。

Description

基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法 技术领域
本发明属于分子医药及代谢性疾病的诊断、预防和治疗领域，具体涉及一种基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法。
背景技术
microRNA(miRNA,miR)是一类新近发现的来源于内源性发夹结构转录本的长度大约为22个核苷酸的内源性小分子非编码单链RNA，通过与靶mRNA的3’非翻译区(3’untranslated region,3’UTR)特异性结合，促进靶mRNA降解，或者抑制靶mRNA翻译，降低编码蛋白质水平，从而实现对基因转录后的表达调控。miRNA与机体的多种疾病联系紧密，包括神经退行性疾病、心脏病、肾病以及肿瘤等。近年来的研究发现，miRNA在糖脂代谢、胰岛素抵抗调控中均有着重要作用。
糖尿病(diabetic mellitus,DM)是严重威胁人类生命健康的重大疾病，目前其全球患病率为4.4％-17.9％，给患者和社会带来了沉重的负担。根据流行病学统计，2011年全球糖尿病患者人数已达3.7亿，其中80％在发展中国家，当年全球共有460万人死于糖尿病，当年全球糖尿病的医疗花费高达4650亿美元。在我国，近30年来糖尿病患病率显著增加。2013年，最新的流行病学调查研究警示，我国成年人糖尿病患病率为11.6％(约1.1亿)，糖尿病前期的患病率高达50.1％(约4.9亿)。
90％以上的糖尿病患者为2型糖尿病，是由胰岛素抵抗和β-细胞功能衰竭等引起的严重糖脂代谢紊乱的状态，并最终导致心脑血管系统和肾脏等多种严重并发症甚至死亡。已有的研究表明，胰岛素抵抗是由多种遗传及环境因素相互作用所导致的糖脂代谢紊乱，包括胰岛素信号传导通路缺陷、胰岛素作用靶点表达异常、与其他激素系统的交叉作用及其他代谢通路失衡等。然而其具体病理生理机制和分子信号网络还远未被完全认识和阐明。目前，糖尿病的药物治疗策略多基于纠正糖脂代谢紊乱，包括促进胰岛素分泌(磺脲类、格列奈类、DPP-4抑制剂)和通过其他机制降糖的药物(双胍类、TZDs、α-糖苷酶抑制剂)。即使患者接受包括注射胰岛素在内的治疗方案，仍只有40％的患者血糖控制良好，而且，糖尿病所致的代谢障碍不能得到有效纠正，因此亟待研发新的治疗措施。另一方面，糖尿病的病因和危险因素并未完全阐明，因此需要寻找新的危险因素和采用更加有效的风险评估方法。
作为重要的内源性调控因子，miRNA广泛参与动植物体内的信号传导途径调控。尽管miRNA的调控机制尚未完全阐明，已有的研究资料表明miRNA在糖脂代谢调控中起到重要作用，不仅可以作为糖尿病的新型诊断标志物，还参与调控糖尿病胰岛素抵抗及其并发症的发生发展。一系列研究发现，与对照人群相比，糖尿病患者外周血循环miRNA表达谱具有显著差异。miRNA-375在胰岛β细胞中特异性表达，并且能通过PI3K/PDK1/PKB等通路参与调控胰岛素的分泌，而miR-375敲除小鼠呈现出糖脂代谢紊乱的表型。全身或胰岛特异性高表达let-7b将导致糖耐量受损及胰岛素分泌减少。高糖状态下，miR-503在内皮细胞中表达增高，并通过Cdc25A和CCNE1介导参与糖尿病血管功能的调控。
RNA干扰技术作为具有突破性临床应用前景的生物技术，从出现开始的短短数年间就有多个产品进入临床试验，获得了巨大的成功。如发明专利201210069179.7中所披露，本发明的申请人在前期研究工作中发现miR-320a在动脉粥样硬化性疾病中具有预测作用，并研究确定了miR-320a的反义序列anti-miR-320a在动脉粥样硬化性疾病中的治疗作用。但本领域尚未发现miR-320a与糖尿病的关系，现有技术在糖尿病的分子诊断和分子治疗方面也缺乏行之有效的产品或药物。
发明内容
本发明正是基于上述本领域的空白和需求，在原创基础工作上进一步确定了miR-320a在糖尿病疾病风险预测中的作用；还有miR-320a参与糖代谢调控及糖尿病发展的作用及anti-miR-320a在糖尿病中的治疗作用。本发明提供了一种内源性的非编码小RNA的新医药用途，更具体地说涉及microRNA-320a(hsa-miR-320a)及其反义核苷酸序列hsa-anti-miR-320a在糖尿病的风险评估、预防和治疗中的用途。本发明还涉及能调控人类miR-320a的重组腺相关病毒重组体(rAAV-miR-320a/rAAV-anti-miR-320a)的构建和制备方法，更具体地说涉及hsa-miR-320a及反义序列anti-hsa-miR-320a的克隆及分别包含hsa-miR-320a和anti-hsa-miR-320a的重组腺相关病毒重组体的包装制备方法，和所述的重组腺相关病毒重组体的药学用途。
本发明的技术方案如下：
本发明一方面提供一种用于诊断和/或早期预警糖尿病的生物标志物，其特征在于，包括：含有hsa-miR-320a在内的序列片段；所述hsa-miR-320a如SEQ ID No.1所示。
所述生物标志物包括：所述hsa-miR-320a。
所述生物标志物为所述hsa-miR-320a。
本发明第二个方面提供一种用于诊断和/或早期预警糖尿病的试剂盒，其特征在于，包括 用于定量检测所述的生物标志物的试剂。
所述试剂盒包括用于定量检测hsa-miR-320a的试剂。
所述用于定量检测hsa-miR-320a的试剂包括所述hsa-miR-320a的特异性引物对。
所述特异性引物对为商购获得的引物产品miRQ0000510-1-1。该引物产品miRQ0000510-1-1购自广州锐博生物公司。
所述用于定量检测hsa-miR-320a的试剂还包括逆转录试剂；和/或逆转录PCR试剂；
优选地，所述逆转录试剂包括：RT Primer Mix、2×TS reaction buffer、RNase free H 2O、TS enzyme；
进一步优选地，所述逆转录PCR试剂包括：2×SYBR Green Mix、RNase free H 2O。
所述定量检测指荧光定量PCR检测；
最优选地，所述试剂盒包括：miR-320a逆转录引物、miR-320a real-time PCR引物F、miR-320a real-time PCR引物R、SYBR Green I、TS reaction enzyme、TS reaction buffer、DEPC ddH 2O。
本发明的第三个方面提供所述的生物标志物在制备糖尿病诊断试剂方面的应用。
本发明的第四个方面提供一种用于防治糖尿病的药物，其特征在于，所述药物的药效成分以hsa-miR-320a为药物靶点，且通过结合、和/或捕获、和/或降解、和/或下调表达所述hsa-miR-320a起到所述防治糖尿病的药效；所述hsa-miR-320a如SEQ ID NO.1所示。
所述药物的药效成分包括可降解、和/或下调表达所述hsa-miR-320a的物质；优选为可下调表达所述hsa-miR-320a的物质。
所述药物的药效成分包含与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a；
优选地，所述反义互补指所述anti-hsa-miR-320a与所述hsa-miR-320a的全长或部分序列呈反向互补的长度为15-25个碱基的序列片段。
所述药物的药效成分为可表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒。
所述药物的药效成分为所述药物的药效成分为重组腺相关病毒质粒pAAV-D(+)-anti-miR-320a，其表达的序列片段anti-hsa-miR-320a与hsa-miR-320a反义互补。
更具体地，所述pAAV-D(+)-anti-miR-320a是通过将序列如SEQ ID NO.4和SEQ ID NO.5所示的引物对插入至腺病毒表达载体pAAV-D(+)中构建而得。
所述药物还包括药学上可接受的辅料，和/或，用于缓冲、培养、和/或扩繁所述重组腺相关病毒质粒pAAV-D(+)-anti-miR-320a的试剂。
本发明的第五个方面提供一种防治糖尿病的药物的筛选方法，其特征在于，检测待选物 质是否能结合、捕获、降解、和/或下调表达hsa-miR-320a；
优选地，筛选出能使hsa-miR-320a表达水平降低的物质。
本发明的第六个方面提供一种防治糖尿病的药物的制备方法，其特征在于，包括：将降解、和/或下调表达所述hsa-miR-320a的物质；
与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a；
表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒；和/或，
表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组腺相关病毒质粒作为抗心衰药物的活性成分。
所述制备方法还包括：将可表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的引物对插入表达载体，从而制备得到可稳定表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒。
可表达所述与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的引物对如SEQ ID NO.4和SEQ ID NO.5所示；所述表达载体为腺相关病毒表达载体pAAV-D(+)。
本发明人通过大量的实验发现，糖尿病患者外周血中miR-320a的表达显著增高。进一步，本发明人根据hsa-miR-320a碱基序列，分别设计并合成了表达hsa-miR-320a和反义互补anti-hsa-miR-320a的序列，并成功分别插入真核表达载体pAAV-D(+)中构成重组质粒pAAV-D(+)-miR-320a和pAAV-D(+)-anti-miR-320a。之后，将以下三种质粒：1)pXX2、pXX8或pXX9质粒、2)phelper质粒、3)pAAV-D(+)-miR-320a或pAAV-D(+)-anti-miR-320a质粒，用钙磷共转染法分别转入293细胞包装制备能分别表达hsa-miR-320a和反义互补anti-hsa-miR-320a的三种血清型重组腺相关病毒(rAAV2、rAAV8和rAAV9)，经纯化后，real-time PCR法测定滴度。下一步将包装制备的同一血清型的两种重组腺相关病毒(rAAV-miR-320a和rAAV-anti-miR-320a)分别经尾静脉注射至db/db糖尿病小鼠中，发现db/db小鼠的血糖受到明显调控，重组腺相关病毒介导的anti-hsa-miR-320a表达能明显改善db/db小鼠的升高的血糖，并且改善心脏功能。而hsa-miR-320a则进一步加重db/db小鼠的血糖增高，并恶化心功能。这些结果进一步支持了anti-hsa-miR-320a对糖尿病及其并发症的治疗作用。
具体的，本发明的第一个目的是提供糖尿病患者的外周血miR-320a表达情况，结果显示糖尿病患者外周血中的miR-320a的表达显著升高，这意味着miR-320a可作为一种早期诊断和预测糖尿病的生物标志物。
本发明的第二个目的是在第一个目的的基础上，本发明人通过实验确证miR-320a的反义核苷酸anti-miR-320a对糖尿病具有保护作用，anti-miR-320a可成为一种治疗糖尿病及其 并发症的药物。
综上，本发明发现的hsa-miR-320a可以做为一种有效的评估糖尿病风险、用于确诊糖尿病的生物标志物，基于hsa-miR-320a的试剂盒是糖尿病诊断领域的一种利用分子生物学手段的高效、准确的产品。而本发明所述基于hsa-miR-320a的糖尿病防治药物为分子生物学领域治疗糖尿病提供了一种行之有效的、安全的药物，为患者和医疗人员提供了一种糖尿病治疗的新选择。
附图说明
从下面给出的说明结合附图，本发明的上面和其他目的和特征将变得明了。其中：
图1显示了糖尿病患者血糖水平与外周血hsa-miR-320a表达水平的相关性；A.Real-time PCR法检测糖尿病患者外周血中hsa-miR-320a的表达情况；以U6为内参，所采用比较方法为2 -△△CT法；B.糖尿病患者空腹血糖值；C.外周血hsa-miR-320a表达与空腹血糖的关联性分析；D.外周血hsa-miR-320a对糖尿病诊断的ROC曲线；其中，**P<0.01与对照组相比(n＝200)；DM：糖尿病患者；Non-DM：正常对照人群。
图2是显示pAAV-D(+)-miR-320a和pAAV-D(+)-anti-miR-320a的质粒构成。
图3是纯化后的病毒转染细胞后荧光显微镜观察GFP(40X，绿色显示的是转染成功的细胞)，其转染效率可达90％以上。
图4是不同microRNA治疗对db/db小鼠空腹血糖的影响，空腹血糖检测显示rAAV-miR-320a和rAAV-anti-miR-320a可明显调控db/db小鼠的糖代谢(前者促进血糖升高，后者使得血糖降低)。
图5是不同microRNA治疗对db/db小鼠心脏射血分数的影响，心脏超声检测显示rAAV-anti-miR-320a可明显改善db/db小鼠的心脏射血分数；而rAAV-miR-320a则明显降低db/db小鼠的心脏射血分数。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
生物材料的来源
C57对照小鼠、db/db糖尿病小鼠来自南京模式动物中心；293T细胞购自武汉大学典型物种保藏中心。
下述任一实施例/实验例中所使用的酶、载体、细胞及各类分子生物学实验常规使用的试剂、试剂盒、耗材均可商购获得。
第1组实施例、本发明糖尿病诊断的生物标志物
本组实施例提供一种用于诊断和/或早期预警糖尿病的生物标志物。在本组所有的实施例中，所述生物标志物都具备如下共同特征：所述生物标志物包括：含有hsa-miR-320a在内的序列片段；所述hsa-miR-320a如SEQ ID No.1所示。
在具体的实施例中，所述生物标志物包括：所述hsa-miR-320a。
更具体的实施例中，所述生物标志物为所述hsa-miR-320a。
第2组实施例、本发明的糖尿病诊断/早期预警/风险评估试剂盒
本组实施例提供一种用于诊断和/或早期预警糖尿病的试剂盒。本组所有的实施例都具有如下共同特征：所述试剂盒包括用于定量检测第1组实施例任一所述的生物标志物的试剂。
进一步的实施例中，所述试剂盒包括用于定量检测hsa-miR-320a的试剂。
在具体的实施例中，所述用于定量检测hsa-miR-320a的试剂包括所述hsa-miR-320a的特异性引物对。
更具体的实施例中，所述特异性引物对为商购获得的引物产品miRQ0000510-1-1。
在优选的实施例中，所述用于定量检测hsa-miR-320a的试剂还包括逆转录试剂；和/或逆转录PCR试剂；
优选地，所述逆转录试剂包括：RT Primer Mix、2×TS reaction buffer、RNase free H 2O、TS enzyme；
进一步优选地，所述逆转录PCR试剂包括：2×SYBR Green Mix、RNase free H 2O。
一些实施例中，所述定量检测指荧光定量PCR检测；
在另一些优选实施例中，所述试剂盒包括：miR-320a逆转录引物、miR-320a real-time PCR引物F、miR-320a real-time PCR引物R、SYBR Green I、TS reaction enzyme、TS reaction buffer、DEPC ddH 2O。
第3组实施例、本发明的生物标志物的应用
本组实施例提供第1组实施例任一所述的生物标志物在制备糖尿病诊断试剂方面的应用。
第4组实施例、本发明的糖尿病防治药物
本组实施例提供一种用于防治糖尿病的药物。本组所有的实施例都具备如下共同特征：所述药物的药效成分以hsa-miR-320a为药物靶点，且通过结合、和/或捕获、和/或降解、和/或下调表达所述hsa-miR-320a起到所述防治糖尿病的药效；所述hsa-miR-320a如SEQ ID NO.1所示。
在一些实施例中，所述药物的药效成分包括可降解、和/或下调表达所述hsa-miR-320a的物质；优选为可下调表达所述hsa-miR-320a的物质。
在本组另一些实施例中，所述药物的药效成分包含与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a；
在优选的实施例中，所述反义互补指所述anti-hsa-miR-320a与所述hsa-miR-320a的全长或部分序列呈反向互补的长度为15-25个碱基的序列片段。
在另一些优选的实施例中，所述药物的药效成分为可表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒。
在一些具体的实施例中，所述药物的药效成分为所述药物的药效成分为重组腺相关病毒质粒pAAV-D(+)-anti-miR-320a，其表达的序列片段anti-hsa-miR-320a与hsa-miR-320a反义互补。
在更具体的实施例中，所述pAAV-D(+)-anti-miR-320a是通过将序列如SEQ ID NO.4和SEQ ID NO.5所示的引物对插入至腺病毒表达载体pAAV-D(+)中构建而得。
在进一步的实施例中，所述药物还包括药学上可接受的辅料，和/或，用于缓冲、培养、和/或扩繁所述重组腺相关病毒质粒pAAV-D(+)-anti-miR-320a的试剂。
第5组实施例、本发明的糖尿病防治药物的筛选方法
本组实施例提供一种防治糖尿病的药物的筛选方法。本组所有实施例的共同特征如下：所述筛选方法具有如下特点：检测待选物质是否能结合、捕获、降解、和/或下调表达hsa-miR-320a；
在本组优选的实施例中，筛选出能使hsa-miR-320a表达水平降低的物质。
第6组实施例、本发明的糖尿病药物的制备方法
本组实施例提供一种防治糖尿病的药物的制备方法。本组所有的实施例中，所述制备方法都具备如下共同特征：所述制备方法包括：将降解、和/或下调表达所述hsa-miR-320a的物质；与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a；表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒；和/或，表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组腺相关病毒质粒作为抗心衰药物的活性成分。
在进一步的实施例中，所述制备方法还包括：将可表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的引物对插入表达载体，从而制备得到可稳定表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒。
在本组具体的实施例中，可表达所述与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的引物对如SEQ ID NO.4和SEQ ID NO.5所示；所述表达载体为腺相关病毒表达载体pAAV-D(+)。
实验例1.糖尿病患者外周血miRNA检测
1.收集200例糖尿病患者和200例正常对照人群外周血。取材后，室温3,500rpm离心6min，取上层血浆，保存于-80℃冰箱。每0.25ml外周血血浆中加入1ml TRIZOL LS(Invitrogen公司)，提取RNA，RNasey Mini Kit(Qiagen)处理样品。使用
Figure PCTCN2020090794-appb-000001
ND-1000检测RNA质量。
2.采用广州锐博公司miRNA检测试剂盒对hsa-miR-320a的表达进行real-time PCR检测：
miRNAs逆转录：
逆转录引物(RT Primer Mix)配置：
miRNA RT Primer 1μl
U6RT Primer 1μl
RNase free H 2O 78μl
逆转录反应体系：
RNA template 2μg
RT Primer Mix 4μl
RNase free H 2O up to 19μl
以上体系混匀后，瞬时离心，70℃孵育10min后，冰育2min，再加入以下试剂：
2×TS reaction buffer 25μl
TS enzyme 2.5μl
RNase free H 2O 3.5μl
逆转录反应程序：
42℃ 60min，70℃ 10min；停止后4℃备用，产物保存于-20℃。
miRNAs real-time PCR：
反应体系：2×SYBR Green Mix 9μl
RT product 2μl
miRNA Forward Primer 2μl(购自广州锐博生物科技有限公司)
miRNA Reverse Primer 2μl(购自广州锐博生物科技有限公司)
RNase-free H 2O 5μl
反应程序：
95℃ 30sec--(95℃ 10sec--60℃ 20sec--70℃ 1sec)×40cycles--Melting Curve
结果显示：糖尿病患者外周血中hsa-miR-320a的表达水平增高(图1A)，空腹血糖也明显增高(图1B)，且hsa-miR-320a的表达水平与空腹血糖水平呈正相关性(图1C)。hsa-miR-320a可用于诊断糖尿病(图1D)。
aaaagcuggguugagagggcga hsa-miR-320a的序列(SEQ ID NO.1)。
实验例2.制备检测糖尿病风险评估的试剂盒
试剂盒成分：检测试剂盒包括用于扩增miR-320a的特异性逆转录引物及real-time PCR引物对一套；用于扩增对照RNA(U6)的特异性逆转录引物及real-time PCR引物对一套及相关试剂，成分和含量如下(100次)，保存于-20度：
Figure PCTCN2020090794-appb-000002
以上试剂均由各来源公司提供，已经商品化。具体检测方法及相关反应参数参照实施例1。
实验例3、本发明糖尿病早期预警/诊断试剂盒的准确性验证：
本领域目前常用的糖尿病诊断方法是空腹血糖检测及OGTT实验，采集100例事先经该方法确诊已是糖尿病患者的血液样品，再利用本发明第1组实施例任一实施例所提供的生物标志物和/或第2组实施例任一实施例所提供的糖尿病早期预警/诊断试剂盒进行分子检测，检测结果的诊断标准为：miR-320a的表达量高于？？(目前尚无大规模人群数据)则确诊为糖尿病；最终结果发现，共有？？例糖尿病患者的血液样品中的miR-320a的表达量高于？？，说明本发明的糖尿病早期预警/诊断试剂盒的准确性为？？％(目前尚无大规模人群数据)。
[援引加入(细则20.6) 03.07.2020]　
本领域目前常用的糖尿病诊断方法是空腹血糖检测及OGTT实验，采集100例事先经该方法确诊己是糖尿病患者的血液样品，再利用本发明第1组实施例任一实施例所提供的生物标志物和/或第2组实施例任一实施例所提供的糖尿病早期预警/诊断试剂盒进行分子检测，检测结果的诊断标准为：miR-320a的表达量高于50nmol/L则确诊为糖尿病；最终结果发现，共有83例糖尿病患者的血液样品中的miR-320a的表达量高于50nmol/L,说明本发明的糖尿病早期预警/诊断试剂盒的准确性为83%。
实验例4.重组腺相关病毒的构建
1.插入片段合成
依据hsa-miR-320a的碱基序列(图2)，分别设计并合成表达hsa-miR-320a及反向互补anti-hsa-miR-320a的两条反向互补链，并用TE溶解。
表达hsa-miR-320a的引物对：
hsa-miR-320a-Sense：
5`agctttcgccctctcaacccagcttttttcaagagaaaaagctgggttgagagggcgaccgc 3`(SEQ ID NO.2)
hsa-miR-320a-Antisense：
3`aagcgggagagttgggtcgaaaaaagttctct ttttcgacccaactctcccgctggcgccgg 5`(SEQ ID NO.3)
表达anti-hsa-miR-320a的引物对：
anti-hsa-miR-320a-Sense：
5`agctt aaaagctgggttgagagggcgattcaagagatcgccctctcaacccagcttttccgc 3`(SEQ ID NO.4)
anti-hsa-miR-320a-Antisense：
3`attttcgacccaactctcccgctaagttctctagcgggagagttgggtcgaaaaggcgccgg 5`(SEQ ID NO.5)
2.按照说明书中体系及温度进行反应：
Nuclease-Free Water 36μl
Annealing Buffer for DNA Oligos(5X) 10μl
DNA oligo A(50μM) 2μl
DNA oligo B(50μM) 2μl
90℃ 3min，37℃ 1hr，4℃保存。
3.载体酶切
采用BamH I和Not I对真核表达载体pAAV-D(+)在37℃下双酶切2hr，体系如下：
10×K Buffer 1μl
BSA 1μl
BamH I 1μl
Not I 1μl
pAAV-D(+) 2μl
ddH 2O 14μl
4.琼脂糖凝胶电泳胶回收
用1％的琼脂糖电泳凝胶电泳双酶切产物，然后使用TaKaRa公司琼脂糖凝胶DNA回收试剂盒TaKaRa Agarose Gel DNA Purification Kit Ver.2.0回收双酶切产物，具体操作步骤如下：
1.制作1×TAE缓冲液琼脂糖凝胶，然后对目的DNA进行琼脂糖凝胶电泳；
2.在紫外灯下切出含有目的DNA的琼脂糖凝胶；
3.称量胶块重量，计算胶块体积，切碎胶块；
4.向胶块中加入3倍体积的胶块融化液DR-I Buffer，75℃加热融化胶块；
5.向胶块融化液中加入DR-I Buffer量的1/2体积量的DR-II Buffer，均匀混合。当分离小于400bp的DNA片段时，应在此溶液中再加入终浓度为20％的异丙醇；
6.将试剂盒中的Spin Column安置于Collection Tube上；
7.将上述操作5的溶液转移至Spin Column中，12,000rpm离心1min，弃滤液；
8.将500μl的Rinse A加入Spin Column中，12,000rpm离心30sec，弃滤液；
9.将700μl的Rinse B加入Spin Column中，12,000rpm离心30sec，弃滤液；
10.重复操作步骤9；
11.将Spin Column安置于Collection Tube上，12,000rpm离心1min；
12.将Spin Column安置于新的1.5ml的离心管上，在Spin Column膜的中央处加入25μl的60℃预热的Elution Buffer，室温静置1min；
13. 12,000rpm离心1min洗脱DNA。
5.载体连接
1.用T4连接酶连接回收的pAAV-D(+)载体和合成的DNA片段，按照下述反应体系16℃孵育过夜：
Figure PCTCN2020090794-appb-000003
2.全量(25μl)加入至100μl DH5α感受态细胞中，冰中放置30min；
3. 42℃加热45sec后，再在冰中放置1min；
4.加入500μl无抗生素LB培养基，37℃ 100rpm振荡培养60min；
5.在Amp +的LB平板培养基上培养，挑选白色单克隆菌落鉴定。
6.质粒小提
挑取单克隆菌落，加入到3ml Amp +的LB液体培养基中，37℃ 280rpm振荡培养过夜。使用北京全式金公司EasyPure Plasmid MiniPrep Kit提取质粒，具体操作步骤如下：
1.取1.5ml过夜培养的细菌10,000g离心1min，尽量吸尽上清；
2.加入250μl无色溶液RB(含RNase A)，震荡悬浮细菌沉淀；
3.加入250μl蓝色溶液LB，温和地上下翻转混合4-6次，使菌体充分裂解，形成蓝色透亮的溶液；
4.加入350μl黄色溶液NB，轻轻混合5-6次，直至形成紧实的黄色凝集块，室温静置2min；
5. 15,000g离心5min，小心吸取上清加入吸附柱中；
6. 15,000g离心1min，弃流出液；
7.加入650μl溶液WB，15,000g离心1min，弃流出液；
8. 15,000g离心2min，彻底去除残留的WB；
9.将吸附柱置于新Ep管中，在柱中央加入20μl 70℃预热的EB，室温静置1min；
10. 10,000g离心1min，洗脱DNA，洗脱出的DNA于-20℃保存。
7.质粒鉴定
采用双酶切和测序对所构建的质粒进行鉴定，得到真核表达质粒pAAV-D(+)-miR-320a和pAAV-D(+)-anti-miR-320a，其结构如图3所示。
8.质粒大提
准备1L的无菌锥形瓶，加入300ml无菌LB培养基，加氨苄青霉素溶液至终浓度为100μg/ml。分别加入50μl所需质粒(pXX2、pXX8或pXX9；phelper；pAAV-D(+)、pAAV-D(+)-miR-320a或pAAV-D(+)-anti-miR-320a)，280rpm，37℃过夜培养。按照OMEGA公司
Figure PCTCN2020090794-appb-000004
Endo-Free Plasmid Maxi Kit说明书操作，提取质粒，具体步骤如下：
1.室温下5000g离心10min收集细菌；
2.弃培养基，加入10ml Solution I/RNase A混合液，漩涡震荡完全重悬；
3.重悬混合液中加入10ml Solution II，轻轻颠倒混匀10-15次后，室温放置2min；
4.加入5ml冰浴的Buffer N3，并温和颠倒数次至形成白色絮状沉淀；
5.将HiBind柱套在收集管中，加入5ml Buffer GPS，室温静置3-10min，5,000g离心5min，弃滤液，将柱子重新放回收集管中；
6.将细菌裂解液倒入针筒过滤器中，静置2min，插入并推动活塞，收集过滤的裂解液；
7.过滤裂解液中加入1/10体积的ETR，颠倒7-10次后，冰浴10min；
8. 42℃水浴5min后，室温5,000g离心5min，转移上清液至新离心管，加入0.5倍体积无水乙醇，混匀后室温静置2min；
9.转移混合液至已激活HiBind柱中，室温5,000g离心5min，弃去滤液；
10.将结合柱重新装回收集管，加入10ml HB Buffer，室温5,000g离心5min，弃去滤液；
11.将结合柱重新装回收集管，加入15ml DNA Wash Buffer，室温5,000g离心5min，弃去滤液；
12.重复上一步骤；
13.弃去滤液，结合柱重新装回收集管，6,000g离心15min；
14.取出结合柱，65℃干燥10min；
15.结合柱装入新离心管，加入70℃预热的1-3ml Endotoxin free Elution Buffer，室温静置2min后，6,000g离心5min以洗脱DNA。
9.rAAV介导的病毒包装
293T细胞(人源胚胎肾上皮细胞)生长至90％，钙磷转染前1-2hr，每个培养皿换新鲜培养基(含血清)12-15ml，在50ml离心管内先加入氯化钙(CaCl 2)，再加入质粒，形成Ca-DNA混合液，充分混匀，向Ca-DNA混合液中缓慢滴加2XHEBS BUFFER，形成Ca-DNA-P混合液，一边加2XHEBS一边振荡离心管，充分混匀形成钙磷颗粒。8-12hr后，换18-20ml无血清培养基，72hr后，吸弃培养基，用PBS洗3遍，每个培养皿中加入Tris+NaCl(pH 8.5)1ml，用刮匙刮取细胞，收集于干净离心管，-80℃冻存。
10.病毒纯化
将冻存于-80℃的细胞取出，在37℃解冻溶解，反复冻融4次，8,000g离心15min，将上清放至干净离心管，弃细胞沉淀。
用-20℃预冷的无水乙醇与rAAV以3：1的体积比充分混匀，-20℃冰箱放置2hr后，4℃，13,000rpm离心15min，弃上清；乙醇挥发后，加入相应体积Tris+NaCl(pH 8.5)溶解沉淀。用Millipore小滤器(0.22μm)过滤。
11.病毒滴度测定
样品处理：rAAV病毒液40μl
蛋白酶K(20mg/ml) 5μl
55℃，反应1hr；
酚：氯仿：异戊醇 45μl
4℃，12,000g离心5min回收水相；
氯仿 45μl
4℃，12,000g离心5min回收水相。
Real-time PCR：
Primer 1(10μm) 0.4μl
Primer 2(10μm) 0.4μl
SYBR Green I Mix 10μl
ddH 2O 8.2μl
Template 1μl
95℃ 30sec---(95℃ 5sec---60℃ 5sec---72℃ 20sec)×40个循环---Melting Curve
12.病毒转染效率
纯化后的病毒转染至293T细胞48小时后，荧光显微镜观察被转染细胞比例，其转染效率可达90％以上(图4)。
实验例5.以rAAV9型表达hsa-miR-320a/hsa-anti-miR-320a的重组腺相关病毒为例检测其对糖尿病及并发症的治疗作用
1.db/db小鼠血糖检测：
我们采用12周龄的C57对照和db/db糖尿病小鼠，通过尾静脉分别注射rAAV-miR-320a和rAAV-anti-miR-320a，病毒滴度1×10 11PFU/每只，至实验终点(12周后)时采尾静脉血法，试纸法检测db/db小鼠的空腹血糖，结果显示：db/db糖尿病小鼠血糖较C57对照小鼠明显升高。rAAV-miR-320a处理可明显促进血糖更为增高，而rAAV-anti-miR-320a处理可明 显降低升高的血糖(图5)。
2.db/db小鼠心功能检测：
实验终点时采用心脏超声检测db/db小鼠心功能，方法如下：
使用仪器为配有30MHz高频探头的超声仪。在使用异氟烷麻醉小鼠后，将小鼠仰卧放置于检测平台，沿小鼠胸骨旁左室乳头肌水平短轴和长轴切面采集左室二维图像，同时在二维图像引导下分别获取5个以上连续心动周期M型超声影像，根据采集的影像使用软件分析结果，得到心脏超声检测心脏血流动力学指标，经相关软件分析后计算出下列指标：包括心率(Heart rate，HR)、左室舒张期内径(Left Ventricular Internal Dimension，diastole，LVIDd)、左室收缩期内径(Left Ventricular Internal Dimension，systole，LVIDs)、左室后壁舒张期厚度(Left Ventricular Posterior Wall，diastole，LVPWd)、左室后壁收缩期厚度(Left Ventricular Posterior Wall，systole，LVPWs)、室间隔舒张期厚度(Interventricular septal thickness，diastole，IVSd)、室间隔收缩期厚度(Interventricular septal thickness，systole，IVSs)、射血分数(Ejection Fraction，EF)以及缩短分数(Fractional Shortening，FS)等。
结果显示：与C57对照小鼠相比，db/db小鼠心脏收缩功能明显受损。rAAV-anti-miR-320a处理可明显改善db/db小鼠受损的心脏功能，而rAAV-miR-320a处理可明显加重已损伤的db/db小鼠的心脏功能(图6)。
实验例6、本发明的糖尿病防治药物的动物临床治疗验证
本发明针对数百只患有糖尿病的小鼠采用本发明的糖尿病防治药物进行了糖尿病治疗,治疗前每只糖尿病小鼠的症状包括:血糖高,糖尿病并发症,例如,心脏收缩功能受损等。
所述糖尿病治疗具体为:采用本发明第4组任一实施例所提供的糖尿病防治药物，和/或，第5组任一实施例所提供筛选方法筛选得到的糖尿病防治药物，和/或，第6组任一实施例所提供的制备方法制备得到的糖尿病防治药物，在db/db小鼠12周龄时通过尾静脉注射一次，病毒滴度1×10 11PFU/只，12周后，检测每只小鼠的血糖及心脏收缩功能，发现全部的糖尿病小鼠经上述治疗后血糖显著降低、心脏收缩功能显著改善(每只小鼠的治疗前后具体的对比数值图表类似于图4、图5所示的结果，为节约篇幅，本文不再一一罗列)，这表明，本发明的糖尿病防治药物的动物临床治疗的准确率为100％。同时，半年后追踪观察上述所有注射药物的小鼠，无一出现异常或其它副作用，同时血糖保持稳定，各并发症症状半年内没有再出现。
Figure PCTCN2020090794-appb-000005
Figure PCTCN2020090794-appb-000006

Claims (20)

  1. 用于防治糖尿病的药物，其特征在于，所述药物的药效成分以hsa-miR-320a为药物靶点，且通过结合、和/或捕获、和/或降解、和/或下调表达所述hsa-miR-320a起到所述防治糖尿病的药效；
    所述hsa-miR-320a如SEQ ID NO.1所示。
  2. 根据权利要求1所述的药物，其特征在于，所述药物的药效成分包括可降解、和/或下调表达所述hsa-miR-320a的物质；优选为可下调表达所述hsa-miR-320a的物质。
  3. 根据权利要求1或2所述的药物，其特征在于，所述药物的药效成分包含与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a；
    优选地，所述反义互补指所述anti-hsa-miR-320a与所述hsa-miR-320a的全长或部分序列呈反向互补的长度为15-25个碱基的序列片段。
  4. 根据权利要求3所述的药物，其特征在于，所述药物的药效成分为可表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒。
  5. 根据权利要求4所述的药物，其特征在于，所述药物的药效成分为所述药物的药效成分为重组腺相关病毒质粒pAAV-D(+)-anti-miR-320a，其表达的序列片段anti-hsa-miR-320a与hsa-miR-320a反义互补。
    更具体地，所述pAAV-D(+)-anti-miR-320a是通过将序列如SEQ ID NO.4和SEQ ID NO.5所示的引物对插入至腺病毒表达载体pAAV-D(+)中构建而得。
  6. 根据权利要求1-5任一所述的药物，其特征在于，所述药物还包括药学上可接受的辅料，和/或，用于缓冲、培养、和/或扩繁所述重组腺相关病毒质粒pAAV-D(+)-anti-miR-320a的试剂。
  7. 一种防治糖尿病的药物的筛选方法，其特征在于，检测待选物质是否能结合、捕获、降解、和/或下调表达hsa-miR-320a；
    优选地，筛选出能使hsa-miR-320a表达水平降低的物质。
  8. 一种防治糖尿病的药物的制备方法，其特征在于，包括：将降解、和/或下调表达所述hsa-miR-320a的物质；
    与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a；
    表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒；和/或，
    表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组腺相关病毒质粒作为抗糖尿病药物的活性成分。
  9. 根据权利要求8所述的制备方法，其特征在于，还包括：
    将可表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的引物对插入表达载 体，从而制备得到可稳定表达与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的重组质粒。
  10. 根据权利要求8或9所述的制备方法，其特征在于，可表达所述与hsa-miR-320a反义互补的序列片段anti-hsa-miR-320a的引物对如SEQ ID NO.4和SEQ ID NO.5所示；
    所述表达载体为腺相关病毒表达载体pAAV-D(+)。
  11. 用于诊断和/或早期预警糖尿病的生物标志物，其特征在于，包括：含有hsa-miR-320a在内的序列片段；所述hsa-miR-320a如SEQ ID No.1所示。
  12. 根据权利要求11所述的生物标志物，其特征在于，包括：所述hsa-miR-320a。
  13. 根据权利要求11或12所述的生物标志物，其特征在于，为所述hsa-miR-320a。
  14. 用于诊断和/或早期预警糖尿病的试剂盒，其特征在于，包括用于定量检测权利要求11-13任一所述的生物标志物的试剂。
  15. 根据权利要求14所述的试剂盒，其特征在于，包括用于定量检测hsa-miR-320a的试剂。
  16. 根据权利要求15所述的试剂盒，其特征在于，所述用于定量检测hsa-miR-320a的试剂包括所述hsa-miR-320a的特异性引物对。
  17. 根据权利要求16所述的试剂盒，其特征在于，所述特异性引物对为商购获得的miRQ0000510-1-1。
  18. 根据权利要求15或16所述的试剂盒，其特征在于，所述用于定量检测hsa-miR-320a的试剂还包括逆转录试剂；和/或逆转录PCR试剂；
    优选地，所述逆转录试剂包括：RT Primer Mix、2×TS reaction buffer、RNase free H2O、TS enzyme；
    进一步优选地，所述逆转录PCR试剂包括：2×SYBR Green Mix、RNase free H2O。
  19. 根据权利要求14-18任一所述的试剂盒，其特征在于，所述定量检测指荧光定量PCR检测；
    最优选地，所述试剂盒包括：miR-320a逆转录引物、miR-320a real-time PCR引物F、miR-320areal-time PCR引物R、SYBR Green I、TS reaction enzyme、TS reaction buffer、DEPC ddH2O。
  20. 权利要求11-13任一所述的生物标志物在制备糖尿病诊断试剂方面的应用。
PCT/CN2020/090794 2019-06-11 2020-05-18 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法 WO2020248771A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP20822198.6A EP3984541A4 (en) 2019-06-11 2020-05-18 METHOD FOR PRODUCING AN EARLY WARNING AND/OR DIAGNOSTIC REAGENT KIT BASED ON HSA-MIR-320A FOR DIABETES, MEDICINAL PRODUCTS FOR PREVENTING DIABETES, SCREENING METHOD FOR MEDICINAL PRODUCTS AND METHOD FOR THE PRODUCTION THEREOF
US17/904,597 US20230313182A1 (en) 2019-06-11 2020-05-18 Preparation method for diabetes early warning and/or diagnostic reagent kit based on hsa-mir-320a, medicament for preventing diabetes, screening method for medicament, and preparation method therefor

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201910500477.9A CN110295227B (zh) 2019-06-11 2019-06-11 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法
CN201910500630.8 2019-06-11
CN201910500477.9 2019-06-11
CN201910500630.8A CN110327363A (zh) 2019-06-11 2019-06-11 基于hsa-miR-320a的防治糖尿病的药物及其筛选方法和制备方法

Publications (2)

Publication Number Publication Date
WO2020248771A1 true WO2020248771A1 (zh) 2020-12-17
WO2020248771A9 WO2020248771A9 (zh) 2021-02-04

Family

ID=73781629

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/090794 WO2020248771A1 (zh) 2019-06-11 2020-05-18 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法

Country Status (3)

Country Link
US (1) US20230313182A1 (zh)
EP (1) EP3984541A4 (zh)
WO (1) WO2020248771A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011154689A1 (en) * 2010-06-07 2011-12-15 King's College London Methods and means for predicting or diagnosing diabetes or cardiovascular disorders based on micro rna detection
CN102965429A (zh) * 2012-03-15 2013-03-13 汪道文 microRNA-320(miR-320a)及其反义核苷酸在诊断、防治心血管疾病中的用途
CN110295227A (zh) * 2019-06-11 2019-10-01 华中科技大学同济医学院附属同济医院 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法
CN110327363A (zh) * 2019-06-11 2019-10-15 华中科技大学同济医学院附属同济医院 基于hsa-miR-320a的防治糖尿病的药物及其筛选方法和制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102970994A (zh) * 2009-12-16 2013-03-13 昆山市工业技术研究院小核酸生物技术研究所有限责任公司 与糖尿病中的mirna相关的组合物和方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011154689A1 (en) * 2010-06-07 2011-12-15 King's College London Methods and means for predicting or diagnosing diabetes or cardiovascular disorders based on micro rna detection
CN102965429A (zh) * 2012-03-15 2013-03-13 汪道文 microRNA-320(miR-320a)及其反义核苷酸在诊断、防治心血管疾病中的用途
CN110295227A (zh) * 2019-06-11 2019-10-01 华中科技大学同济医学院附属同济医院 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法
CN110327363A (zh) * 2019-06-11 2019-10-15 华中科技大学同济医学院附属同济医院 基于hsa-miR-320a的防治糖尿病的药物及其筛选方法和制备方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AUDREY VILLARD ET AL.: "Diagnostic Value of Cell-free Circulating MicroRNAs for Obesity and Type 2 Diabetes: A Meta-analysis", J MOL BIOMARK DIAGN, vol. 6, no. 6, 30 November 2015 (2015-11-30), XP055764461, DOI: 20200702142008X *
DWI SETYOWATI KAROLIN ET AL.: "MicroRNA 144 Impairs Insulin Signaling by Inhibiting the Expression of Insulin Receptor Substrate 1 in Type 2 Diabetes Mellitus", PLOS ONE, vol. 6, no. 8, 31 August 2011 (2011-08-31), XP055027307, DOI: 20200702135730X *
ELENA FLOWERS ET AL.: "Circulating MicroRNA-320a and MicroRNA-486 Predict Thiazolidinedione Response: Moving Towards Precision Health for Diabetes Prevention", METABOLISM, vol. 9, no. 64, 30 September 2015 (2015-09-30), XP055764457, DOI: 20200702140752X *
HE MENG-YING;YIN ZHONG-WEI;ZHAO YAN-RU;LI HUA-PING;WEN ZHENG;JIANG JIAN-GANG: "MiR-320a mediates doxorubicin-induced cardiotoxicity by targeting VEGF signal pathway", MOLECULAR CARDIOLOGY OF CHINA, vol. 18, no. 5, 25 October 2018 (2018-10-25), pages 2594 - 2599, XP055874563, ISSN: 1671-6272, DOI: 10.16563/j.cnki.1671-6272.2018.10.003 *
LING, HONGYAN: "The Screening and Function Study of miRNAs Related with Insulin Resistance in 3T3-L1 Adipocytes", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, no. 02, 15 February 2010 (2010-02-15), pages 1 - 151, XP055874545 *
ZHU, Z. ET AL.: "Role of MicroRNAs in the Treatment of Type 2 Diabetes Mellitus with Roux-en-Y Gastric Bypass", BRAZ J MED BIOL RES, vol. 50, no. 3, 31 December 2017 (2017-12-31), XP055764464, DOI: 20200702142433X *

Also Published As

Publication number Publication date
EP3984541A1 (en) 2022-04-20
WO2020248771A9 (zh) 2021-02-04
EP3984541A4 (en) 2023-09-06
US20230313182A1 (en) 2023-10-05

Similar Documents

Publication Publication Date Title
Chun et al. Translational advances in the field of pulmonary hypertension. Translating MicroRNA biology in pulmonary hypertension. It will take more than “miR” words
JP5871791B2 (ja) 心不全または心不全の危険性を中和、予防および／または決定する手段および方法
US20230287419A1 (en) DUCHENNE MUSCULAR DYSTROPHY-RELATED EXONIC SPLICING ENHANCER, sgRNA AND GENE EDITING TOOL, AND APPLICATIONS
US20190153446A1 (en) Mir-149-3p and method for treating metabolic disease using the same
Yang et al. Cardiac hypertrophy and dysfunction induced by overexpression of miR-214 in vivo
Zhou et al. Novel lncRNA XLOC_032768 alleviates cisplatin-induced apoptosis and inflammatory response of renal tubular epithelial cells through TNF-α
WO2023227061A1 (zh) 一种环状RNA-circ-Magi1及其应用
Zhu et al. miR-340-5p mediates cardiomyocyte oxidative stress in diabetes-induced cardiac dysfunction by targeting Mcl-1
CN110295227B (zh) 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法
Yan et al. Effect of miR-23a on anoxia-induced phenotypic transformation of smooth muscle cells of rat pulmonary arteries and regulatory mechanism
WO2020248771A1 (zh) 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法
CN107252491B (zh) 用于治疗心力衰竭的药物及其筛选方法和制备方法
CN111110691A (zh) 人参皂苷Rb2在制备预防和/或治疗动脉粥样硬化药物中的应用
CN110327363A (zh) 基于hsa-miR-320a的防治糖尿病的药物及其筛选方法和制备方法
AU2020384026B2 (en) Use of LncRNA-266 in preparation of a medicament for inducing differentiation of brown adipose cells
CN112746101A (zh) 一种骨质疏松诊断标志物及促进骨质疏松骨再生的核酸药物
CN113186224B (zh) 具有抑制乙肝病毒复制活性的microRNA-27a及其应用
CN115838725B (zh) 在哺乳动物心脏中特异性启动基因的启动子序列及其应用
WO2019112489A1 (en) Methods and agents for treatment of aging and aging related conditions and diseases
CN111118139B (zh) 一种骨质疏松的分子靶标及其应用
CN111763735B (zh) 肿瘤差异表达基因及其应用
CN111808954B (zh) lncRNA及其在疾病中的应用
CN110652522B (zh) miR-2052在制备抗肝癌药物中的应用
EP4009051A1 (en) Use of dkk1 inhibitor in prevention and/or treatment of tumor cachexia and diseases associated with diabetes
CN107653319B (zh) 胶质瘤诊断标志物circ8:61680968|61684188及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20822198

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020822198

Country of ref document: EP

Effective date: 20220111