CN105400893A - Intervertebral disc degenerative change miRNA marker - Google Patents

Intervertebral disc degenerative change miRNA marker Download PDF

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CN105400893A
CN105400893A CN201510983002.1A CN201510983002A CN105400893A CN 105400893 A CN105400893 A CN 105400893A CN 201510983002 A CN201510983002 A CN 201510983002A CN 105400893 A CN105400893 A CN 105400893A
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microrna
mirna
mir
cell
intervertebral disk
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杨承刚
边洋
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Beijing Medintell Bioinformatic Technology Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention discloses application of miR-1288 as an intervertebral disc degenerative change diagnosis and treatment marker. The miR-1288 is used for developing a product for diagnosing the intervertebral disc degenerative change and developing a drug for treating the intervertebral disc degenerative change. The research result provides a theoretic basis for clinicians to formulate an individual-based treatment scheme, and can provide a new drug target for developing the drug for the intervertebral disc degenerative change.

Description

Intervertebral disk retrogression pathology miRNA marker
Technical field
The invention belongs to biomedicine field, relate to the purposes of miR-1288 in diagnosis and treatment intervertebral disk retrogression pathology.
Background technology
MiRNA is the non-coding RNA molecule of the natural 21-22nt be present in body, is the RNA that a class is regulated expression of target gene by PTGS.According to estimates, the gene of 1/3 is about had in organism by the regulation and control of miRNA.The complex body of miRNA and RISC can be combined with the complementary sequence in target gene mRNA5 '-UTR or 3 '-UTR by base pairing, and arrestin matter is translated, or causes mRNA degraded, thus the expression of negative regulation target gene.
The expression level detecting miRNA can provide reference for the clinical diagnosis of disease.And the unconventionality expression of miRNA directly causes the abnormal expression of some and disease generation genes involved, the generation induced an illness.Have been reported and prove that miRNA by the expression of regulation and control target gene mRNA, can occur in disease, develops and play a significant role in transfer.In future clinical treatment, miRNA not only can become the new disease early diagnosis marker relevant with disease process, and is expected to by the change expression of miRNA or the expression treatment disease of its target gene.Find and identify that the clinical treatment that relevant miRNA and target gene thereof are miRNA occurs to disease provides basic.
Summary of the invention
An object of the present invention is to provide a kind of Microrna marker that can be used for early diagnosis intervertebral disk retrogression pathology.
Two of object of the present invention is the purposes providing above-mentioned Microrna.
To achieve these goals, present invention employs following technical scheme:
The invention provides a kind of Microrna and preparing the application in intervertebral disk retrogression pathological changes diagnosis instrument, described Microrna is selected from following group: initial miRNA, precursor miRNA, ripe miRNA; Initial miRNA can be sheared and be expressed as ripe miRNA in people's cell; Precursor miRNA can be sheared and be expressed as ripe miRNA in people's cell; Described Microrna is miR-1288.
It should be known that Microrna of the present invention comprises the function equivalent of composing type nucleic acid molecule, i.e. variant, it shows the identical function of complete Microrna nucleic acid molecule, although they are suddenlyd change by the disappearance of nucleotide residue, displacement or insertion.
Those skilled in the art should understand, and in order to ensure the stability of Microrna, can increase protectiveness base, as TT, also can modify Microrna base, but above-mentioned modification does not affect the function of Microrna in one end of Microrna or two ends.Therefore, those skilled in the art know, and under the condition not affecting miR-1288 function, carry out base modification or be included in equally within protection scope of the present invention in the sequence that two ends increase base obtains miR-1288.
In concrete embodiments more of the present invention, described miR-1288 is ripe miR-1288.
Although the ripe miRNA that uses in some embodiment, but those skilled in the art it is expected to, initial miRNA, precursor miRNA can obtain the technique effect same with ripe miRNA, because cell has the ability further initial miRNA, precursor miRNA to be processed as ripe miRNA.
Microrna nucleic acid molecule of the present invention can exist with the form of strand or double-strand.Ripe miRNA is mainly in single stranded form, and precursor miRNA is part complementation certainly, to form duplex structure.Nucleic acid molecule of the present invention can be the form of RNA, DNA, PNA, LNA.
Further, above-mentioned diagnostic tool includes but not limited to, chip, test kit, test paper, high-flux sequence platform.Described diagnostic tool comprises the reagent of the expression level for detecting miR-1288.
Further, described test kit comprises primer for miR-1288 and/or probe; Described chip comprises solid phase carrier; And the oligonucleotide probe be fixed on described solid phase carrier, described oligonucleotide probe comprises the part or all of sequence corresponding to miR-1288 specifically; Described test paper comprises primer for miR-1288 and/or probe; Described high-flux sequence platform comprises primer for miR-1288 and/or probe.
The invention provides a kind of diagnostic tool of intervertebral disk retrogression pathology, described diagnostic tool comprises the reagent detecting miR-1288 expression level.
Further, described diagnostic tool comprises test kit, chip, test paper, high-flux sequence platform.
Further, described test kit comprises primer for miR-1288 and/or probe; Described chip comprises solid phase carrier; And the oligonucleotide probe be fixed on described solid phase carrier, described oligonucleotide probe comprises the part or all of sequence corresponding to miR-1288 specifically; Described test paper comprises primer for miR-1288 and/or probe; Described high-flux sequence platform comprises primer for miR-1288 and/or probe.
Further, the primer for miR-1288 in described test kit and/or probe also can comprise the primer and/or the probe that can be used for detecting foregoing microrna expression level for having reported in prior art.The detection primer of multiple Microrna and/or probe are placed in same reagent box and are also contained within protection scope of the present invention by the situation detecting multiple Microrna index Combining diagnosis intervertebral disk retrogression pathology.
Further, fixing on described chip described oligonucleotide probe also can comprise the oligonucleotide probe that can be used for the expression level detecting miR-1288 for having reported in prior art.The detection probes of multiple miRNA is placed and is also contained within protection scope of the present invention by detecting multiple miRNA index Combining diagnosis intervertebral disk retrogression pathology on the same chip.
Further, described solid phase carrier comprises the various common used materials that described solid phase carrier can adopt gene chip field, such as but not limited to nylon membrane, the slide, plastic sheet etc. of the slide modified through active group (as aldehyde radical, amino etc.) or silicon chip, unmodified.
The preparation of described miRNA chip can adopt the common manufacturing method of biochip known in the art, such as, if what solid phase carrier adopted is modify slide or silicon chip, 5 ' end of probe is containing amido modified poly-dT string, oligonucleotide probe can be mixed with solution, then employing point sample instrument is by its point on modification slide or silicon chip, is arranged in predetermined sequence or array, then spent the night by placement and fix, just can obtain miRNA chip of the present invention.If nucleic acid is not containing amido modified, then its preparation method also can refer to: " the gene diagnosis technology-on-radiation operational manual " of Wang Shenwu chief editor; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploringthemetabolicandgeneticcontrolofgeneex pressiononagenomicscale.Science, 1997; 278:680 and Ma Li people, Jiang Zhonghua edits. biochip. and Beijing: Chemical Industry Press, 2000,1-130.
MiR-1288 of the present invention can be natural or synthetic, or uses the vector-transfected cell can expressing the DNA fragmentation of miR-1288 to obtain.Described carrier comprises virus vector, eukaryotic vector.
Virus vector can be any suitable carrier, includes but not limited to retroviral vector, adenovirus carrier, adeno-associated virus (AAV) carrier, simplexvirus (such as hsv, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any suitable expression vector, include but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEFBos expression vector, pTet expression vector, pTRE expression vector or the carrier through transforming on the basis of known expression vector, such as pBin438, pCAMBIA1301 etc.
The DNA fragmentation can expressing Microrna can obtain in the following way: find the position of Microrna on genome and concrete sequence information from (http://microrna.sanger.ac.uk/sequences/) miRNA database, the position of initial miRNA is determined according to genome sequence, in the upstream and downstream 500-800bp interval of initial miRNA position, design Auele Specific Primer, the sequence in the middle of amplimer can obtain the DNA fragmentation of expressing Microrna.
Drug screening: after the Close relation obtaining the foregoing miR-1288 of cicada and intervertebral disk retrogression pathology, can screen based on this feature the material promoting that miR-1288 expresses.Afterwards, can find from described material for the really useful medicine for the treatment of intervertebral disk retrogression pathology.
Therefore, present invention also offers a kind of method of screening the potential material for the treatment of intervertebral disk retrogression pathology, described method comprises: by candidate substances process intervertebral disk retrogression pathology relevant cell system, if described candidate substances can promote expression or the activity of foregoing miR-1288, then show that this candidate substances is the potential material for the treatment of intervertebral disk retrogression pathology.Described cell system can be ubcellular system, solution system, organizational framework, organ systems or animal system (as animal model, the animal model of preferred non-human mammal, as mouse, rabbit, sheep, monkey etc.) etc.Preferably, further cell experiment and/or animal experiment are carried out to the potential material obtained, to select further and to determine for the really useful material for the treatment of intervertebral disk retrogression pathology.
Present invention also offers the application of miR-1288 in the medicine of preparation treatment intervertebral disk retrogression pathology.
Further, described pharmaceutical composition comprises the inhibitor of effective dose.The stability that described inhibitor can suppress the expression of miR-1288, maybe can suppress the activity of miR-1288, maybe can shorten the effective acting time of miR-1288, maybe can suppress miR-1288.The target of described inhibitor is not limited to miR-1288 itself, also comprises the upstream and downstream of miR-1288, such as: the genome sequence of coding miR-1288, and the target gene of miR-1288, the albumen of regulation and control miR-1288 or gene.
Further, miR-1288 inhibitor comprises albumen, oligonucleotide, micromolecular compound, oligonucleotide expression vector.
The described carrier for oligonucleotide expression comprises virus vector, eukaryotic vector.
Virus vector can be any suitable carrier, includes but not limited to retroviral vector, adenovirus carrier, adeno-associated virus (AAV) carrier, simplexvirus (such as hsv, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any suitable expression vector, include but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEFBos expression vector, pTet expression vector, pTRE expression vector or the carrier through transforming on the basis of known expression vector, such as pBin438, pCAMBIA1301 etc.
Preferably, described miR-1288 inhibitor is antisense oligonucleotide (anti-miR-1288) or the miRNA-1288 stand-in of miRNA-1288.
Easily design its antisense oligonucleotide according to miRNA-1288 sequence, transferred to by antisense oligonucleotide after in human body, they obviously can lower the expression of miRNA-1288." antisense oligonucleotide (antisense-oligonucleotides; AS-Ons or ASO) " is also called " antisense nucleotide ", refers to that length is about the DNA molecular of 18-26nt (more particularly about 19-22nt) or RNA molecule or its analogue.
Described " antisense oligonucleotide " also comprises the modified antisense nucleotide adopted as obtained based on means such as nucleic acid lock or nucleic acid chains backbone modification technology, described modification does not change the activity of antisense oligonucleotide substantially, more preferably, described modification can improve the stability of antisense oligonucleotide, activity or result for the treatment of.Nucleic acid lock (lockednucleicacid, LNA) typically refers to the modification technique 2 ' of ribose Sauerstoffatom and 4 ' carbon atom coupled together by a methylene bridge.The antisense drug developed based on the modification technique of nucleic acid chains skeleton is in solubility, and the aspects such as nuclease-resistant degraded are improved greatly, and are easy to a large amount of synthesis.The backbone modification method of oligonucleotide has multiple, comprises sulfo-method, such as, be sulfo-deoxynucleotide chain by deoxynucleotide chain thio-modification.The method is substituted by the Sauerstoffatom sulphur atom of the phosphate bond on DNA skeleton, can resist nuclease degradation.Should be understood that and anyly the major part of described antisense oligonucleotide or all active modification can be kept to be included in the present invention.
The medicine for the treatment of intervertebral disk retrogression pathology of the present invention also comprises acceptable carrier on pharmacology, and described carrier includes but not limited to: thinner, buffer reagent, suspensoid, emulsion, granule, encapsulation agents, vehicle, weighting agent, tackiness agent, sprays, cutaneous permeable agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, tinting material, correctives or absorption carrier.
Described medicine can be made and include but not limited to microinjection agent, be suitable for the formulation of transfection, injection liquid, tablet, pulvis, granula, capsule.The medicine of above-mentioned various formulation all can be prepared according to the ordinary method of pharmaceutical field.
Described medicine can be used separately; Or the medicine can treating intervertebral disk retrogression pathology with other carries out combined administration.
Described medicine can be used in vitro: imported in vitro by the expression vector of anti-miR-1288 or transfection human body self or variant cell (or heterogenous cell), after vitro cell expansion, and defeated the Huis' body.
Described medicine can be used in body: directly imported in body by the expression vector of anti-miR-1288.This carrier can be virus type or non-viral, or even naked DNA or RNA.
Described experimenter can be the mankind or other Mammalss.More specifically, experimenter is organ, tissue, cell.
" significant quantity " that the present invention uses refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.The significant quantity of miR-1288 inhibitor of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio, metabolism, transformation period etc. of described miRNA promotor; The severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.
The method analyzing miRNA express spectra includes but not limited to following several: inverse transcription polymerase chain reaction method (RT-PCR), Fluorescent quantitative PCR method (Real-timePCR), Northern hybridization blot assays (Northernblotting), rnase protection analysis method (RNaseprotectionassay), Solexa sequencing technologies (Solexasequencingtechnology) and biochip.In the specific embodiment of the present invention, have employed Solexa sequencing technologies.
" Microrna " and " miRNA ", " miR " that use in the present invention is general.
" the diagnosis intervertebral disk retrogression pathology " that use in the present invention comprises the anticipation to intervertebral disk retrogression pathology, namely judge whether experimenter exists the risk suffering from intervertebral disk retrogression pathology, also the diagnosis to intervertebral disk retrogression pathology is comprised, namely judge whether experimenter has suffered from intervertebral disk retrogression pathology, also comprise the judgement to the prognosis of intervertebral disk retrogression pathology, namely judge whether experimenter exists the possibility of recurrence or judge that experimenter is recurred.
" the treatment intervertebral disk retrogression pathology " that use in the present invention comprises the healing of disease, the improvement of disease.
The present invention uses the nucleus pulposus cell of vitro culture to study the therapeutic action of miR-1288 to intervertebral disk retrogression pathology.Those skilled in the art are known, the important physiological characteristic that intervertebral disk retrogression pathology occurs be nucleus pulposus cell proliferation slowed down, old and feeblely to accelerate, apoptosis aggravation, therefore the present invention utilizes that nucleus pulposus cell grows, the change of apoptosis index to be to study the relation of miR-1288 and intervertebral disk retrogression lesions treatment.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention miR-1288 is to intervertebral disk retrogression pathology relevant, by detecting the expression of experimenter miR-1288, can judge whether experimenter suffers from intervertebral disk retrogression pathology or judge whether experimenter exists the risk suffering from intervertebral disk retrogression pathology, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-miR-1288, compare traditional detection means, small diagnosis more in time, more special, sensitiveer, the early diagnosis of intervertebral disk retrogression pathology can be realized, thus reduce the mortality ratio of intervertebral disk retrogression pathology.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of miR-1288 in intervertebral disk retrogression pathological tissues;
Fig. 2 display utilizes QPCR to detect miR-1288 process LAN situation.
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is that following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, person skilled in art can make some nonessential improvement and adjustment according to the invention described above content to the present invention.In following embodiment, if not specially show, reagent used is analytical pure, and agents useful for same all can obtain from commercial channel.The experimental technique of unreceipted actual conditions in literary composition, the condition described in " Molecular Cloning: A Laboratory guide " book that the Science Press that conveniently condition is write as J. Pehanorm Brooker etc. usually publishes for 2002, or according to the condition that manufacturers advises.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.
The miRNA that embodiment 1 cDNA microarray is relevant to intervertebral disk retrogression pathology
1, sample collection
People's normal disc tissue 8 example, takes from wound and causes vertebral burst fracture patient.Get disc tissue 8 example of intervertebral disk retrogression disease surgery patient, 75 years old mean age, according to Gr1es (5) standards of grading, be severe regression.Patient and family members' agreement thereof is all obtained during sample collection.
2, nucleus pulposus extracts
Normal nucleus pulposus: operation wins normal disc tissue close to complete, can see the tremelloid nucleus pulposus of fibrous ring and center of periphery white.Soak intervertebral disk 10 minutes with the physiological saline containing dual anti-(a blue or green Streptomycin sulphate), gently nucleus pulposus be separated from intervertebral disk with curet, dual anti-physiological saline soaking flushing till without obvious bloodstain, about 3-4 time.
Regression nucleus pulposus: operation, can not complete taking-up intervertebral disk all from rear side approach.When separated fiber ring tissue and nucleus pulposus, naked eyes, feel etc. is relied on to distinguish.Nucleus pulposus is gelatin translucent, and fibrous ring is white pliable and tough shape.Nucleus pulposus eye scissors easily shreds, immalleable.
3, RNA is organized to extract
Every 100mg tissue adds 1mLTrizol, fully grinds tissue block by liquid nitrogen grinding.Add the chloroform of about 1/5 volume, fully mixing about 1 minute of turning upside down, left at room temperature 5 minutes.4 DEG C, 12,000rpm carefully shifts supernatant liquor and enters new 1.5ml centrifuge tube after centrifugal 15 minutes, add isopyknic Virahol, put upside down mixing gently, room temperature leaves standstill 10 minutes.4 DEG C, 12000rpm, after centrifugal 10 minutes, removes supernatant, and in precipitation, add 70% ethanol of 2/5 volume, 4 DEG C, 12000rpm centrifuge washing precipitates 5 minutes.Remove supernatant, add after precipitation room temperature is dried and fully dissolve without the water of RNA enzyme in right amount, measure OD260 and OD280 value.Adopt the DNA enzymatic I process without RNA enzyme, QIAGENRNeasy kits total serum IgE, detailed principle of operation and method are shown in test kit specification sheets.Agarose gel electrophoresis evaluates total serum IgE quality, gel imaging instrument is observed, takes pictures, and preserves image, it is generally acknowledged 28S:18S >=2 can preliminary judgement total serum IgE quality better.
3, the extraction of miRNA and mark
3.1 obtain miRNA with the miRNAs extraction agent box extracting of Ambion company, and concrete operations are according to respective description book.The sample method of T4RNA ligase enzyme markers step according to Thomson.MiRNA marking method is roughly as follows: 1.4 μ gmiRNA and 500ng5 '-phosphoric acid salt-cytosine(Cyt)-uridylic cy3-3 ' (Dharmacon, Chicago, USA) and 2 unit T4RNAligase (NEB, Ipswich, USA), 2 hours are hatched in 4 DEG C.The corresponding negative control of equivalent all established by every part of miRNA sample.
The RNA 0.3M sodium-acetate of 3.2 marks and the ethanol of 2.5 times of volumes precipitate, resuspended containing the hybridization solution of 3 × SSC, 0.2%SDS and 15% methane amide with 15 μ l again, all hybridization repeats twice, hybridization uses LifterSlipTM (Erie, PAUSA) to ensure hybridization solution Uniform Flow between chip and cover plate.
Hybridization chamber is placed on (CapitalBioCorp, Beijing, China) on hybridization instrument BioMixerTMII by 3.3, in 42 DEG C of water-bath hybridized overnight, washes twice by washing lotion afterwards.
4, miRNA chip operation:
MiRNA chip, adopt the miRNA chip of expression spectrum (single passage chip) of Boao Biological Co., Ltd, the detection of miRNA express spectra is carried out in instruction to specifications.
5, result:
Analyze the detected result of miRNA chip express spectra, known miRNA-1288 there are differences expression in normal control tissue and intervertebral disk retrogression pathology, significantly raises in the level of intervertebral disk retrogression pathological tissues miR-1288.
Embodiment 2QPCR verifies the miR-1288 of differential expression
1, miRNA-1288 is selected to carry out large sample QPCR checking according to the detected result of miRNA chip.According to the sample collection way selection normal control tissue in embodiment 1 and each 50 examples of intervertebral disk retrogression pathological tissues.
2, RNA leaching process is with embodiment 1.
3, reverse transcription: the total serum IgE template of 10pg-1 μ g is mixed with 2 μ l10* damping fluids, 2 μ ldATP (10mM), 0.5 μ lpolyA polymerase, 0.5 μ l rnase (RNase) inhibitor and deoxyribonuclease water (RNasefreewater), volume is finally 20 μ l, hatches 1h for 37 DEG C.Then add 1 μ l0.5 μ g/ μ lOligo (dT) specific RT primer in reaction tubes, 70 DEG C hatch 5min after hatch at least 2min on ice at once, interrupt the secondary structure of RNA and primer.Finally, by above-mentioned 20 μ l reaction mixtures and 4 μ l5* damping fluids, 1 μ ldNTP (10mM), 0.5 μ lM-MLV reversed transcriptive enzyme, 0.5 μ l rnase (RNase) inhibitor, 10 μ lpolyA reaction mixtures and the mixing of 4 μ l deoxyribonucleases water (RNasefreewater), hatch 1h for 42 DEG C.
4, QPCR reaction: adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, all amplified reactions are above to ensure the reliability of result all in triplicate.Prepare following reaction system: SYBRGreen polymerase chain reaction system 12.5 μ l, forward primer (5 μMs/μ l) 1 μ l, reverse primer (5 μMs/μ l) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l.Operations is all in carrying out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 20s, 60 DEG C of 55s) * 50 circulation.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument.The forward primer sequence of amplification miRNA-1288 is: 5 '-TGGACTGCCCTGATCTGGAGA-3 ' (SEQIDNO.1), reverse primer is general reverse primer (purchased from Beijing Quanto Biotechnology Co., Ltd.).Using snRNAU6 as reference gene, its upstream primer sequence is: 5 '-CTCGCTTCGGCAGCACA-3 ' (SEQIDNO.2); Downstream primer sequence is: 5 '-AACGCTTCACGAATTTGCGT-3 ' (SEQIDNO.3).By melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
5, result
As shown in Figure 1, compared with normal control tissue, in intervertebral disk retrogression pathological tissues, the expression level of miRNA-1288 significantly raises, consistent with miRNA chip results.
Embodiment 3miR-1288 process LAN
1, miRNA synthesis
Synthesize contrast miRNA (miRNA-NC), miR-1288 by Shanghai Ji Ma company, wherein the sequence of miR-1288 is 5 '-UGGACUGCCCUGAUCUGGAGA-3 ' (SEQIDNO.4).
2, cell cultures
Normal disc nucleus pulposus puts into the 100ml beaker filling 10m12%II Collagenase Type, magnetic stirrer about 60 minutes.After organizing and dissolving completely, centrifugal 10 minutes of 1000r/min, sucking-off supernatant liquor, cell is dispelled gently with the DMEM substratum lml containing 10% foetal calf serum, be drawn in 50ml culturing bottle, add the DMEM substratum 6-8ml of 10% foetal calf serum, be statically placed in 37 DEG C, saturated humidity, 5%CO 2cultivate 3 days in incubator.After 3 days, inverted microscope observation of cell adherent growth situation, changes liquid every other day.
3, cell transfecting
S-generation nucleus pulposus cell is divided into 2 groups, is respectively control group (miR-NC), miR-1288 process LAN group, is used by nucleus pulposus cell transfection reagent LipofectamineTM2000 to carry out transfection, transfection method is with reference to specification sheets.The working concentration of miRNA is 10 μMs.After transfection, 48h collects each group of cell for subsequent experimental.
4, QPCR experiment
Cell total rna extracts and conventionally carries out.
Reverse transcription and PCR process are with embodiment 2.
5, result
As shown in Figure 2, compared with miR-NC group, in the cell of miR-1288 process LAN group, the level of miR-1288 significantly raises, and shows the success of miR-1288 process LAN.
The impact that embodiment 4miR-1288 process LAN is bred nucleus pulposus cell
Use CellCountingkit-8 (cck-8) test kit for detecting nucleus pulposus cell propagation
1, step
Cultivation and the transfection of nucleus pulposus cell is carried out, after transfection 24h, with 2 × 10 according to the method for embodiment 3 5/ ml density is inoculated in 96 porocyte culture plates, and each experimental group design three wells, every hole 100 μ l, is positioned over 37 DEG C, 5%CO 2hatch in incubator, after 24h cell attachment, in the culture hole of required detection, every hole adds 10 μ lCK-8 solution respectively, continues to hatch 1h in cell culture incubator, measures each hole, 450nm place absorbance (OD value).
2, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
3, result
Result is as shown in table 1, and compared with miR-NC group cell, the cell proliferation of miR-1288 process LAN group is slow, and difference has statistical significance (P<0.05).Above-mentioned experimental result points out us, and the expression of interference miR-1288 can accelerate the propagation of nucleus pulposus cell.
Table 1 nucleus pulposus cell OD value
Experimental group OD value (optical density(OD))
miR-NC 0.1953±0.005
miR-1288 0.1014±0.001
Embodiment 5miR-1288 process LAN is on the impact of nucleus pulposus cell apoptosis
1, cell cultures and transfection
Step is with embodiment 3.
2, apoptosis experiment: after cell transfecting 72h, uses precooling PBS washed cell, then uses 0.25% trypsin digestion cell, stop digestion, and using PBS resuspended in the cell of collected by centrifugation, is 1 × 10 by cell quantification 6individual/ml, gets the above-mentioned cell suspension of 200 μ L and is placed in Appendorf pipe, add 10 μ LAnnexin-V-FITC and mix, and dyeing 15min is hatched in room temperature dark place, and before upper machine, 5min adds 10mg/L iodate third ingot (PI) and to dye 5 μ L.The cell of untransfected is used for standard quantitative with Annexin-V-FITC and PI dyeing respectively.Two Colour Fluorescence cell cytometry is carried out, observing apoptosis cell percentages with FACS flow cytometer.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, the t inspection that difference between the two adopts, think to there is statistical significance as P<0.05.
4, result:
The apoptosis rate of miR-NC group is (7.14 ± 0.08) %, the apoptosis rate of miR-1288 process LAN group is (52.15 ± 0.61) %, above-mentioned difference has statistical significance (P<0.05), the above results shows, miR-1288 process LAN then can cause apoptosis to aggravate, and the enlightenment giving us is that the mode can expressed by interference miR-1288 carrys out inhibited apoptosis.
Embodiment 5miR-1288 process LAN is on the impact of nucleus pulposus cell aging
The old and feeble situation of SA-β-gal staining examine nucleus pulposus cell.SA-β-gal is a kind of biological markers identifying senile cell.Measure test kit (Biovision company) according to cell aging and detection is described, observe and have blue percentage of cells in tenuigenin under counting microscope, judging the situation of cell aging.Often group establishes 3 multiple holes, each experiment repetition 3 times.
Cell cultures and transfection are with embodiment 3.
Carry out SA-β-gal after cell transfecting 48h to dye.
Result shows, transfection miR-NC groups of cells blue cell average percentage is 11%, transfection miR-1288 groups of cells blue cell average percentage is 37%, difference has statistical significance (P<0.05), the above results shows that miR-1288 process LAN can accelerate the aging course of nucleus pulposus cell, and the enlightenment giving us is that the mode can expressed by interference miR-1288 carrys out T suppression cell aging.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.

Claims (10)

1. Microrna is preparing the application in intervertebral disk retrogression pathological changes diagnosis instrument, it is characterized in that, described Microrna is selected from following group: initial miRNA, precursor miRNA, ripe miRNA; Initial miRNA can be sheared and be expressed as ripe miRNA in people's cell; Precursor miRNA can be sheared and be expressed as ripe miRNA in people's cell; Described Microrna is miR-1288.
2. application according to claim 1, is characterized in that, described Microrna is ripe miR-1288.
3. application according to claim 1 and 2, is characterized in that, described instrument comprises test kit, chip, test paper, high-flux sequence platform.
4. a diagnostic tool for intervertebral disk retrogression pathology, is characterized in that, described diagnostic tool comprises the reagent that test right requires the microrna expression level described in 1.
5. diagnostic tool according to claim 4, is characterized in that, described diagnostic tool comprises test kit, chip, test paper, high-flux sequence platform.
6. diagnostic tool according to claim 5, is characterized in that, described test kit comprises primer for Microrna according to claim 1 and/or probe; Described chip comprises solid phase carrier, and is fixed on the oligonucleotide probe on described solid phase carrier, and described oligonucleotide probe comprises the part or all of sequence corresponding to Microrna according to claim 1 specifically; Described test paper comprises primer for Microrna according to claim 1 and/or probe; Described high-flux sequence platform comprises primer for Microrna according to claim 1 and/or probe.
7. the application of Microrna according to claim 1 in the medicine of preparation treatment intervertebral disk retrogression pathology.
8. application according to claim 7, is characterized in that, described pharmaceutical pack is containing the inhibitor of the Microrna described in claim 1.
9. application according to claim 8, it is characterized in that, described inhibitor can suppress the expression of Microrna according to claim 1 or can suppress the stability of Microrna according to claim 1 or can suppress the activity of Microrna according to claim 1 or can shorten action time of Microrna according to claim 1.
10. application according to claim 8 or claim 9, it is characterized in that, described inhibitor is selected from following group: albumen, oligonucleotide, micromolecular compound, oligonucleotide expression vector.
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