WO2022052678A1 - miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒 - Google Patents

miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒 Download PDF

Info

Publication number
WO2022052678A1
WO2022052678A1 PCT/CN2021/110486 CN2021110486W WO2022052678A1 WO 2022052678 A1 WO2022052678 A1 WO 2022052678A1 CN 2021110486 W CN2021110486 W CN 2021110486W WO 2022052678 A1 WO2022052678 A1 WO 2022052678A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
mir
olanzapine
seq
rna
Prior art date
Application number
PCT/CN2021/110486
Other languages
English (en)
French (fr)
Inventor
秦胜营
孙竞
贺林
Original Assignee
上海交通大学
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
Application filed by 上海交通大学 filed Critical 上海交通大学
Priority to US18/025,639 priority Critical patent/US20230332233A1/en
Publication of WO2022052678A1 publication Critical patent/WO2022052678A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biotechnology, in particular, to an application of a miRNA marker in the preparation of a product for evaluating the efficacy of olanzapine in the treatment of schizophrenia, and a kit, in particular to an application of a miRNA marker for the efficacy of olanzapine in the treatment of schizophrenia
  • Schizophrenia is one of the most serious mental disorders, mainly characterized by positive symptoms such as hallucinations, delusions, and psychomotor excitement and/or negative symptoms such as emotional retardation, apathy, social withdrawal, and decreased willpower. Clinical features. Jingfen is currently facing two major dilemmas: "difficult to diagnose and difficult to treat", which brings a heavy burden to individuals, families and society. Effective prevention and treatment of this disease is imminent.
  • schizophrenia patients can be diagnosed early and receive effective treatment in a timely manner, they can significantly improve their psychological, physical, social functions and work performance, and greatly reduce medical expenses and disability rates.
  • the current diagnosis of schizophrenia still relies on the subjective assessment of its clinical symptoms, and there is no effective "gold standard" for clinical diagnosis, so the incidence of clinical misdiagnosis and mistreatment of schizophrenia is high.
  • the optimal therapeutic drug can only be found clinically through continuous trial administration. This not only affects the treatment effect of the disease, reduces the patient's medication compliance, but also causes a huge waste of medical resources. Therefore, it is necessary to develop a sensitive and specific biomarker to improve the diagnostic reliability of schizophrenia, so as to facilitate targeted drug intervention and clinical scientific research.
  • Olanzapine also known as olanzapine, is a second-generation atypical antipsychotic drug that has become a first-line clinical drug since it was approved for marketing in 1996.
  • olanzapine has better curative effect on schizophrenia, but some adverse reactions are still inevitable.
  • Clinical studies have pointed out that long-term use of olanzapine can induce energy metabolism syndromes such as lethargy, weight gain, and hyperglycemia in patients to a certain extent.
  • Schizophrenia is a complex neuropsychiatric disorder involving disturbances in neural circuits and synaptic function.
  • the delicate network structure and the ability of neurons to re-establish postsynapses require the coordination of a complex intracellular molecular signal transduction system.
  • the redundancy of these networks means that many combinations of genetic variants have the potential to contribute to systemic dysfunction manifesting as associated neurobehavioral syndromes.
  • miRNAs post-transcriptional gene regulation and associated microRNAs
  • miRNAs exhibit complex spatial expression patterns in the mammalian brain and act as specific factors for intracellular gene silencing mechanisms, potentially regulating thousands of target genes.
  • the purpose of the present invention is to provide an application and a kit of a miRNA marker in the preparation of a product for evaluating the efficacy of olanzapine in treating schizophrenia.
  • a first aspect of the present invention provides an application of a miRNA marker in the preparation of a product for evaluating the efficacy of olanzapine in treating schizophrenia, wherein the miRNA marker is miR-143-3p.
  • the method for detecting the expression level of the miRNA marker miR-143-3p specifically includes the following steps:
  • Step 1 processing of serum samples, blood collection, centrifugation, and storage for future use; the serum samples are the peripheral blood of schizophrenia patients whose olanzapine treatment is ineffective and schizophrenia patients whose olanzapine treatment is effective;
  • the specific treatment of serum samples is as follows: all the included people took 3ml of fasting venous blood in the morning, and immediately centrifuged at 2000rpm for 8min in a horizontal centrifuge to separate the serum. The aspirated supernatant was placed in a new centrifuge tube and stored in a -80°C refrigerator for total RNA extraction.
  • Step 2 Extraction of total RNA
  • RNA was precipitated from the aqueous layer with isopropanol. The precipitated RNA was washed with ethanol to remove impurities, and then resuspended in RNase-free water to obtain the extracted total RNA, which was stored at -80°C.
  • Step 3 use an ultra-micro spectrophotometer to detect the total amount of RNA; preferably, use an ultra-micro spectrophotometer to detect the absorbance at 260 mm to calculate the total amount of RNA.
  • Absorbance detection method Take 2 ⁇ l of dissolved RNA, and measure the absorbance at 260 mm with an ultra-micro spectrophotometer to calculate the yield of RNA.
  • Step 4 Perform reverse transcription to obtain cDNA, and perform fluorescence quantitative detection on the obtained cDNA; -3p downstream primer and stem-loop RT1 primer, the miR-143-3p upstream primer is shown in SEQ ID NO: 1:
  • the miR-143-3p downstream primer is shown in SEQ ID NO: 2:
  • the primers of the stem-loop RT1 are shown in SEQ ID NO: 3:
  • the internal reference primer for detecting the expression level of the miRNA marker including the internal reference upstream primer, the internal reference downstream primer and the primer of stem-loop RT2, the internal reference is U6 RNA, and the upstream primer of the U6 RNA is such as SEQ ID NO: 4 shows:
  • the downstream primer of the U6 RNA is shown in SEQ ID NO: 5:
  • the primers of the stem-loop RT2 are shown in SEQ ID NO: 6:
  • reverse transcription is as follows: Thaw 2 ⁇ miRNA L-RT Solution mix and mix well, put miRNA L-RT Enzyme Mix in ice for later use, and add 2 ⁇ miRNA L-RT Solution mix to a reaction tube pre-cooled on ice 10 ⁇ l, 1.5 ⁇ L of miRNA L-RT Enzyme Mix, 4 ⁇ g of total RNA, 1 ⁇ l of Stem-loop primer (10 ⁇ M), and RNase-Free dd H 2 O to a total volume of 20 ⁇ l, and then reacted, and the obtained cDNA was subjected to fluorescence quantitative detection.
  • Real-time fluorescent quantitative PCR reaction of miRNA operate according to the instructions of SYBR Green Masrer kit, set 3 sub-wells for each experiment, and use LightCycler 96 real-time fluorescent quantitative PCR instrument for detection.
  • Step 5 Use the relative quantitative method to analyze the test results, and determine whether olanzapine is effective for patients with schizophrenia by detecting the expression level of miR-143-3p in the sample.
  • the increased expression level of miR-143-3p indicates that olanzapine is Ping is effective for patients with schizophrenia.
  • the miRNA marker miR-143-3p exists in peripheral blood and nerve cells.
  • the expression level of miR-143-3p in peripheral blood and nerve cells is up-regulated. .
  • the second aspect of the present invention provides a kit for evaluating the efficacy of olanzapine in the treatment of schizophrenia, the kit includes a primer for the miRNA marker miR-143-3p and an internal reference for detecting the expression level of miR-143-3p primers.
  • the primers for the miRNA marker miR-143-3p include miR-143-3p upstream primers, miR-143-3p downstream primers and stem-loop RT1 primers, and the miR-143-3p upstream primers are such as SEQ ID NO: 1, the miR-143-3p downstream primer is shown in SEQ ID NO: 2, and the stem-loop RT1 primer is shown in SEQ ID NO: 3; used to detect the miRNA marker miR
  • the internal reference primer of the expression level of 143-3p including the internal reference upstream primer, the internal reference downstream primer and the primer of stem-loop RT2, the internal reference is U6 RNA, and the upstream primer of the U6 RNA is as shown in SEQ ID NO: 4.
  • the downstream primer of the U6 RNA is shown in SEQ ID NO: 5
  • the primer of the stem-loop RT2 is shown in SEQ ID NO: 6.
  • the kit includes the following reagents for a 20 ⁇ L PCR reaction system: 2 ⁇ miRNA L-RT Solution mix 10 ⁇ L, miRNA L-RT Enzyme Mix 1.5 ⁇ L, total RNA 4 ⁇ g, 10 ⁇ M Stem-loop primer 1 ⁇ L, RNase -Free dd H2O to a total volume of 20 ⁇ L.
  • the kit determines whether olanzapine is effective for patients with schizophrenia by detecting the expression level of miR-143-3p in the sample, and whether the patients taking olanzapine will have the risk of developing metabolic syndrome.
  • the expression of miR-143-3p is up-regulated in patients with schizophrenia, and the up-regulation of miR-143-3p increases the risk of metabolic syndrome, such as increased blood sugar and other side effects.
  • the kit includes the following reagents for a 20 ⁇ L PCR reaction system: 2 ⁇ miRNA L-RT Solution mix 10 ⁇ L, miRNA L-RT Enzyme Mix 1.5 ⁇ L, total RNA 4 ⁇ g, 10 ⁇ M Stem-loop primer 1 ⁇ L, RNase -Free dd H2O to a total volume of 20 ⁇ L.
  • biomarker is any gene whose expression level in a tissue or cell is altered compared to that in normal or healthy cells or tissue.
  • the present invention can utilize any method known in the art to measure gene expression. As will be understood by those skilled in the art, the means of measuring gene expression is not an important aspect of the present invention.
  • the expression levels of biomarkers can be detected at the transcriptional level.
  • the present invention has the following beneficial effects:
  • the present invention finds for the first time that miR-143-3p is differentially expressed in the curative effect of olanzapine in the treatment of schizophrenia. By detecting the expression level of miR-143-3p, the curative effect of olanzapine in the treatment of schizophrenia can be judged.
  • the present invention provides a precise medical method for schizophrenia, which can treat schizophrenia patients by increasing the transcription level of miR-143-3p in the patients.
  • the present invention provides a molecular marker for schizophrenia, which can be used as a detection or treatment index in clinical applications, and provides a theoretical basis for the mechanism research and clinical application of schizophrenia.
  • the present invention provides a diagnostic kit, which provides a basis for realizing the early diagnosis of olanzapine treatment efficacy evaluation for schizophrenia.
  • FIG. 1 is a schematic diagram of a detection method for a peripheral blood miRNA marker detection kit for evaluating the efficacy of olanzapine in treating schizophrenia provided in an embodiment of the present invention
  • Figure 2 is the use of miRNA sequencing to detect gene markers associated with the efficacy of olanzapine in the treatment of schizophrenia;
  • Figure 3 is a boxplot of the expression of miR-143-3p in the neural stem cells of schizophrenia patients who are effective and ineffective in olanzapine treatment by qRT-PCR;
  • Figure 4 is a boxplot of the expression of miR-143-3p in peripheral blood mononuclear cells of schizophrenia patients who are effective and ineffective in olanzapine treatment by qRT-PCR;
  • Figure 5 is a graph showing the expression of miR-143-3p in SH-SY5Y cells detected by olanzapine by qRT-PCR.
  • the present invention discovers for the first time that miR-143-3p is differentially expressed in the curative effect of olanzapine in treating schizophrenia, and by detecting the expression level of miR-143-3p, the curative effect of olanzapine in treating schizophrenia can be judged.
  • Example 1 High-throughput sequencing screening of miRNA markers associated with the efficacy of olanzapine in the treatment of schizophrenia
  • the peripheral blood of 10 patients with schizophrenia who were ineffective in olanzapine treatment and 10 patients with schizophrenia who were effective in olanzapine treatment were collected, and the patients gave informed consent. All the above specimens were obtained with the consent of the organizational ethics committee. 3ml of fasting venous blood in the morning was taken from all the included people, and immediately centrifuged at 2000rpm for 8min in a horizontal centrifuge to separate the serum, the supernatant was placed in a centrifuge tube, centrifuged at 14000rpm for 10min at 4°C, and the supernatant was set fresh. In a centrifuge tube, it was stored in a -80°C freezer for the extraction of total RNA.
  • RNA was precipitated from the aqueous layer with isopropanol. The precipitated RNA was washed with ethanol to remove impurities, and then resuspended in RNase-free water to obtain the extracted total RNA, which was stored at -80°C.
  • RNA integrity Use agarose gel electrophoresis technology and AStraGene technology to test the quality of RNA to ensure RNA quality.
  • T4 RNA ligase 2 (NEB company) to connect the 3' end linker and the RNA small fragment with a length of 15-50 nt after cutting (using UltraTM RNA Library Prep Kit for Standard operating procedure); use T4 RNA ligase 1 to connect the 5' end ligation linker to the above small RNA fragment (using T4 RNA ligase 1) UltraTM RNA Library Prep Kit for Standard operating procedure); reverse transcribing the ligated RNA fragments to obtain cDNA; amplify the reverse transcription product by PCR to obtain a cDNA library; purify the PCR product of 140-150 bp by 12% PAGE gel electrophoresis, and perform subsequent sequencing.
  • T4 RNA ligase 2 NEB company
  • the purified cDNA library was subjected to miRNA high-throughput sequencing and data filtering through the IIIumina HiseqXten platform. And use Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) for miRNA sequencing data processing.
  • human peripheral blood mononuclear cells were transferred to fresh medium without antibiotics, preheated in a 37°C incubator, counted, determined cell density, centrifuged at 3000 rpm for 5 min at room temperature, and discarded the supernatant. Resuspend the cell pellet with electroporation buffer to obtain a cell suspension to make the cell density 1.0 ⁇ 10 4 /ml, add an appropriate amount of Episomal iPSC reprogramming vector to the Neon Transfection System 100 ⁇ L Kit transfection reagent system, and mix gently.
  • the cell suspension was added to the transfection system for electroporation, and the pulsed samples were immediately transferred to the prepared culture plate containing human embryonic stem cell culture medium (hESC medium), and the culture plate was placed in the incubator for culture, about 20 days later. Observed iPSCs clones.
  • pluripotent stem cells When pluripotent stem cells form, aspirate the medium and add the minimum amount of Accutase solution required to cover the surface of the dish. The dishes were incubated at 37°C for 15-30 min and observed under the microscope. When all cells appeared single cells, collect clones in fresh hESC medium. The cells in the dish were triturated using a Pasteur pipette and filtered through a 40 ⁇ m cell strainer to remove debris and cell clumps. Cells were washed and centrifuged twice with 160 g hESC medium for 5 min each to remove any remaining Accutase solution.
  • CM containing ROCK inhibitor was added to the appropriate cell concentration to plate cells on Matrigel-coated petri dishes at 10,000 cells/cm and grown in CM containing 10 ng ml-1 FGF cell.
  • the medium was replaced with medium containing 10 ⁇ M SB431542 and 500 ng ml-1 KSR. Change the medium and slowly switch from KSR to N 2 on days 2, 3, 5, 7, 9 and 11 and observe for 2 weeks to obtain iPSCs-NSCs.
  • RNA was precipitated from the aqueous layer with isopropanol. The precipitated RNA was washed with ethanol to remove impurities, and then resuspended in RNase-free water to obtain the extracted total RNA, which was stored at -80°C.
  • T4 RNA ligase 2 (NEB company) to connect the 3' end linker and the RNA small fragment with a length of 15-50nt after cutting; use T4 RNA ligase 1 ligates the 5'-end ligation linker with the above small RNA fragments; reverse-transcribes the ligated RNA fragments to obtain cDNA; the reverse transcription product is amplified by PCR to obtain a cDNA library; 12% PAGE gel electrophoresis PCR products of 140-150 bp were purified and sequenced.
  • the purified cDNA library was subjected to miRNA high-throughput sequencing and data filtering through the IIIumina HiseqXten platform. And use Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) for miRNA sequencing data processing.
  • Example 1 the peripheral blood of schizophrenia patients with ineffective olanzapine treatment and schizophrenia patients with effective olanzapine treatment were collected from 4 cases each, and the remaining operations were the same as those in Example 1;
  • RNA Take 2ul of dissolved RNA, and use an ultra-micro spectrophotometer to detect the absorbance at 260mm to calculate the yield of RNA.
  • Primers for the miRNA marker miR-143-3p used in reverse transcription including miR-143-3p upstream primers, miR-143-3p downstream primers and stem-loop RT1 primers, the miR-143-3p upstream primers are such as SEQ ID NO: 1 shows:
  • the miR-143-3p downstream primer is shown in SEQ ID NO: 2:
  • the primers of the stem-loop RT1 are shown in SEQ ID NO: 3:
  • the internal reference primer for detecting the expression level of the miRNA marker including the internal reference upstream primer, the internal reference downstream primer and the primer of stem-loop RT2, the internal reference is U6 RNA, and the upstream primer of the U6 RNA is such as SEQ ID NO: 4 shows:
  • the downstream primer of the U6 RNA is shown in SEQ ID NO: 5:
  • the primers of the stem-loop RT2 are shown in SEQ ID NO: 6:
  • the reverse transcription steps are as follows: Thaw 2 ⁇ miRNA L-RT Solution mix and mix well, put miRNA L-RT Enzyme Mix on ice for later use, add 10 ⁇ l of 2 ⁇ miRNA L-RT Solution mix to the reaction tube pre-cooled on ice, 1.5 ⁇ L of miRNA L-RT Enzyme Mix, 4 ⁇ g of total RNA, 1 ⁇ l of Stem-loop primer (10 ⁇ M), and RNase-Free dd H 2 O to a total volume of 20 ⁇ l were reacted, and the obtained cDNA was subjected to fluorescence quantitative detection.
  • Real-time fluorescent quantitative PCR reaction of miRNA operate according to the instructions of SYBR Green Masrer kit, set 3 sub-wells for each experiment, and use LightCycler 96 real-time fluorescent quantitative PCR instrument for detection.
  • the experiments were repeated three times, and the results were expressed in the form of mean ⁇ standard deviation.
  • SPSS18.0 statistical software was used for statistical analysis. The difference between the two was tested by t test. Statistical significance was established when P ⁇ 0.05.
  • RT-PCR test results showed that compared with schizophrenia patients who responded to olanzapine therapy (OLA-R), the miR-143-3p gene was found in schizophrenia patients who did not respond to olanzapine therapy (OLA-non-R)
  • OVA-R olanzapine therapy
  • the expression was down-regulated in neural stem cells (Fig. 3) and peripheral blood mononuclear cells (Fig. 4), and the expression level was significantly decreased (Fig. 3 and Fig. 4, *P ⁇ 0.05, **P ⁇ 0.01), and the difference was statistically significant ( P ⁇ 0.05), consistent with the miRNA-seq results in Example 1.
  • miR143-3p has the same trend in the expression of this marker in human peripheral blood and neural tissue, and is a candidate molecular marker that can be used to characterize the difference in the efficacy of olanzapine in the treatment of schizophrenia.
  • Example 3 Study on the differential expression of miR-143-3p gene in other neural cell lines
  • SH-SY5Y cells Human neuroblastoma cells (SH-SY5Y cells) were seeded in 6-well cell culture plates, and MEM:F12 medium containing 10% fetal bovine serum was added, and placed in an incubator at 37°C, 5% CO2 and saturated humidity , cells in logarithmic growth phase were used for experiments. When the confluence is about 80%. SH-SY5Y cells were treated with olanzapine at concentrations of 0 ⁇ M (control), 1 ⁇ M, 10 ⁇ M for 24 hours.
  • Primers for the miRNA marker miR-143-3p used in reverse transcription including miR-143-3p upstream primers, miR-143-3p downstream primers and stem-loop RT1 primers, the miR-143-3p upstream primers are such as SEQ ID NO: 1 shows:
  • the miR-143-3p downstream primer is shown in SEQ ID NO: 2:
  • the primers of the stem-loop RT1 are shown in SEQ ID NO: 3:
  • the internal reference primer for detecting the expression level of the miRNA marker including the internal reference upstream primer, the internal reference downstream primer and the primer of stem-loop RT2, the internal reference is U6 RNA, and the upstream primer of the U6 RNA is such as SEQ ID NO: 4 shows:
  • the downstream primer of the U6 RNA is shown in SEQ ID NO: 5:
  • the primers of the stem-loop RT2 are shown in SEQ ID NO: 6:
  • the reverse transcription steps are as follows: Thaw 2 ⁇ miRNA L-RT Solution mix and mix well, put miRNA L-RT Enzyme Mix on ice for later use, add 10 ⁇ l of 2 ⁇ miRNA L-RT Solution mix, 1.5 ⁇ L of miRNA L-RT Enzyme Mix, 4 ⁇ g of total RNA, 1 ⁇ l of Stem-loop primer (10 ⁇ M), and RNase-Free dd H 2 O to a total volume of 20 ⁇ l, the reaction was carried out, and the obtained cDNA was subjected to fluorescence quantitative detection.
  • Real-time fluorescent quantitative PCR reaction of miRNA operate according to the instructions of SYBR Green Masrer kit, set 3 sub-wells for each experiment, and use LightCycler 96 real-time fluorescent quantitative PCR instrument for detection.
  • Real-time fluorescent quantitative PCR reaction of miRNA operate according to the instructions of SYBR Green Masrer kit, set 3 sub-wells for each experiment, and use LightCycler 96 real-time fluorescent quantitative PCR instrument for detection.
  • the experiments were repeated three times, and the results were expressed in the form of mean ⁇ standard deviation.
  • SPSS18.0 statistical software was used for statistical analysis. The difference between the two was tested by t test. Statistical significance was established when P ⁇ 0.05.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了一种miRNA标志物miR-143-3p的检测试剂在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒。通过检测miR-143-3p的表达水平可以判断患者对奥氮平治疗精神分裂症疗效的高低，可作为检测或治疗指标应用于临床，为精神分裂症的机理研究以及临床应用提供理论基础。

Description

miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒 技术领域
本发明涉及生物技术领域，具体地，涉及一种miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒，尤其是一种用于奥氮平精神分裂症疗效评估的外周血miRNA标志物miR-143-3p的应用、检测方法及试剂盒。
背景技术
精神分裂症(schizophrenia,SZ)是最为严重的精神障碍之一，以幻觉、妄想、精神运动性兴奋等阳性症状和(或)以情感迟钝、情感淡漠、社交退缩、意志减退等阴性症状为主要临床特征。精分目前面临“诊断难，治疗难”两大困境，给个人、家庭和社会都带来了极为沉重的负担，有效防治这一疾病迫在眉睫。
已有研究表明，精神分裂症病人若能早期明确诊断并及时得到有效治疗，可以显著改善患者的心理、生理、社会功能以及工作表现，并大大减少医疗开支，降低致残率。然而，目前对精神分裂症的诊断仍是依靠其临床症状的主观评估，缺乏有效的临床诊断“金标准”，因而精神分裂症的临床误诊、误治发生率较高。另一方面，由于事先未知患者对治疗药物反应特点，临床上只能通过不断尝试给药，才可能找到最佳治疗药物。这不仅影响该病的治疗效果，降低患者服药依从性，并造成医疗资源的巨大浪费。因此，有必要研发一种具有敏感性和特异性的生物标志物，来提高精神分裂症的诊断可靠性，以便于有针对性地进行药物干预及开展临床科学研究。
奥氮平(Olanzapine)又称奥兰扎平，属于第二代非典型抗精神病药物，自1996年批准上市以来，已成为临床一线用药。研究指出，奥氮平可通过作用NRG-1对抗PCP诱导的前额皮质神经元突触生成降低。在耐受性和安全性方面，奥氮平与传统抗精神病药物相比，对精分疗效更好，但仍然不可避免地出现一些不良反应。临床研究指出，长期服用奥氮平会一定程度诱发患者嗜睡、体重增加、高血糖症等能量代谢综合征，不仅如此，部分早期用药有效患者在连续用药一段时间后，出现阳性症状反弹情况，且增加给药剂量仍无法逆转，即发生奥氮平耐受，因此，有 必要研发一种具有敏感性和特异性的生物标志物，来提高精神分裂症的诊断可靠性，以便于有针对性地进行药物干预及开展临床科学研究。
精神分裂症是一种复杂的神经精神疾病，包括神经回路和突触功能的紊乱。精细的网络结构和神经元突触后重建的能力需要一个复杂的细胞内分子信号转导系统的协调。这些网络的冗余意味着许多基因变异组合有可能导致系统功能障碍，表现为相关的神经行为综合征。最近的研究表明，转录后基因调控和相关的microRNA(miRNA)可能是形成这些网络的重要因素。miRNA在哺乳动物大脑中表现出复杂的空间表达模式，作为细胞内基因沉默机制的特异性因子，有可能调控数千个靶基因。它们正在成为许多神经发育和神经过程的关键调节者，因为它们的失调可能导致发育过程中网络结构的普遍变化，以及在精神分裂症的病理生理学中高度重要的成熟大脑的变化。值得注意的是，这些miRNA表达和相关机制的变化可能代表着药物开发的新靶点，精神分裂症的miRNA生物标志物也可能为新的临床诊断提供基础。这些发展具有巨大的潜力，并突出了这一研究途径的重要性。
发明内容
针对现有技术中的缺陷，本发明的目的是提供一种miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒。
本发明的目的是通过以下方案实现的：
本发明的第一方面提供一种miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，所述miRNA标志物为miR-143-3p。
其中，miRNA标志物miR-143-3p表达量的检测方法，具体包括以下步骤：
步骤一：血清样本的处理，采血、离心、保存备用；所述血清样本为奥氮平治疗无效的精神分裂症患者与奥氮平治疗有效的精神分裂症患者的外周血；
血清样本的处理具体为：所有纳入人群均取清晨空腹静脉血3ml，并立即用水平离心机以2000rpm离心8min分离血清，吸出的上清置于离心管中，于4℃条件下14000rpm离心10min，吸出的上清液置新的离心管中，放到-80℃冰箱低温保存以便用于总RNA的提取。
步骤二：总RNA的提取；
具体步骤为：
用TRIzol TM试剂将样品匀浆后，加入氯仿，使匀浆分离成透明的上水层(含有RNA)、间相和红色的下有机层(含有DNA和蛋白质)。用异丙醇将RNA从水层析出。 沉淀的RNA用乙醇清洗以去除杂质，然后再用RNase-free water重悬即为提取的总RNA，放置在-80℃保存。
步骤三：使用超微量分光光度计检测RNA的总量；优选地，用超微量分光光度计检测在260mm处的吸光度，计算RNA的总量。吸光度检测方法：取2μl溶解的RNA，用超微量分光光度计检测在260mm处的吸光度计算RNA的产量。
步骤四：进行反转录获得cDNA，将得到的cDNA进行荧光定量检测；优选地，反转录所用的miRNA标志物miR-143-3p的引物，包括miR-143-3p上游引物、miR-143-3p下游引物和stem-loop RT1的引物，所述miR-143-3p上游引物如SEQ ID NO：1所示：
F：CGCGTGAGATGAAGCACTG；
所述miR-143-3p下游引物如SEQ ID NO：2所示：
R：AGTGCAGGGTCCGAGGTATT；
所述stem-loop RT1的引物如SEQ ID NO：3所示：
RT1：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGCTA。
用于检测所述的miRNA标志物表达量的内参引物，包括内参上游引物、内参下游引物和stem-loop RT2的引物，所述内参为U6 RNA，所述U6 RNA的上游引物如SEQ ID NO：4所示：
F：AGAGAAGATTAGCATGGCCCCTG；
所述U6 RNA的下游引物如SEQ ID NO：5所示：
R：ATCCAGTGCAGGGTCCGAGG；
所述stem-loop RT2的引物如SEQ ID NO：6所示：
RT2：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA。
优选地，反转录为：解冻2×miRNA L-RT Solution mix并混匀，miRNA L-RT Enzyme Mix放于冰中备用，在冰上预冷的反应管内加入2×miRNA L-RT Solution mix 10μl、miRNA L-RT Enzyme Mix 1.5μL，总RNA 4μg，Stem-loop primer(10μM)1μl、RNase-Free dd H 2O至总体积20μl后进行反应，得到的cDNA进行荧光定量检测。miRNA的实时荧光定量PCR反应：按照SYBR Green Masrer kit说明书进行操作，每次实验设置3个副孔，使用LightCycler 96实时荧光定量PCR仪进行检测。
步骤五：采用相对定量法对检测结果进行分析，通过检测样本中miR-143-3p的表达水平来判断奥氮平是否对精神分裂症患者有效，miR-143-3p的表达水平上调说明奥氮平对精神分裂症患者有效。
优选地，所述步骤五中，miRNA表达量以2 -ΔCt循环值表示，其中ΔCt＝(Ct miR-143-3p 实验组-Ct u6)。
优选地，所述miRNA标志物miR-143-3p存在于外周血及神经细胞中，当奥氮平对精神分裂症患者有效时，外周血及神经细胞中的miR-143-3p表达水平均上调。
本发明的第二方面提供一种用于奥氮平治疗精神分裂症疗效评估的试剂盒，所述试剂盒包括miRNA标志物miR-143-3p的引物和检测miR-143-3p表达量的内参引物。
优选地，所述miRNA标志物miR-143-3p的引物，包括miR-143-3p上游引物、miR-143-3p下游引物和stem-loop RT1的引物，所述miR-143-3p上游引物如SEQ ID NO：1所示，所述miR-143-3p下游引物如SEQ ID NO：2所示，所述stem-loop RT1的引物如SEQ ID NO：3所示；用于检测miRNA标志物miR-143-3p表达量的内参引物，包括内参上游引物、内参下游引物和stem-loop RT2的引物，所述内参为U6 RNA，所述U6 RNA的上游引物如SEQ ID NO：4所示，所述U6 RNA的下游引物如SEQ ID NO：5所示，所述stem-loop RT2的引物如SEQ ID NO：6所示。
优选地，所述试剂盒包括以下用于20μL PCR反应体系的试剂：2×miRNA L-RT Solution mix 10μL、miRNA L-RT Enzyme Mix 1.5μL，总RNA 4μg，10μM的Stem-loop primer 1μL、RNase-Free dd H 2O至总体积20μL。
所述试剂盒通过检测样本中miR-143-3p的表达水平来判断奥氮平是否对精神分裂症患者有效，和患者服用奥氮平是否会有产生代谢综合症的风险。其中，miR-143-3p在精神分裂症患者中表达上调，且miR-143-3p上调会增加代谢综合症的风险，例如血糖的升高等副作用。
优选地，所述试剂盒包括以下用于20μL PCR反应体系的试剂：2×miRNA L-RT Solution mix 10μL、miRNA L-RT Enzyme Mix 1.5μL，总RNA 4μg，10μM的Stem-loop primer 1μL、RNase-Free dd H 2O至总体积20μL。
在本发明中，术语“生物标志物”是其在组织或细胞中的表达水平与正常或健康细胞或组织的表达水平相比发生改变的任何基因。
本领域技术人员将认识到，本发明的实用性并不局限于对本发明的标志物基因 的任何特定变体的基因表达进行定量。
本发明可以利用本领域内已知的任何方法测定基因表达。本领域技术人员应当理解，测定基因表达的手段不是本发明的重要方面。可以在转录水平上检测生物标志物的表达水平。
与现有技术相比，本发明具有如下的有益效果：
1、本发明首次发现了miR-143-3p在奥氮平治疗精神分裂症疗效中差异表达，通过检测miR-143-3p的表达水平可以判断患者对奥氮平治疗精神分裂症疗效的高低。
2、本发明提供了一种精神分裂症的精准医疗手段，通过提高患者中miR-143-3p的转录水平，从而治疗精神分裂症患者。
3、本发明提供了一种精神分裂症的分子标记物，可作为检测或治疗指标应用于临床，为精神分裂症的机理研究以及临床应用提供理论基础。
4、本发明提供了一种诊断试剂盒，为实现奥氮平治疗精神分裂症疗效评估的早期诊断提供基础。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1是本发明实施例提供的奥氮平治疗精神分裂症疗效评估的外周血miRNA标志物检测试剂盒检测方法示意图；
图2是利用miRNA测序检测与奥氮平治疗精神分裂症疗效相关的基因标志物；
图3是利用qRT-PCR验证miR-143-3p在奥氮平治疗有效的和无效的精神分裂症患者神经干细胞中的表达情况箱线图；
图4是利用qRT-PCR验证miR-143-3p在奥氮平治疗有效的和无效的精神分裂症患者外周血单核细胞中的表达情况箱线图；
图5是利用qRT-PCR检测奥氮平对SH-SY5Y细胞中miR-143-3p表达情况图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变化和改进。这些都属于 本发明的保护范围。
本发明首次发现了miR-143-3p在奥氮平治疗精神分裂症疗效中差异表达，通过检测miR-143-3p的表达水平可以判断患者对奥氮平治疗精神分裂症疗效的高低。
接下来结合具体实施例对本发明技术方案做进一步详细描述。
实施例1：高通量测序筛选与奥氮平治疗精神分裂症疗效相关miRNA标志物
一、外周血中与奥氮平治疗精神分裂症疗效相关miRNA标志物的筛选
1.1、血清样本的处理，采血、离心、保存备用
收集奥氮平治疗无效的精神分裂症患者与奥氮平治疗有效的精神分裂症患者的外周血各10例，患者均知情同意，上述所有标本的取得均通过组织伦理委员会的同意。所有纳入人群均取清晨空腹静脉血3ml，并立即用水平离心机以2000rpm离心8min分离血清，吸出的上清置于离心管中，于4℃条件下14000rpm离心10min，吸出的上清液置新的离心管中，放到-80℃冰箱低温保存以便用于总RNA的提取。
1.2、总RNA的提取
用TRIzol TM试剂将样品匀浆后，加入氯仿，使匀浆分离成透明的上水层(含有RNA)、间相和红色的下有机层(含有DNA和蛋白质)。用异丙醇将RNA从水层析出。沉淀的RNA用乙醇清洗以去除杂质，然后再用RNase-free water重悬即为提取的总RNA，放置在-80℃保存。
1.3、构建miRNA文库
利用琼脂糖凝胶电泳技术和AStraGene技术对RNA进行质量检测，确保RNA质量，具体标准为：分别采用琼脂糖凝胶电泳分析样品RNA完整性及是否存在DNA污染；NanoPhotometer spectrophotometer检测RNA纯度(OD260/280及OD260/230比值)；Agilent 2100 bioanalyzer精确检测RNA完整性。检测后无DNA污染结构完整，OD260/280＝1.8-2.0，OD260/230大于等于2，认为是符合检测质量的总RNA。再使用T4 RNA连接酶2(NEB公司)连接3’端接头与切割后长度为15～50nt的RNA小片段(采用
Figure PCTCN2021110486-appb-000001
UltraTM RNA Library Prep Kit for
Figure PCTCN2021110486-appb-000002
标准操作步骤)；使用T4 RNA连接酶1将5’端连接接头与上述小RNA片段连接(采用
Figure PCTCN2021110486-appb-000003
UltraTM RNA Library Prep Kit for
Figure PCTCN2021110486-appb-000004
标准操作步骤)；将连接好的RNA片段进行逆转录，得到cDNA；逆转录产物通过PCR进行扩增得到cDNA文库；通过12％PAGE凝胶电泳纯化140～150bp的PCR产物，进行后续的测序。
1.4、miRNA高通量测序及数据处理:
将纯化后的cDNA文库通过IIIumina HiseqXten平台进行miRNA高通量测序和数据过滤。并利用Illumina’s Sequencing Control Studio software version 2.8(SCS v2.8)进行miRNA测序数据处理。
二、神经干细胞中与奥氮平治疗精神分裂症疗效相关miRNA标志物的筛选
2.1、iPSCs重编程
重编程前，将人外周血单核细胞转移至不含抗生素的新鲜培养基中，放于37℃培养箱预热，细胞计数，确定细胞密度，室温下3000rpm离心5min，弃上清。用电转缓冲液重悬细胞沉淀得到细胞悬液，使细胞密度为1.0×10 4/ml，将适量Episomal iPSC重编程载体加入到Neon Transfection System 100μL Kit转染试剂系统中，轻轻混匀。将细胞悬液加入转染系统进行电转，将脉冲好的样品立即转移至准备好的装有人胚胎干细胞培养基(hESC培养基)的培养板中，将培养板放到培养箱培养，约20天后观察得到iPSCs克隆体。
2.2、iPS定向分化神经干细胞
当多功能干细胞形成时，吸出培养基并加入覆盖培养皿表面所需的最小量Accutase溶液。将培养皿在37℃孵育15-30min并在显微镜下观察。当所有细胞都呈现单细胞时，在新鲜的hESC培养基中收集克隆。使用巴斯德吸管研磨培养皿中的细胞，并采用40μm细胞过滤器过滤除去碎片和细胞团。使用160g hESC培养基洗涤并离心细胞两次，每次5min，以除去任何剩余的Accutase溶液。将细胞重悬于含有ROCK抑制剂的hESC培养基中，并且以大于200,000个细胞每平方厘米的密度涂布在多聚赖氨酸包被的培养皿上。在培养箱中于37℃孵育培养皿30min。收集非粘附细胞，重悬含有ROCK抑制剂的CM中的细胞。细胞计数并将含有ROCK抑制剂的CM加入适当的细胞浓度，以10,000个细胞/cm 2将基质胶涂覆的陪替氏培养皿上的细胞铺板，在含有10ng ml-1 FGF的CM中生长细胞。当细胞开始分化时，用含有10μM SB431542和500ng ml-1 KSR培养基更换培养基。在第2，3，5，7，9和11天更换介质并慢慢从KSR切换到N 2，观察2周，得到iPSCs-NSC。
2.3、总RNA的提取
用TRIzol TM试剂将样品匀浆后，加入氯仿，使匀浆分离成透明的上水层(含有RNA)、间相和红色的下有机层(含有DNA和蛋白质)。用异丙醇将RNA从水层析出。沉淀的RNA用乙醇清洗以去除杂质，然后再用RNase-free water重悬即为提取的总RNA，放置在-80℃保存。
2.4、构建miRNA文库
利用琼脂糖凝胶电泳技术和AStraGene技术对RNA进行质量检测，确保RNA质量；再使用T4 RNA连接酶2(NEB公司)连接3’端接头与切割后长度为15～50nt的RNA小片段；使用T4 RNA连接酶1将5’端连接接头与上述小RNA片段连接；将连接好的RNA片段进行逆转录，得到cDNA；逆转录产物通过PCR进行扩增得到cDNA文库；通过12％PAGE凝胶电泳纯化140～150bp的PCR产物，进行后续的测序。
2.5、miRNA高通量测序及数据处理:
将纯化后的cDNA文库通过IIIumina HiseqXten平台进行miRNA高通量测序和数据过滤。并利用Illumina’s Sequencing Control Studio software version 2.8(SCS v2.8)进行miRNA测序数据处理。
结果分析：
结果如图2A和2B所示，外周血PBMCs和iPS分化神经干细胞的miRNA-seq测序结果分别显示，相比于有效患者，无效患者中分别有55和46个miRNA分子发生了显著变化(P<0.05)，其中有3个miRNA分子在以上两个体系中均具有显著性变化(图2C)。综合发生显著性变化分子在以上两个研究体系中的一致性情况，以及显著性，miR143-3p因其在外周血和神经干细胞中均发生显著下调表明其很可能是一种与奥氮平抗精神分裂症药效相关的重要生物标记分子。
实施例2：qRT-PCR验证miR-143-3p的表达差异
2.1、血清样本的处理，采血、离心、保存备用
收集实施例1患者中，奥氮平治疗无效的精神分裂症患者与奥氮平治疗有效的精神分裂症患者的外周血各4例，其余操作同实施例1；
2.2、总RNA的提取
总RNA提取步骤同实施例1；
2.3、使用超微量分光光度计检测RNA的质量
取2ul溶解的RNA，用超微量分光光度计检测在260mm处的吸光度计算RNA的产量。
2.4、进行反转录获得cDNA，将得到的cDNA进行荧光定量检测；
反转录所用的miRNA标志物miR-143-3p的引物，包括miR-143-3p上游引物、miR-143-3p下游引物和stem-loop RT1的引物，所述miR-143-3p上游引物如SEQ ID NO：1所示：
F：CGCGTGAGATGAAGCACTG；
所述miR-143-3p下游引物如SEQ ID NO：2所示：
R：AGTGCAGGGTCCGAGGTATT；
所述stem-loop RT1的引物如SEQ ID NO：3所示：
RT1：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGCTA。
用于检测所述的miRNA标志物表达量的内参引物，包括内参上游引物、内参下游引物和stem-loop RT2的引物，所述内参为U6 RNA，所述U6 RNA的上游引物如SEQ ID NO：4所示：
F：AGAGAAGATTAGCATGGCCCCTG；
所述U6 RNA的下游引物如SEQ ID NO：5所示：
R：ATCCAGTGCAGGGTCCGAGG；
所述stem-loop RT2的引物如SEQ ID NO：6所示：
RT2：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA。
反转录步骤为：解冻2×miRNA L-RT Solution mix并混匀，miRNA L-RT Enzyme Mix放于冰中备用,在冰上预冷的反应管内加入2×miRNA L-RT Solution mix 10μl、miRNA L-RT Enzyme Mix1.5μL，总RNA 4μg，Stem-loop primer(10μM)1μl、RNase-Free dd H 2O至总体积20μl后进行反应，得到的cDNA进行荧光定量检测。
miRNA的实时荧光定量PCR反应：按照SYBR Green Masrer kit说明书进行操作，每次实验设置3个副孔，使用LightCycler 96实时荧光定量PCR仪进行检测。
2.5、采用相对定量法对检测结果进行分析
采用相对定量法进行分析，miRNA表达量以2 -△Ct表示；2 -ΔCt循环值计算，ΔCt＝(Ct miR-143-3p实验组-Ct u6内参)。实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS18.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
2.6、结果分析
RT-PCR试验结果显示，与奥氮平治疗有效的精神分裂症患者(OLA-R)相比，miR-143-3p基因在奥氮平治疗无效(OLA-non-R)的精神分裂症患者神经干细胞(图3)以及外周血单核细胞(图4)中表达下调，表达水平显著性降低(图3和图4，*P＜0.05， **P＜0.01)，差异具有统计学意义(P<0.05)，同实施例1中miRNA-seq结果一致。以上结果表明miR143-3p在人体外周血和神经组织中该标记物表达变化趋势一致，是一个可用于表征奥氮平治疗精神分裂症疗效差异的候选分子标记物。
实施例3：miR-143-3p基因在其他神经细胞系中的差异表达情况研究
3.1、细胞培养
将人神经母细胞瘤细胞(SH-SY5Y细胞)接种于6孔细胞培养板中，加入含有10％胎牛血清的MEM:F12培养基，置于37℃，5％CO2及饱和湿度的培养箱中，取对数生长期细胞用于实验。当汇合度约为80％。将SH-SY5Y细胞用浓度为0μM(对照组)、1μM、10μM的奥氮平处理24小时。
3.2、RNA的提取
(1)吸去孔板中培养基，用PBS清洗两遍，每个孔加入1mL Trizol反复吹打10-15次后，室温静置5min；
(2)将细胞悬液收集到1.5mL无酶EP管中，涡旋振荡10s后，冰上静置5min；每管加入200μL预冷氯仿，手动振荡15s(手指顶着管盖)，室温静置5min；
(3)4℃，13000rpm离心15min；
(4)吸取上层水相至干净的1.5mL无酶EP管中(约400μL，不能吸到中间层或者下层红色液体)；
(5)每管加入等体积4℃预冷的异丙醇，上下缓慢颠倒10-15次后，室温静置10min；
(6)4℃,13000rpm离心10min；
(7)弃去上清，向每管加入1000μL预冷的75％乙醇，轻轻上下颠倒；
(8)4℃，13000rpm离心5min；
(9)倒掉乙醇，室温晾＜10min至管内近干，每管加入100μL RNase-free water溶解，测定浓度和纯度，-80℃保存。
3.3、使用超微量分光光度计检测RNA的质量
具体步骤同实施例2。
3.4、进行反转录获得cDNA，将得到的cDNA进行荧光定量检测；
反转录所用的miRNA标志物miR-143-3p的引物，包括miR-143-3p上游引物、miR-143-3p下游引物和stem-loop RT1的引物，所述miR-143-3p上游引物如SEQ ID NO：1所示：
F：CGCGTGAGATGAAGCACTG；
所述miR-143-3p下游引物如SEQ ID NO：2所示：
R：AGTGCAGGGTCCGAGGTATT；
所述stem-loop RT1的引物如SEQ ID NO：3所示：
RT1：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGCTA。
用于检测所述的miRNA标志物表达量的内参引物，包括内参上游引物、内参下游引物和stem-loop RT2的引物，所述内参为U6 RNA，所述U6 RNA的上游引物如SEQ ID NO：4所示：
F：AGAGAAGATTAGCATGGCCCCTG；
所述U6 RNA的下游引物如SEQ ID NO：5所示：
R：ATCCAGTGCAGGGTCCGAGG；
所述stem-loop RT2的引物如SEQ ID NO：6所示：
RT2：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA。
反转录步骤为：解冻2×miRNA L-RT Solution mix并混匀，miRNA L-RT Enzyme Mix放于冰中备用，在冰上预冷的反应管内加入2×miRNA L-RT Solution mix 10μl、miRNA L-RT Enzyme Mix 1.5μL，总RNA 4μg，Stem-loop primer(10μM)1μl、RNase-Free dd H 2O至总体积20μl后进行反应，得到的cDNA进行荧光定量检测。miRNA的实时荧光定量PCR反应：按照SYBR Green Masrer kit说明书进行操作，每次实验设置3个副孔，使用LightCycler 96实时荧光定量PCR仪进行检测。
miRNA的实时荧光定量PCR反应：按照SYBR Green Masrer kit说明书进行操作，每次实验设置3个副孔，使用LightCycler 96实时荧光定量PCR仪进行检测。
3.5、采用相对定量法对检测结果进行分析
采用相对定量法进行分析，miRNA表达量以2 -△Ct表示；2 -ΔCt循环值计算，ΔCt＝(Ct miR-143-3p实验组-Ct u6内参)。实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS18.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
3.6、结果分析
结果如图5所示。与对照组(Contral)相比，1μM和10μM奥氮平会显著升高miR-143-3p表达水平，且随剂量的升高而升高(图5，*P＜0.05，**P＜0.01)。结果显 示，治疗浓度的奥氮平会升高miR-143-3p的表达水平。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变化或修改，这并不影响本发明的实质内容。在不冲突的情况下，本申请的实施例和实施例中的特征可以任意相互组合。

Claims (9)

  1. miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，其特征在于，所述miRNA标志物为miR-143-3p。
  2. 根据权利要求1所述的miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，其特征在于，包括对miRNA标志物miR-143-3p表达量进行检测，所述检测方法，具体包括以下步骤：
    步骤一：血清样本的处理，采血、离心、保存备用；所述血清样本为奥氮平治疗无效的精神分裂症患者与奥氮平治疗有效的精神分裂症患者的外周血；
    步骤二：总RNA的提取；
    步骤三：使用超微量分光光度计检测RNA的总量；
    步骤四：进行反转录获得cDNA，将得到的cDNA进行荧光定量检测。
  3. 根据权利要求2所述的miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，其特征在于，所述步骤三中，用超微量分光光度计检测在260mm处的吸光度，计算RNA的总量。
  4. 根据权利要求2所述的miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，其特征在于，步骤四中，反转录所用的miRNA标志物miR-143-3p的引物，包括miR-143-3p上游引物、miR-143-3p下游引物和stem-loop RT1的引物，所述miR-143-3p上游引物如SEQ ID NO：1所示，所述miR-143-3p下游引物如SEQ ID NO：2所示，所述stem-loop RT1的引物如SEQ ID NO：3所示。
  5. 根据权利要求2所述的miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，其特征在于，步骤四中，反转录时，用于检测所述的miRNA标志物表达量的内参引物，包括内参上游引物、内参下游引物和stem-loop RT2的引物，所述内参为U6 RNA，所述U6 RNA的上游引物如SEQ ID NO：4所示，所述U6 RNA的下游引物如SEQ ID NO：5所示，所述stem-loop RT2的引物如SEQ ID NO：6所示。
  6. 根据权利要求2所述的miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用，其特征在于，所述miRNA标志物miR-143-3p存在于外周血及神经细胞中，当奥氮平对精神分裂症患者有效时，外周血及神经细胞中的miR-143-3p表达水平均上调。
  7. 一种用于奥氮平治疗精神分裂症疗效评估的试剂盒，其特征在于，所述试剂盒包 括miRNA标志物miR-143-3p的引物和所述检测miRNA标志物表达量的内参引物。
  8. 根据权利要求7所述的用于奥氮平治疗精神分裂症疗效评估的试剂盒，其特征在于，所述miRNA标志物miR-143-3p的引物，包括miR-143-3p上游引物、miR-143-3p下游引物和stem-loop RT1的引物，所述miR-143-3p上游引物如SEQ ID NO：1所示，所述miR-143-3p下游引物如SEQ ID NO：2所示，所述stem-loop RT1的引物如SEQ ID NO：3所示；用于检测miRNA标志物miR-143-3p表达量的内参引物，包括内参上游引物、内参下游引物和stem-loop RT2的引物，所述内参为U6 RNA，所述U6 RNA的上游引物如SEQ ID NO：4所示，所述U6 RNA的下游引物如SEQ ID NO：5所示，所述stem-loop RT2的引物如SEQ ID NO：6所示。
  9. 根据权利要求7所述的用于奥氮平治疗精神分裂症疗效评估的试剂盒，其特征在于，所述试剂盒包括以下用于20μL PCR反应体系的试剂：2×miRNA L-RT Solution mix 10μL、miRNA L-RT Enzyme Mix 1.5μL，10μM的Stem-loop primer 1μL、RNase-Free dd H 2O至总体积20μL。
PCT/CN2021/110486 2020-09-09 2021-08-04 miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒 WO2022052678A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/025,639 US20230332233A1 (en) 2020-09-09 2021-08-04 USE OF microRNA (miRNA) MARKER IN PREPARATION OF PRODUCT FOR EVALUATING THERAPEUTIC EFFECT OF OLANZAPINE IN TREATMENT OF SCHIZOPHRENIA (SZ) AND KIT

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010942097.3 2020-09-09
CN202010942097.3A CN111909997B (zh) 2020-09-09 2020-09-09 miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒

Publications (1)

Publication Number Publication Date
WO2022052678A1 true WO2022052678A1 (zh) 2022-03-17

Family

ID=73267569

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/110486 WO2022052678A1 (zh) 2020-09-09 2021-08-04 miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒

Country Status (3)

Country Link
US (1) US20230332233A1 (zh)
CN (1) CN111909997B (zh)
WO (1) WO2022052678A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111909997B (zh) * 2020-09-09 2021-09-28 上海交通大学 miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒
CN112725430B (zh) * 2020-11-16 2022-12-09 首都医科大学附属北京朝阳医院 奥氮平药效评估的生物标志物、方法及应用
CN112710523B (zh) * 2020-12-28 2023-09-19 山西省农业科学院农作物品种资源研究所 一种适用于分子标记物检测的收集装置
CN112680509A (zh) * 2021-01-20 2021-04-20 河南省中医院（河南中医药大学第二附属医院） 一种评估冠心病预后分子标志物miR-302e及其逆转录引物、扩增引物和应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101985659A (zh) * 2010-11-13 2011-03-16 上海交通大学 检测精神分裂关联基因的试剂盒及其制备方法
CN104120172A (zh) * 2013-04-28 2014-10-29 上海交通大学 精神分裂症的基因标志物及其使用方法和应用
CN104862310A (zh) * 2015-05-04 2015-08-26 张理义 精神分裂症生物标记物、筛选方法及试剂盒
CN105755167A (zh) * 2016-05-21 2016-07-13 陈亮 用于尿道癌诊断的miRNA生物标志物及检测试剂盒
CN105950730A (zh) * 2016-05-21 2016-09-21 崔学俊 用于甲状腺癌诊断的miRNA生物标志物及检测试剂盒
CN109423519A (zh) * 2017-09-01 2019-03-05 安科默(北京)生物技术有限公司 早期胰腺癌标记物及其检测方法
WO2019046303A1 (en) * 2017-08-28 2019-03-07 The Trustees Of Columbia University In The City Of New York METHOD FOR PREDICTING PATIENT RESPONSE TO SLC MODULATOR PROCESSING
CN109890394A (zh) * 2016-08-30 2019-06-14 耶鲁大学 作为子宫内膜异位症的生物标志物的微小rna
CN111909997A (zh) * 2020-09-09 2020-11-10 上海交通大学 miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101985659A (zh) * 2010-11-13 2011-03-16 上海交通大学 检测精神分裂关联基因的试剂盒及其制备方法
CN104120172A (zh) * 2013-04-28 2014-10-29 上海交通大学 精神分裂症的基因标志物及其使用方法和应用
CN104862310A (zh) * 2015-05-04 2015-08-26 张理义 精神分裂症生物标记物、筛选方法及试剂盒
CN105755167A (zh) * 2016-05-21 2016-07-13 陈亮 用于尿道癌诊断的miRNA生物标志物及检测试剂盒
CN105950730A (zh) * 2016-05-21 2016-09-21 崔学俊 用于甲状腺癌诊断的miRNA生物标志物及检测试剂盒
CN109890394A (zh) * 2016-08-30 2019-06-14 耶鲁大学 作为子宫内膜异位症的生物标志物的微小rna
WO2019046303A1 (en) * 2017-08-28 2019-03-07 The Trustees Of Columbia University In The City Of New York METHOD FOR PREDICTING PATIENT RESPONSE TO SLC MODULATOR PROCESSING
CN109423519A (zh) * 2017-09-01 2019-03-05 安科默(北京)生物技术有限公司 早期胰腺癌标记物及其检测方法
CN111909997A (zh) * 2020-09-09 2020-11-10 上海交通大学 miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DONG LI-CAI, LU TIAN-LAN，YUE WEI-HUA: "Detection of differentially expressed miＲNAs in serum of patients with schizophrenia by ＲT-PCＲ", CHINESE MENTAL HEALTH JOURNAL, vol. 29, no. 9, 10 September 2015 (2015-09-10), XP055911736, ISSN: 1000-6729 *
M.M.J. VAN DEN BERG ET AL.: "Circulating microRNAs as potential biomarkers for psychiatric and neurodegenerative disorders", PROGRESS IN NEUROBIOLOGY, vol. 185, 7 December 2019 (2019-12-07), XP085996530, ISSN: 0301-0082, DOI: 10.1016/j.pneurobio.2019.101732 *

Also Published As

Publication number Publication date
CN111909997B (zh) 2021-09-28
US20230332233A1 (en) 2023-10-19
CN111909997A (zh) 2020-11-10

Similar Documents

Publication Publication Date Title
WO2022052678A1 (zh) miRNA标志物在制备奥氮平治疗精神分裂症疗效评估方面的产品中的应用及试剂盒
Ertekin et al. Evaluation of circulating miRNAs in wet age-related macular degeneration
Qu et al. Expression signature of lncRNAs and their potential roles in cardiac fibrosis of post-infarct mice
CN107385035B (zh) 与2型糖尿病视网膜病变相关的血清/血浆miRNA标志物及其应用
Chen et al. Increased salivary microRNAs that regulate DJ-1 gene expression as potential markers for Parkinson’s disease
Zeng et al. Potential microRNA biomarkers for acute ischemic stroke
Zhong et al. Umbilical cord blood-derived exosomes from very preterm infants with bronchopulmonary dysplasia impaired endothelial angiogenesis: roles of exosomal microRNAs
Wu et al. Discovery and validation of hsa_circ_0001953 as a potential biomarker for proliferative diabetic retinopathy in human blood
CN104862310A (zh) 精神分裂症生物标记物、筛选方法及试剂盒
CN110423811A (zh) 脓毒症诊断标志物
Li et al. Expression profile and potential functions of circulating long noncoding RNAs in acute ischemic stroke in the southern Chinese Han population
Qiao et al. The Novel Regulatory Role of the lncRNA–miRNA–mRNA Axis in Chronic Inflammatory Airway Diseases
Zhou et al. Oxygen‑induced circRNA profiles and coregulatory networks in a retinopathy of prematurity mouse model
Wang et al. Differentially expressed miRNAs in oxygen‑induced retinopathy newborn mouse models
WO2019143828A9 (en) Biomarkers of cardiovascular status and uses therof
Drekolia et al. Unravelling the impact of aging on the human endothelial lncRNA transcriptome
CN112143790B (zh) 基于NRIP1 mRNA的m6A甲基化修饰评估唐氏综合征风险的方法及其应用
CN107326076A (zh) 一种脊柱侧弯早期辅助检测试剂盒及其应用
Sun et al. Differential expression profiles and functional prediction of circular RNAs in pediatric dilated cardiomyopathy
Sheikh et al. The mir-21 inhibition enhanced HUVEC cellular viability during hypoxia-reoxygenation injury by regulating PDCD4
CN112852950A (zh) 急性心肌梗死生物标志物及其应用
CN111471682A (zh) miR-23a作为诊断和治疗胃癌伪管生成标志物的应用
Zhao et al. A 3′ Untranslated Region Polymorphism of CTNNB1 (Rs2953) Alters MiR-3161 Binding and Affects the Risk of Ischemic Stroke and Coronary Artery Disease in Chinese Han Population
Hao et al. Correlation between Platelet miRNA Expression and Coagulation Function in Children with Severe Pneumonia
CN110408693A (zh) Loc105373033的新用途

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: 21865734

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21865734

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21865734

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 21.09.2023)