CN107190066A - A kind of urine excretion body hsa microRNA486 3p and its application - Google Patents
A kind of urine excretion body hsa microRNA486 3p and its application Download PDFInfo
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Abstract
The present invention discloses a kind of urine excretion body hsa microRNA486 3p and its application, belongs to pharmaceutical technology field.The present invention is to be based on excretion body fluid body Biopsy, using the sequencing technologies of future generation of Illumina Hiseq2500 microarray datasets, the microRNA (s) for finding differential expression in steroid femur head necrosis in urine excretion body；QRT PCR verify the microRNA (s) of differential expression, obtain the obvious potential mark of differential expression：Urine excretion body hsa miR486 3p, its sequence such as SEQ ID NO：Shown in 1.Urine excretion body is extracted, the change of the hsa miR486 3p in urine excretion body is analyzed using sequencing technologies of future generation, steroid femur head necrosis can be found earlier than MRI；It is cheap；It is easy to carry and promotes.
Description
Technical field
The invention belongs to pharmaceutical technology field, it is related to the biomarker that a kind of extreme early diagnoses steroid femur head necrosis
Urine excretion body hsa-microRNA486-3p, more particularly to a kind of urine excretion body hsa-microRNA486-3p and its should
With.
Background technology
Caput femoris necrosis (ONFH) is the orthopaedic disease with high disability rate, is apt to occur in the young and the middle aged of 30-50 Sui, easily makes
Into patient's labor capacity is lost, white elephant [1,2] is brought to family and society.If it find that not in time, failing to take
Intervening measure, 80% ONFH meeting rapid progress finally receives total hip replacement and controlled to the late period (arthritis phase) of osteonecrosis
Treat [3].Therefore early utilization Non-invasive detection means find caput femoris necrosis as far as possible, carry out early intervention, delay or even avoid
The problem of joint replacement is urgent need to resolve in clinical position [4].Diagnostic mode non-invasive at present includes common X-ray plain film, meter
Calculation machine tomoscan (CT), Magnetic resonance imaging (MRI), bone scanning etc..It is compared to other Non-invasive detection means, MRI inspections
Survey is considered as current diagnosis ONFH " goldstandard ".But MRI is only capable of to occurring the stock that organic (femur head morphology) changes
Bone provides information, it is impossible to which the stage for sexually revising (cartilage degeneration, cartilage gauffer etc.) in femoral head generating function makes diagnosis
[5]；And the factors such as MRI is expensive, the patient that is not used to Metallic orthopaedic implants, limit MRI bad in femoral head
The dead Clinical practice value for occurring early stage.
Liquid biopsy (Liquid biopsy) is a kind of new detection method, enters body by capturing and accurately analyzing
The other cells or DNA of liquid, so as to make early diagnosis to disease, are widely used to the diagnosis [6] of kinds cancer at present.Should
Detection method is first time non-invasi, obtains sample to be checked repeatablely, and doctors are so as to setting up to the disease
The gene expression profile of disease, targeting mutation medication, the quick development for judging therapeutic effect and real-time monitoring of diseases and adjustment for the treatment of
Scheme [7,8].In recent years, as the important component of liquid biopsy, one kind is called excretion body (exosomes) vesicles
Just by more and more extensive concern [9].
The encytosis that excretion body is mediated by non-grid albumen or clathrin forms body in primary, the primary interior body of acidifying
And late endosomes are obtained, and then interior body film caves in form many vesica bodies.There are intracavitary vesicles in multivesicular body, have many in it
Content includes ribonucleic acid (miRNAs, mRNAs), albumen (acceptor, enzyme etc.), also lipid, virion.Many vesica bodies lead to
Cross lysosomal degradation or by merging release with film, that is, discharge excretion body (see Fig. 1) [10].
As living cells secretion come film vesica, excretion body be naturally occurring in body fluid (blood, saliva, urine etc.) and
In cell culture medium [11].2007, Valadi etc. find cell secretion excretion body in the mRNAs containing bioactivity,
MicroRNAs (miRNAs), lipid and protein, this causes researcher to increase sharply [12] to the research enthusiasm of excretion body.With
Progress of research, scientist has found that excretion body not only plays special make in the regulation of cell-cell communication and local microenvironment
With, and important function (see Fig. 2) [13,14] is also functioned in blood coagulation in the blood vessels, the management of internal rubbish.
MiRNAs is important a member in non-coding RNA (non-coding RNAs) family, about 17-24 nucleosides
Sour length, regulates and controls the gene silencing after translation, and then suppress by being combined with the 3 ' non-translational regions (3 ' UTR) on target mRNA
The expression [15] of related gene.MiRNAs is the main tiny RNA in excretion body, and they are thin by surrounding or other distal sites
After born of the same parents' " swallowing ", and then the function of recipient cell is adjusted, affect the Biological signaling network in human body, participated in cell and breed, carefully
Born of the same parents' differentiation, cell transfer, the progress [16-20] of the generation of disease and disease.
Increasing evidence shows that miRNAs can avoid degraded under the protection of excretion body bilayer lipid membrane, keep it
Bioactivity, can be stable in the presence of in body fluid (blood, saliva, milk etc.) [21-23].Importantly, in excretion body
MiRNAs quantity and composition has obvious difference in physiological status and pathological state, and these all turn into for excretion body miRNAs
Early diagnose disease, indicate that the non-invasive biomarker of disease change provides advantage [24].For example, a series of next
From the miRNAs in human saliva, including miRNAs：Hsa-miR-150, hsa-miR-379, hsa-miR-888 are used as diagnosis
The potential mark [25] of Sjogren syndromes；And hsa-miR-132, hsa-miR-155, hsa-miR-21, miR-331-5p
Deng in the lung cancer of recurrence be in high expression status [26]；As can be seen here, excretion body miRNAs has been used to a variety of diseases, especially
The early diagnosis of cancer.As described in the Jeanne Tie of Australian Walter and Eliza Hall Institute for Medical Research,
" these new marks (excretion body, ctDNA etc.) manage cancer there is provided a surprising chance.In the past, we are always
Lag behind the step of disease one.There are these more specific, more sensitive instruments, we can stride forward a step finally ".Although in cancer
Disease field, the development of excretion body miRNAs marks is like a raging fire, but the application in diagnosis orthopaedic disease is still in beginning
Stage.
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Chen W-M.Etiologies and outcome of osteonecrosis of the femoral head:Etiology
and outcome study in a Taiwan population.Journal of the Chinese Medical
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[3].Mont MA,Hungerford DS.Non-traumatic avascular necrosis of the
femoral head.Journal of Bone and Joint Surgery(American Volume).1995,77(3):
459-474.
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R.Classification,functions,and clinical relevance of extracellular
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Expression of oncogenic and tumor-suppressive microRNAs and their target
genes in human adenocarcinoma alveolar basal epithelial cells.2016.
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bioinformatics.2015,13(1):17-24.
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[19].Kota J,Chivukula RR,O'Donnell KA,Wentzel EA,Montgomery CL,Hwang
H-W,Chang T-C,Vivekanandan P,Torbenson M,Clark KR.Therapeutic
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model.Cell.2009,137(6):1005-1017.
[20].Ma L,Teruya-Feldstein J,Weinberg RA.Tumour invasion and
metastasis initiated by microRNA-10b in breast cancer.Nature.2007,449(7163):
682-688.
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detectable in serum and saliva is concentrated in exosomes.PloS One.2012,7
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cancer.Journal of Clinical Oncology.2010,28(10):1721-1726.
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X.Immune-related microRNAs are abundant in breast milk exosomes.International
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Alevizos I.Exosomes from human saliva as a source of microRNA biomarkers.Oral
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recurrent lung cancer.Tumor Biology.2016:1-12.
[27] is in snowy peak, Li Hongtao, Sun Guicai, the big of Fan Xiang<" preventiveed treatment of disease " theoretic discussion hormonal femoral head from the traditional Chinese medical science
The prevention .pdf of necrosis>Medical information .2013,26 (12)
[28] the grandson He Ying traditional Chinese medical science " preventiveing treatment of disease " and the bright traditional Chinese medical science .2009 of genetic test, (6):1137-1138.
[29] Yuan Hao<" preventiveing treatment of disease " theoretical application .pdf in caput femoris necrosis preventing and treating>Jiangsu traditional Chinese medicine .2008,
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Control .pdf>Journal of Traditional Chinese Medicine .2005,45 (4)
The content of the invention
In order to overcome shortcoming and deficiency of the prior art：1. fallen ill when making a definite diagnosis；2. it is expensive, spend the time long；
3.MRI equipment is expensive in itself, it is impossible to popularize；It is an object of the invention to provide a kind of urine excretion body hsa-microRNA486-
3p。
Another object of the present invention is to provide above-mentioned urine excretion body hsa-microRNA486-3p application.
The purpose of the present invention is achieved through the following technical solutions：
The present invention provides a kind of urine excretion body hsa-microRNA486-3p, abbreviation hsa-miR486-3p, its sequence
For：5′-CGGGGCAGCUCAGUACAGGAU-3′.
The present invention provides a kind of kit for being used to diagnose steroid femur head necrosis, including above-mentioned urine excretion body hsa-
microRNA486-3p。
The present invention provides above-mentioned urine excretion body hsa-microRNA486-3p and is preparing the reagent of diagnosis caput femoris necrosis
In application；Application especially in the reagent for preparing diagnosis steroid femur head necrosis.
Further, above-mentioned urine excretion body hsa-microRNA486-3p is being prepared earlier than nuclear magnetic resonance (MRI) diagnosis
Application in the reagent of caput femoris necrosis；Especially preparing earlier than nuclear magnetic resonance (MRI) diagnosis steroid femur head necrosis
Application in reagent.
The urine excretion body hsa-microRNA486-3p of the present invention high expression can be used as potential molecular biology mark
Will thing diagnoses steroid femur head necrosis earlier than nuclear magnetic resonance (MRI).
The present invention has the following advantages and effect relative to prior art：
(1) present invention is that (liquid biopsy is a kind of new detection method, by catching based on excretion body fluid body Biopsy
Obtain and accurately analysis, into the other cells or DNA of body fluid, so as to make early diagnosis to disease, is widely used to many at present
Plant the diagnosis of cancer), using the sequencing technologies of future generation (NGS) of Illumina Hiseq2500 microarray datasets, for finding urine
In liquid excretion body in steroid femur head necrosis differential expression microRNA (s)；QRT-PCR checking differential expressions
MicroRNA (s), confirms that it can indicate the generation of steroid femur head necrosis, obtains the obvious potential mark of differential expression
Thing：Urine excretion body hsa-miR486-3p.
(2) urine excretion body is extracted, analyzes the hsa-miR486-3p's in urine excretion body using sequencing technologies of future generation
Change, can find steroid femur head necrosis earlier than MRI；It is cheap；It is easy to carry and promotes.
Brief description of the drawings
Fig. 1 is the generation schematic diagram of excretion body.
Fig. 2 is excretion body function schematic diagram.
Fig. 3 is that excretion body is extracted, miRNAs builds the flow chart of storehouse, sequencing and data analysis.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
(1) examination of urine excretion body candidate markers
1. the acquisition and identification of urine excretion body
A. acquisition methods：
Caput femoris necrosis, steroid femur head necrosis (ARCO IV phases) needs do not occur after collecting Healthy People, Corticosteroids
The urine of row half-sib recurrent selection patient, then extracts the excretion body in urine using exosomes separating kits.
Use Qiagen companies exoRNeasy Serum/Plasma Kits excretion body extracts kit (article No.s：77064)
Or Guangzhou Rui Bo bio tech ltd RiboTM Exosome Isolation Kit excretion body extracts kit (article No.s：
C10110), concrete operation step is with reference to kit specification.Step is summarized as follows：
(1) urine specimen is taken out from -80 DEG C, be placed in standby on ice；
(2) 20min (14,000rpm) is centrifuged at room temperature, to remove residual cell and fragment；
(3) transfer supernatant adds the RiboTM exosome Isolation reagent (for of 1/3 volume to new pipe
plasma or serum)；
(4) mixing or pipettor mixing are overturned, until mixing sample completely；(solution will present muddy)
(5) 4 DEG C of refrigerators are put into and stand 30min；
(6) 4 DEG C, centrifugation 2min (14,000rpm)；
(7) supernatant is carefully sucked with pipettor, sediment is the excretion body of acquisition.
(8) excretion body quantitatively using micro BCA protein assay (Thermo Fisher Scientific KK,
Tokyo,Japan)。
B. identification (the reference of excretion body：Cancer Exosomes Perform Cell-Independent MicroRNA
Biogenesis and Promote Tumorigenesis.Cancer Cell)
The excretion body of extraction is identified using the method for document report, is summarized as follows：
(1) ESEM is detected, a diameter of 50~140nm of excretion body；
(2) printing and dyeing detection is immunized, excretion body specific antigen TSG101, CD9, and CD63 are detected using antigen-antibody reaction
Expression.
(3) flow cytomery, after excretion body is combined with latex bead, using flow cytometry analysis TSG101, CD9,
CD63 and flotillin1 and expression.
2. urine excretion body total RNAs extraction and detection (reference：Estrogen-Related Receptor
Alpha Confers Methotrexate Resistance via Attenuation of Reactive Oxygen
Species Production and P53Mediated Apoptosis in Osteosarcoma Cells.BioMed
Research International)
A. urine excretion body total RNAs extraction
Trizol (Life Technology) fully cracking, homogenate are proportionally added into, chloroform is added and is stored at room temperature 10 points
Clock, takes supernatant, (Thermo Kingfisher Duo instruments, orthopedics and traumatology of Chinese medicine is real after 4 DEG C of 12000rpm centrifugations 15 minutes
Room is tested to have bought) paramagnetic particle method automatically extracts total serum IgE, the dissolving of DEPC water, -80 DEG C of preservations.
B. the concentration of total serum IgE, purity and integrity detection
I) method one：By RNA sample after melting on ice, centrifuge and fully mix, take appropriate amount of sample to add
Agilent2100 detected, RIN values >=7, rRNA Ratio [28s/18s] >=1, and the supreme lift of baseline, is high-quality RNA,
Storehouse sequencing is built available for follow-up RNA-seq.
Ii) method two：Take 2 μ L RNA solutions to add in Multiskan GO all band ELIASA μ Drop quartz plates to determine
OD260,0D280 and OD320 numerical value, OD260/OD280 ≈ 2, OD260/OD230 >=2, RNA purity, concentration are preferable；According to
The distribution and brightness of 28s, 18s and 5s under 1% agarose gel electrophoresis judge RNA integrality.
3.miRNAs builds storehouse, sequencing and data analysis and completed by Guangzhou Rui Bo bio tech ltd.Flow chart is sequenced
As shown in Figure 3.
Sequencing technologies of future generation (NGS) based on Illumina Hiseq2500 microarray datasets, for finding hormonal stock
The microRNA (s) of differential expression in head necrosis.
4. analyzing the above results using bioinformatics technique, the obvious potential mark of differential expression is obtained：Outside urine
Secrete body hsa-miR486-3p.
(2) real-time q-PCR verify potential mark hsa-miR486-3p
The urine of clinical patient is collected, is extracted after the total serum IgE in excretion body, according to Applied Biosystems companies
TaqMan MicroRNA Reverse Transcription Kit reagent require to carry out q-PCR operations, checking hsa-
Expressions of the miR486-3p after patient uses hormone, and adhere to MRI comparing, it is found that hsa-miR486-3p expression becomes
Steroid femur head necrosis can be found earlier than MRI by changing.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>The First Affiliated Hospital of Guangzhou University of Traditional Chinese Med
<120>A kind of urine excretion body hsa-microRNA486-3p and its application
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<223>Urine excretion body hsa-microRNA486-3p sequence
<400> 1
cggggcagcu caguacagga u 21
Claims (6)
1. a kind of urine excretion body hsa-microRNA486-3p, it is characterised in that：Described hsa-microRNA486-3p's
Sequence is：5′-CGGGGCAGCUCAGUACAGGAU-3′.
2. a kind of kit for being used to diagnose steroid femur head necrosis, it is characterised in that：Including the urine described in claim 1
Excretion body hsa-microRNA486-3p.
3. the urine excretion body hsa-microRNA486-3p described in claim 1 is in the reagent for preparing diagnosis caput femoris necrosis
Application.
4. application according to claim 3, it is characterised in that：Described urine excretion body hsa-microRNA486-3p exists
Prepare the application in the reagent of diagnosis steroid femur head necrosis.
5. application according to claim 3, it is characterised in that：Described urine excretion body hsa-microRNA486-3p exists
Prepare earlier than the application in the reagent of nuclear magnetic resonance diagnosis caput femoris necrosis.
6. the application according to claim 4 or 5, it is characterised in that：Described urine excretion body hsa-microRNA486-
Applications of the 3p in the reagent earlier than nuclear magnetic resonance diagnosis steroid femur head necrosis is prepared.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108410977A (en) * | 2018-05-22 | 2018-08-17 | 陈镇秋 | Alcoholic Femoral Head Necrosis patients serum's miRNAs extreme early detection kits |
| CN109652527A (en) * | 2018-12-13 | 2019-04-19 | 中国医学科学院北京协和医院 | A kind of kit of screening steroid femur head necrosis |
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| CN106701922A (en) * | 2016-12-05 | 2017-05-24 | 边焱焱 | Application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid |
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| CN106701922A (en) * | 2016-12-05 | 2017-05-24 | 边焱焱 | Application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410977A (en) * | 2018-05-22 | 2018-08-17 | 陈镇秋 | Alcoholic Femoral Head Necrosis patients serum's miRNAs extreme early detection kits |
| CN108410977B (en) * | 2018-05-22 | 2022-03-15 | 陈镇秋 | Ultra-early detection kit for serum miRNAs of alcoholic femoral head necrosis patient |
| CN109652527A (en) * | 2018-12-13 | 2019-04-19 | 中国医学科学院北京协和医院 | A kind of kit of screening steroid femur head necrosis |
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