CN106701922A - Application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid - Google Patents
Application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid Download PDFInfo
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Abstract
The invention discloses application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid. The invention discloses an early diagnostic kit for the femoral head necrosis caused by the glucocorticoid, and the kit comprises a reagent used for detecting the expression level of miR-16-5p. The invention further discloses medicine for treating the femoral head necrosis caused by the glucocorticoid, and the medicine contains an miR-16-5p inhibitor. When the miRNA is utilized to detect the femoral head necrosis, early detection can be quickly and effectively obtained; furthermore, a therapeutic target and an important basis are also provided for clinical application of gene therapy, drug therapy and the like.
Description
Technical field
The present invention relates to biomolecular science and medical domain, and in particular to utilize the microRNA related to caput femoris necrosis
As application of the molecular marked compound in the caput femoris necrosis for preventing or treating glucocorticoid to cause.
Background technology
Caput femoris necrosis is obstructed because of its main Department of Pathology's femoral head blood fortune, the head sclerotin ischemic for wrecking and causing,
Therefore it is referred to as avascular necrosis of femoral head or aseptic necrosis of head of femur more.Avascular necrosis of femoral head (avascular
Necrosis of the femoral head, ANFH) it is orthopaedics common disease, frequently-occurring disease, early diagnosis is difficult, and therapeutic effect is owed
It is good.Long-term, high-dose application hormone causes avascular necrosis of femoral head, as clinical common drug induced disease.Reported according to the country
Road, it is 20%, the incidence of disease of heavy dose of short application use that low dose of prolonged application glucocorticoid causes the incidence of disease of caput femoris necrosis
It is 40% or so.
Caput femoris necrosis pathology mechanism is complicated, it is characterized in that because blood circulation disorder causes ANFH, due to
Body has nature repair ability to necrotic area, when newborn osteocyte grows with new vessels to necrotic area and forms the same of new bone
When, downright bad bone trabecula will progressively be absorbed.The mechanical property of bone substantially weakens in the process, and normal heavy burden can cause femoral head
Collapse, joint space narrows, finally result in osteoarthritis, make patient's hip joint function obstacle and paralysis of disabling.Femoral head pulp cavity
Interior bone marrow steatosis are steroid femur head necrosis (Steroid induced Avascular Necrosis of Femoral
Head, SANFH) early stage important pathological change.Recent study proves that hormone can cause marrow stromal cell to be largely divided into
Fat cell, causes marrow fat cell hypertrophy to pile up, and causes iop to increase, and makes microcirculation disorder in femoral head and causes a large amount of bones
Cell ischemic necrosis.Gegenbaur's cell is the chief functional cells of bon e formation.There is dynamic change with the age in the development of bone, its
In the bon e formation that is guided by Gegenbaur's cell and the dynamic equilibrium of bone remoulding is constituted by the bone information that osteoclast is guided.Work as skeletonization
During cell prosoplasia, the generation of corresponding bone metabolic disease, such as osteoporosis, caput femoris necrosis, joint degeneration are may result in
Change, caput femoris necrosis etc..
MicroRNA (miRNAs) is that a class is about 22~25 endogenous non-coding single strand RNA molecules of nucleotides, can
With reference to the 3 ' noncoding regions (3 ' TTR) of target gene by way of base pair complementarity, so as to suppress to translate or degrade target gene.
MicroRNA is highly conserved during evolution, widely distributed in vivo, and participates in the biological process of many complexity, such as
Embryonic development, cell propagation, Apoptosis, fat metabolism, bon e formation etc..There are some researches show：MiRNAs is by regulating and controlling skeletonization point
Change related gene expression to be played a crucial role in bon e formation, such as miR-216 promotes the mescenchymal stem cell of Human Adipose Tissue-derived
To Osteoblast Differentiation, miR-494 by adjusting the expression inhibiting osteoblast differentiation etc. of Runx2.
The diagnosis and treatment of early stage are the keys for treating SANFH.With continuing to develop for bioinformatics, more and more
The gene related to SANFH is seen in report, it was confirmed that several genes, in the developing effect of generation of ANFH, are the morning of SANFH
Phase diagnoses and treatment provides theoretical foundation.
Therefore this area is examined in the urgent need to carrying out the specific miRNA being associated with steroid femur head necrosis as early stage
The research of disconnected label, to provide new effective prevention and treatment target spot.
The content of the invention
In order to realize the early detection of the caput femoris necrosis that glucocorticoid causes, early intervention, the purpose of the present invention exists
In a kind of new miRNAs related to the caput femoris necrosis that glucocorticoid causes of offer.
Draw in prevention or treatment glucocorticoid the present invention also aims to provide caput femoris necrosis correlation miRNAs
Application in the caput femoris necrosis for rising.
To achieve the above object, present invention firstly provides a kind of molecule for detecting the caput femoris necrosis that glucocorticoid causes
Label, the molecular marked compound is miR-4289, miR-378g, miR-378f, miR-378d, miR-196b-5p, miR-
One or more in 196a-5p, miR-16-5p, miR-1268b, miR-1268a, miR-107 and miR-103a-3p
miRNAs。
The present invention is further study show that the Patients with Aseptic Necrosis of Femoral that causes in glucocorticoid of described molecular marked compound
Up-regulated in biological sample.
Further, the invention provides described molecular marked compound in the femur for preventing or treating glucocorticoid to cause
Application in head necrosis.
Preferably, the glucocorticoid includes：Dexamethasone, betamethasone, metacortandracin, prednisolone, methylprednisolone,
By Anxi dragon, hydrocortisone and cortisone.
Further, the invention provides a kind of medicine for treating the caput femoris necrosis that glucocorticoid causes, its feature
It is that described medicine includes miR-16-5p inhibitor.
Preferably, the miR-16-5p inhibitor is the ASON or antagonist of miR-16-5p, described
The Antisensedigonucleotsequence sequence of miR-16-5p is SEQ ID NO.4.
Preferably, described medicine can suppress the apoptosis of mesenchymal stem cells MSCs.
Further, the invention provides a kind of reagent for diagnosing the caput femoris necrosis that glucocorticoid causes, the examination
Agent box includes the reagent of the expression for detecting miR-16-5p.
Preferably, described reagent includes the primer or probe of specific amplification miR-16-5p.
Preferably, described primer sequence includes forward primer sequence as shown in SEQ ID NO.1 and reverse primer is logical
Use primer.
Beneficial effects of the present invention are as follows：
The invention discloses the molecular marked compound related to the caput femoris necrosis that glucocorticoid causes, and it is further characterized by
Molecular marked compound up-regulated in the Patients with Aseptic Necrosis of Femoral biological sample that glucocorticoid causes.Using these points
The caput femoris necrosis that son mark analyte detection glucocorticoid causes can not only fast and effectively accomplish early detection, and be base
Because the clinical practices such as treatment, drug therapy provide therapy target and important evidence.
Brief description of the drawings
Fig. 1 miR-16-5p expressions in the MSCs that various concentrations dexamethasone stimulates.
Influence of Fig. 2 miR-16-5p inhibitor to MSCs Apoptosis.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, reagent used can be commercially available.
The experimental technique of unreceipted actual conditions in embodiment, usually this area conventional method, such as according to normal condition
Such as Sambrook et al., molecular cloning, the condition in laboratory manual (third edition) (Science Press, 2002), or according to
Condition proposed by reagent manufacturing firm.
The present inventor carries out high-flux sequence to 25 Patients with Aseptic Necrosis of Femoral samples and 22 check samples, knot
Closing bioinformatics method carries out the screening of differential expression miRNA, then carries out GO functions enrichment conjunction and signal path analysis, chooses
The miRNAs for having selected differential expression to raise：miR-4289、miR-378g、miR-378f、miR-378d、miR-196b-5p、miR-
196a-5p, miR-16-5p, miR-1268b, miR-1268a, miR-107 and miR-103a-3p.On not having in existing research
The miRNAs reports related to caput femoris necrosis is stated, further, inventor has carried out molecular biology method checking, using RT-
PCR method detects expression of the above-mentioned miRNAs in the MSCs that various concentrations dexamethasone stimulates, and demonstrates above-mentioned miRNAs
The up-regulated in the MSCs that high concentration dexamethasone stimulates.Additionally demonstrating miR-16-5p can cause Apoptosis, its
Related preparations can be used to treat the caput femoris necrosis that glucocorticoid causes.
When " caput femoris necrosis " used herein is not explained, hormonal ischemic necrosis of femoral head is typically refered in particular to.
" glucocorticoid " used herein be by a class steroid hormone of adrenocortical secretion, at present also can be by chemistry
Method is artificial synthesized.Glucocorticoid can be used for the illness less than general antibiotic or antiphlogistic, such as SARS, septicemia
Deng the effect with regulation sugar, fat, the biosynthesis of protein and metabolism, also with antiinflammatory action.Representational sugared cortex
Hormone example includes (but being not limited to):Dexamethasone, betamethasone, metacortandracin, prednisolone, methylprednisolone, by Anxi dragon,
Hydrocortisone and cortisone.One class to the use of the related common adverse effect of glucocorticoid medicine is that can cause unfavorable bone
Disease, including (but being not limited to):Osteoporosis, bone marrow steatosis, caput femoris necrosis.
" dexamethasone " used herein is a kind of artificial synthesized cortex hormone of aadrenaline, and its derivative has the hydrogenation can ground
Pine, metacortandracin etc., with anti-inflammatory, antiendotoxin, suppress the pharmacological action such as immune, Hemorrhagic shock and enhancing stress reaction, extensively should
Various diseases, such as autoimmune disease, allergy, inflammation, asthma, and dept. of dermatology, ophthalmology disease are treated for each section.
The pharmacological action of dexamethasone mainly includes:Antiinflammatory action and immunosuppressive action.Antiinflammatory action shows, mitigates
With prevent reaction of the tissue to inflammation, suppress macrophage and leucocyte gathering in inflammation part, suppress phagocytosis, lyase
The release of body enzyme and the synthesis and release of inflammation chemistry intermediary, mitigate and prevent reaction of the tissue to inflammation, so as to mitigate
The performance of inflammation.Its immunosuppressive action shows:Cell-mediated immune response is prevented or suppresses, the allergy of retardance is anti-
Should, T lymphocytes, monocyte, the number of acidophic cell are reduced, reduce the combination of immunoglobulin and cell surface receptor
Ability, suppresses the synthesis and release of interleukin, reduces T lymphocytes to lymphoblastic transformation, and through primary immune response
Extension；Immune complex can be reduced by basilar memebrane, and the concentration of Complement and immunoglobulin can be reduced.Clinical discovery,
Long-term use dexamethasone (Dex) can cause serious caput femoris necrosis.
" mesenchymal stem cells MSCs " used herein and " BMSCs " implication are identical, can be with used interchangeably.
Mesenchymal stem cells MSCs is a kind of mescenchymal stem cell being present in marrow, with orientation or Multidirectional Differentiation
Potential.Gegenbaur's cell and fat cell all derive from mesenchymal stem cells MSCs (BMSCs), and the differentiation of the two has one kind
Emulative balance, thus occur substantial amounts of fat prompting BMSCs steering Adipocyte Differentiations in caput femoris necrosis patient's marrow
And Osteoblast Differentiation is suppressed.BMSCs has powerful self duplication ability and differentiation multipotency, can be by adjusting to skeletonization
Direction breaks up.The differentiation direction of BMSCs is subject to the strict regulation and control of transcription factor, and Runx2 is the key for starting osteoblast differentiation
Transcription factor.
Medicine of the invention can as needed be prepared into various formulations.Including but not limited to, percutaneous, mucous membrane, buccal, sublingual
Or oral tablet, solution, granule, patch, paste, capsule, aerosol or the suppository for using.
The route of administration of medicine of the invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to orally, intravenous injection, intramuscular injection, hypodermic injection, sublingual administration, rectal perfusion, nasal spray, mouth
Chamber spraying, local skin or whole body transdermal administration.
The dosage of medicine of the invention can change with mode of administration and disease degree to be treated etc., can be according to disease
The appropriate determination such as shape, sex, age.The dosage of preferred medicine of the invention or prophylactic agent can be used to disease
The therapeutic effect or preventive effect of disease determine as index.
" biological sample " used herein includes but is not limited to blood, serum, saliva, urine, synovia, cartilage, synovial tissue
Deng sample.Any tissue sample (such as interverbebral disc, articular process, the ridge of any subject are derived from for biological sample of the invention
Marrow, paraspinal muscle or blood sample) or cell sample (such as Gegenbaur's cell, cartilage cell or blood cell samples) can be according to present invention side
Method is used.These samples can therefrom be obtained and included but do not limited using subject's example of these samples according to the inventive method
Subject, clinical diagnosis in asymptomatic subject, performance or more caput femoris necrosis symptom are receiving with caput femoris necrosis
Examination person, the subject for being susceptible to suffer from caput femoris necrosis (if any the subject of caput femoris necrosis family history, have caput femoris necrosis inheritance susceptible
Property subject and life style make its susceptible caput femoris necrosis or raising suffer from caput femoris necrosis possibility subject), bosom
Doubt with caput femoris necrosis subject, just receiving the subject of femoral head necrosis therapeutic, with caput femoris necrosis and non-receiving
The subject of femoral head necrosis therapeutic, practitioner (such as doctor) are defined as health or the subject without caput femoris necrosis (i.e. just
Often), the subject that has cured caput femoris necrosis, the subject for controlling its caput femoris necrosis and caput femoris necrosis is had not been diagnosed as
Subject.
" control " used herein refers to and does not show any caput femoris necrosis symptom (including traumatic property caput femoris necrosis and non-
Traumatic femoral head necrosis) and have not been diagnosed as caput femoris necrosis individuality or group of individuals.Preferably, the control individuality does not make
With the medicine of influence caput femoris necrosis.It is highly preferred that control is individual to have similar sex compared with test sample, age and body
Weight index (BMI).According to the present invention, " control " also refers to the sample for being isolated from normal individual, including is isolated from the total of normal individual
RNA or mRNA.Taking from the individual sample of control may include to be isolated from the RNA of sample tissue, and wherein RNA is isolated from sample tissue,
The sample tissue separates when self-organizing is removed and has not been diagnosed as caput femoris necrosis and do not show any caput femoris necrosis symptom
It is individual.
It is preceding being used according to the inventive method, optionally (but preferably) biological sample of collection is stored in low temperature such as 4
At DEG C.In some embodiments, the very first time according to the inventive method using sample a part, and by one or more
Remaining sample part preserves a period of time for future use.This time can be 1 hour or longer, 1 day or longer, 1 week or
Longer, January or it is longer, 1 year it is longer or uncertain.For long-term preservation, it is possible to use store method well known in the art,
For example preserved in cryogenic temperature (as being less than -80 DEG C).
The high-flux sequence of embodiment 1 screens difference expression gene
1st, sample
The Patients with Aseptic Necrosis of Femoral myeloid tissue sample of 25 customary total hip replacement operations is collected, sampling derives from 2012
The patient gone to a doctor in BJ Union Hospital's orthopaedics during October in December, 2015 in year, all patients are diagnosed as by long-term or big
Dose application glucocorticoid causes avascular necrosis of femoral head.The comminuted bone of hip joint that control is in hospital from same time orthopaedics
Myeloid tissue sample at the hip joint of patient is rolled over, 22 are collected altogether.The sample of all research objects is gathered, is numbered rearmounted -80 DEG C
Low temperature refrigerator is preserved.All clinical samples of this research, to patient know the inside story and inform and logical through this Hospital Ethical Committee
Cross.
2nd, Total RNAs extraction is carried out to tissue samples
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation
Carried out by product description, concrete operations are as follows：
Frozen after collection sample and be ground in tissue is put into the mortar of precooling after liquid nitrogen, taking-up, sample to be organized
After this is powdered：
1. Trizol, room temperature preservation 5 minutes are added；
2. the imitative 0.2mL of chlorination, uses forced oscillation centrifuge tube, fully mixes, and places -10 minutes 5 minutes at room temperature；
3. 12000rpm high speed centrifugations draw upper strata aqueous phase (inhale 70%) in another new centrifuge tube pipe after 15 minutes, note
The protein substance between two-layer water phase should not be drawn onto.New pipe is moved into, -20 DEG C of isometric pre- cold isopropanols are added, it is fully reverse
Mix, be placed in 10 minutes on ice；
4. 12000rpm carefully discarded supernatant after 15 minutes at a high speed, and 75% is added in the ratio of I mL/mL Trizol
DEPC ethanol washes paint precipitation (4 DEG C of preservations), washes paint sediment, vibration mixing, 12000rpm high speed centrifugations 5 minutes at 4 DEG C；
5. ethanol liquid is discarded, 5 minutes is placed at room temperature fully to dry precipitation, add the treated water dissolves of DEPC to sink
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometers, is frozen in -80 DEG C.RNA mass is sentenced
Calibration is accurate：The OD260/OD280 values of RNA samples are between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S band；
70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern and water-bath insulation before collection of illustrative plates no significant difference.
4th, high-flux sequence
Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of IllTmina companies, carries out high flux transcript profile depth
Sequencing, we use Fast-QC (http after sequencing://www.bioinformatics.babraham.ac.Tk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, including base quality Distribution value, the position point of mass value
Cloth, G/C content, PCR dTplication contents, freqTency of kmer etc..In differential genes expression analysis, according to obtaining
FPKM values, differential screening is carried out using internationally recognized algorithm EBSeq.Wherein, during screening, LOG2FC>1 or<- 1, FDR<
0.05.In order to be better understood from the function of difference expression gene, we difference expression gene has been carried out Gene Ontology and
Signal path is analyzed, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of above number
According to the result of analysis, with reference to document, we have screened differential expression miRNAs:miR-4289、miR-378g、miR-378f、miR-
378d, miR-196b-5p, miR-196a-5p, miR-16-5p, miR-1268b, miR-1268a, miR-107 and miR-103a-
3p, the miRNAs up-regulated in Patients with Aseptic Necrosis of Femoral tissue samples.
The cell has-miR-16 expressions of the RT-PCR of embodiment 2 detection various concentrations dexamethasone treatment
1st, sample
The Bone Marrow of Patients sample of 3 customary total hip replacement operations is collected, sampling derives from October, 2012 to 2015
The Hip osteoarthritis patient gone to a doctor in BJ Union Hospital's orthopaedics during December.The sample of all research objects is gathered,
Rearmounted -80 DEG C of low temperature refrigerators of numbering are preserved.All clinical samples of this research, to patient know the inside story and inform and through this hospital
Ethics Committee passes through.
2nd, the separation of mesenchymal stem cells MSCs and culture
(1) myeloid tissue of femur side seam is extracted using aseptic marrow puncture needle and syringe.
(2) in myeloid tissue test tube of the immigration containing culture medium and anticoagulant heparin agent (4000T/ml) that will be extracted, with etc.
The PBS of volume is diluted.30s is stood, 1.077 μ g/ml lymphocyte separation mediums of addition equivalent after precipitation are discarded, with
778xg is centrifuged 20 minutes, in white intermediate layer collecting monocytic cell.
(3) monocyte is washed with 10ml D-Hanks liquid, and 280xg is collected after being centrifuged 6 minutes.Add containing 58% DMEM
DMEM/F12 nutrient solutions, 40%MCDB-201,2% hyclone, 10ng/ml EGF, 10ng/ml PDGF, 1X insulin-
Transferrins-selenium additive, 1X linoleic acid-bovine serum albumin(BSA), 50 μM of beta -mercaptoethanols, 2mM Glus, 100 μ g/
Ml penicillin and 100 μ g/ml streptomycin sulphate cultures, cell density is 2x106/ml。
(4) nutrient solution is changed within every 2 days, after cell confluency is in blocks and adherent rate reaches 70-80%, plus 0.25% pancreas
Protease and 0.01% EDTA are made cell suspension, then according to 1:3 ratio carries out Secondary Culture.
(5) when culture is to the third generation, by MSCs with 2 × 104/cm2It is inoculated in 25CM2(T25) in blake bottle.When they reach
To 70%-80% when converging rate, osteogenic induction nutrient solution is added：Dexamethasone containing 10% hyclone, 10nm, 0.2mM
The H-DMEM culture mediums of ascorbic acid and 10mM sodium β-glycerophosphates.The concentration of dexamethasone is respectively adopted 10-7Mol/L and 10- 9Two kinds of concentration packets of mol/L are cultivated, and change a nutrient solution within every 2 days.
3rd, RT-PCR detections
In 6 days after osteogenic induction, using in the MSCs after RT-PCR two groups of various concentrations dexamethasone stimulation process of detection
The expression of has-miR-16.
3.1 Total RNAs extractions
RNA extractions are carried out with the method for embodiment 1.
3.2 reverse transcriptions
Use One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Code
No.D350A reverse transcription) is carried out, experimental implementation is carried out by product description, using 20 μ L reaction systems, it is total that each sample takes 1 μ g
RNA is used as template.It is standby that -20 DEG C of refrigerators are put in the cDNA preservations of acquisition.It is standby that -20 DEG C of refrigerators are put in the cDNA preservations of acquisition.
3.3 Real-Time PCR
Expanded using SYBR PrimeScipt miRNA RT-PCR Kit (Takara, Code No.RR716), it is real
Test operation is carried out by product description.Reaction system is as shown in table 1.
The RT-PCR reaction systems of the miRNA of table 1
| Component | Addition |
| SYBR Premix Ex TaqII(2×) | 10μL |
| PCR Forward Primer(10μM) | 0.5μL |
| Tni-miR qPCR Primer(10μM) | 0.5μL |
| ROX Reference Dye II(50×) | 2μL |
| Template (cDNA solution) | 2μL |
| dH2O | To 20 μ L |
The detection of expression of miRNAs sets 3 parallel tube reactions every time.Amplification miR-16-5p forward primers sequence such as SEQ
Shown in ID NO.1, using snRNA T6 as internal reference, its sense primer is that shown in SEQ ID NO.2, anti-sense primer is SEQ ID
Shown in NO.3.Amplification program：95℃30s；40x (95 DEG C of 5s, 60 DEG C of 34s).
3.4 experimental results
With the type quantitative real time PCR Instruments of ABI 7500, using 2-△△CT methods carry out the relative quantitative assay of data.Real-time quantitative
PCR amplification curve flex points understand, amplification curve entirety collimation is good, shows that the amplification efficiency of each reaction tube is close, the limit it is flat and
Without raising up now, exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production solubility curve be all it is unimodal,
Illustrate that amplified production only has one, be specific amplification；Relative quantification formula according to qRT-PCR：2-△△Ct × 100%, than
Compared with has-miR-16 10-7With 10 in the MSCs that mol/L dexamethasone stimulates-9Table in the MSCs that mol/L dexamethasone stimulates
Up to level.Result shows：MiR-16-5p is 10-7Up-regulated in the MSCs that mol/L dexamethasone stimulates, as shown in Figure 1.
Embodiment 3 suppresses miR-16-5p expression
1st, cell derived
The mesenchymal stem cells MSCs of the culture of embodiment 2.
2nd, ASON (anti-miR-16-5p) of the design synthesis for miR-16-5p
Sequence information according to miR-16-5p synthesizes miR-16-5p by the design of Dalian treasured biotinylated biomolecule Technology Co., Ltd.
Antisensedigonucleotsequence sequence and random controls sequence, the Antisensedigonucleotsequence sequence of miR-16-5p is SEQ ID NO.4.
3rd, transfect
Transiently transfected using cationic-liposome method, operated according to LipofectamineTM2000
Transfection Reagent reagents specifications are carried out.The good cell of growth conditions is inoculated into 6 orifice plates by 24h before transfection
In, cell count about 5 × 104, cellar culture is tested to the same day, cell fusion degree is transfected when being 50-60%.By 80nM
Anti-miR-16-5p is added in 100TL DMEM culture mediums, soft to mix；Another 100TL DMEM culture mediums dilute 2TL
LipofectaminTM2000 liposomes, it is soft to mix, it is incubated at room temperature 5min；Mixing DMEM- liposomes and DMEM-miRNAs,
Incubation at room temperature 20min, to form transfection composite；Then said mixture is added in cell culture medium, is gently mixed, make him
Be sufficiently mixed.Wherein, used as negative control (anti-NC), the same period is provided with blank control group to non-specific sequences.Culture 48h
Extracting cell total rna afterwards carries out next step experiment.
4th, anti-miR-16-5p is for effect that mesenchymal stem cells MSCs miR-16-5p is expressed：
Cell total rna is extracted and PCR steps are with embodiment 2.
Real-time PCR results show that negative control group is to miR-16-5p expression in mesenchymal stem cells MSCs without bright
Aobvious inhibitory action, and blank control group no difference of science of statistics, the anti-miR-16-5p groups of transfection are to mesenchymal stem cells MSCs
It is 54% that certain inhibitory action inhibiting rate is played in middle miR-16-5p expression.
Influence of the miR-16-5p inhibitor of embodiment 4 to apoptosis of mesenchymal stem cell
Method according to embodiment 3 carries out the transfection of anti-miR-16-5p and anti-NC to mesenchymal stem cells MSCs.
After cell transfecting 48h, by collected by trypsinisation (note of the cell without EDTA：Pancreatin digestion time be difficult it is long, otherwise easily
Cause false positive)；1~5 × 10 are collected with PBS washed cells secondary (2000rpm is centrifuged 5min)5Cell；According to Annexin
The operating instruction of the double transfection reagent boxes of V-FITC/PI (being purchased from Invitrogen companies) carries out cell treatment.Add 500 μ L's
Binding BTffer (Kai Ji, Nanjing) suspension cell；After adding 5 μ LAnnexin V-FITC (Kai Ji, Nanjing) to mix, add
5 μ LPropidiTm Iodide (Kai Ji, Nanjing), mix；The inspection of flow cytometer is carried out after room temperature, lucifuge, reaction 5-15min
Survey.Statistics apoptosis rate, experiment is all completed according to being repeated 3 times, and result data is all with mean+SD
Mode represents that using SPSS19.0 statistical softwares carry out statistical analysis, difference between the two is checked using t, it is believed that
Work as P<There is statistical significance when 0.05.
Result is as shown in Fig. 2 compared with anti-NC groups of cells is transfected, transfect the apoptosis rate of anti-miR-16-5p
Reduce, difference has statistical significance (P<0.05).
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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<120>Applications of the MicroRNA in the caput femoris necrosis for preventing or treating glucocorticoid to cause
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Claims (10)
1. it is a kind of to detect the molecular marked compound of caput femoris necrosis that glucocorticoid causes, it is characterised in that the molecular labeling
Thing be miR-4289, miR-378g, miR-378f, miR-378d, miR-196b-5p, miR-196a-5p, miR-16-5p,
One or more miRNAs in miR-1268b, miR-1268a, miR-107 and miR-103a-3p.
2. molecular marked compound as claimed in claim 1, it is characterised in that one or more described molecular marked compounds are in sugared skin
Up-regulated in the Patients with Aseptic Necrosis of Femoral biological sample that matter hormone causes.
3. molecular marked compound as claimed in claim 1 or 2 is in the caput femoris necrosis for preventing or treating glucocorticoid to cause
Application.
4. application as claimed in claim 3, it is characterised in that the glucocorticoid includes：Dexamethasone, betamethasone,
Metacortandracin, prednisolone, methylprednisolone, by Anxi dragon, hydrocortisone and cortisone.
5. a kind of medicine for treating the caput femoris necrosis that glucocorticoid causes, it is characterised in that described medicine includes miR-
16-5p inhibitor.
6. medicine as claimed in claim 5, it is characterised in that the miR-16-5p inhibitor is that the antisense of miR-16-5p is few
Nucleotides or antagonist, the Antisensedigonucleotsequence sequence of described miR-16-5p is SEQ ID NO.4.
7. the medicine as described in claim 5 or 6, it is characterised in that described medicine can suppress mesenchymal stem cells MSCs
Apoptosis.
8. it is a kind of to diagnose the kit of caput femoris necrosis that glucocorticoid causes, it is characterised in that the kit includes using
In the reagent of the expression of detection miR-16-5p.
9. the kit as described in right wants 8, it is characterised in that described reagent includes drawing for specific amplification miR-16-5p
Thing or probe.
10. the kit as described in right wants 9, it is characterised in that described primer sequence includes forward primer sequence such as SEQ
ID NO.1 are shown and reverse primer is universal primer.
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| CN201611102118.0A CN106701922A (en) | 2016-12-05 | 2016-12-05 | Application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190066A (en) * | 2017-06-07 | 2017-09-22 | 广州中医药大学第附属医院 | A kind of urine excretion body hsa microRNA486 3p and its application |
| 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 |
| CN109762895A (en) * | 2019-01-07 | 2019-05-17 | 中国医学科学院北京协和医院 | Application of the miR-146a in preparation diagnosis steroid femur head necrosis product |
| CN112280742A (en) * | 2020-11-05 | 2021-01-29 | 北京欣颂生物科技有限公司 | Anti-aging pharmaceutical composition or health product prepared from stem cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104667294A (en) * | 2013-11-29 | 2015-06-03 | 中国医学科学院基础医学研究所 | Application of miR-216 in preparation of medicines for promoting osteogenesis differentiation |
| CN106011264A (en) * | 2016-06-30 | 2016-10-12 | 西北工业大学 | Application of miR-489-3p to preparation of medicines for diagnosing and treating human osteoporosis |
-
2016
- 2016-12-05 CN CN201611102118.0A patent/CN106701922A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104667294A (en) * | 2013-11-29 | 2015-06-03 | 中国医学科学院基础医学研究所 | Application of miR-216 in preparation of medicines for promoting osteogenesis differentiation |
| CN106011264A (en) * | 2016-06-30 | 2016-10-12 | 西北工业大学 | Application of miR-489-3p to preparation of medicines for diagnosing and treating human osteoporosis |
Non-Patent Citations (2)
| Title |
|---|
| BIAN Y.等: "Pathogenesis of glucocorticoid-induced avascular necrosis:A microarray analysis of gene expression in vitro", 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》 * |
| KRICHEVSKY A.M.等: "A microRNA array reveals extensive regulation of microRNAs during brain development", 《RNA》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190066A (en) * | 2017-06-07 | 2017-09-22 | 广州中医药大学第附属医院 | A kind of urine excretion body hsa microRNA486 3p and its application |
| 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 |
| CN109762895A (en) * | 2019-01-07 | 2019-05-17 | 中国医学科学院北京协和医院 | Application of the miR-146a in preparation diagnosis steroid femur head necrosis product |
| CN112280742A (en) * | 2020-11-05 | 2021-01-29 | 北京欣颂生物科技有限公司 | Anti-aging pharmaceutical composition or health product prepared from stem cells |
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