CN105420376A - MicroRNA relevant to disc degeneration - Google Patents
MicroRNA relevant to disc degeneration Download PDFInfo
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Abstract
The invention relates to microRNA relevant to disc degeneration and discloses the application of miR-744 in preparing disc degeneration diagnosis tools. Sequencing and QPCR experiments prove that expression of miR-744 in normal control tissue differs from expression of miR-744 in disc degeneration tissue, and therefore miR-744 is deemed the molecular marker for diagnosis of disc degeneration. In-vitro cultured cell experiments prove that multiplication of nucleus pulposus cells can be restrained through interference with miR-744 expression, senescence and apoptosis of the cells are promoted, and therefore miR-744 is deemed the drug target for treating disc degeneration. As a new molecular marker for diagnosis of disc degeneration, the molecular marker has broad application prospects clinically.
Description
Technical field
The invention belongs to biomedicine field, relate to the purposes of miR-744 in diagnosis and treatment intervertebral disk retrogression pathology.
Background technology
A series of investigation shows: in western industrial society, the people of 12%-35% suffers from low back pain, wherein 10% disability.U.S.'s recent statistics display, the direct medical expense caused because of low back pain and indirect loss conservative estimation are annual 19600000000 dollars, bring heavy burden to social medical treatment.Degeneration of intervertebral disc is the major cause causing low back pain, it and sciatica and the disease relationship such as protrusion of intervertebral disc, prolapsus close.
People's miRNA, English microRNA by name is the non-coding strand small ribonucleic acid molecules that a class is about 19 to 23 Nucleotide.They are high conservative in evolution, and with many normal physiological activity of animal, as closely related in biont growths, tissue differentiation, apoptosis and energy metabolism etc., while, also also exists with the generation of numerous disease and development and contacts closely.The expression level of several miRNAs that nearest research finds in lymphocytic leukemia and Burkitt lymphoma all has downward (LawrieCH in various degree, GalS, DunlopHMetal.Detectionofelevatedlevelsoftumor-associated microRNAsinserumofpatientswithdiffuselargeB-celllymphoma .BrJHaematol2008; 141:672-675); When miRNA in com-parison and analysis people lung cancer, breast cancer tissue is expressed, the expression level finding that there is some tissue specificity miRNAs there occurs change (GarofaloM relative to healthy tissues, QuintavalleC, DiLevaGetal.MicroRNAsignaturesofTRAILresistanceinhumanno n-smallcelllungcancer.Oncogene2008).Also there are some researches prove that miRNA have impact on generation and the development of the cardiovascular disordeies such as myocardial hypertrophy, heart failure, atherosclerosis, and there is close association (TryndyakVP with metabolic diseases such as type ii diabetes, RossSA, BelandFA, PogribnyIP.Down-regulationofthemicroRNAsmiR-34a, miR-127, andmiR-200binratliverduringhepatocarcinogenesisinducedby amethyl-deficientdiet.MolCarcinog.2008Oct21).These experimental results prompting miRNA is expressed and there is positive connection between specific variations and Occurrence and development of disease.
Owing to playing vital role beyond imagination in the expression regulation of miRNA after genetic transcription, therefore there is following cognation in it and disease: first, the change of miRNA may be the cause of disease, this is because the supressor of disease and promotive factor may be all the target sites of miRNA, when miRNA itself first there occurs disorderly expression, the miRNA expression amount of disease promotive factor was such as originally suppressed to reduce, or suppress the miRNA expression amount of disease supressor to increase, the entirety of the change that its net result all can cause downstream series of genes to be expressed and some path is disorderly, and then the generation that induces an illness, secondly, the change of miRNA also may be the result of disease, this is because when disease occurs, the violent amplification of the loss of chromosome segment, the sudden change of gene or chromosome segment can be caused, if miRNA is just in time positioned at this varied sections, so generation changes by its expression amount extremely significantly.
Therefore, miRNA molecule completely can as the new disease markers of a class in theory, and its specific variations is inevitable be produced development with disease and be associated.MiRNA as potential drug target, by suppressing the miRNA of miRNA or the process LAN downward of raising in lysis, likely will can also greatly alleviate generation and the development of disease simultaneously.
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-744.
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-744 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-744.
In concrete embodiments more of the present invention, described miR-744 is ripe miR-744.
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-744.
Further, described test kit comprises primer for miR-744 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-744 specifically; Described test paper comprises primer for miR-744 and/or probe; Described high-flux sequence platform comprises primer for miR-744 and/or probe.
The invention provides a kind of diagnostic tool of intervertebral disk retrogression pathology, described diagnostic tool comprises the reagent detecting miR-744 expression level.
Further, described diagnostic tool comprises test kit, chip, test paper, high-flux sequence platform.
Further, described test kit comprises primer for miR-744 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-744 specifically; Described test paper comprises primer for miR-744 and/or probe; Described high-flux sequence platform comprises primer for miR-744 and/or probe.
Further, the primer for miR-744 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-744 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-744 of the present invention can be natural or synthetic, or uses the vector-transfected cell can expressing the DNA fragmentation of miR-744 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-744 of cicada and intervertebral disk retrogression pathology, can screen based on this feature the material promoting that miR-744 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-744, 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-744 in the medicine of preparation treatment intervertebral disk retrogression pathology.
Further, described pharmaceutical composition comprises the promotor of effective dose.The stability that described promotor can promote the expression of miR-744, maybe can promote the activity of miR-744, maybe can promote the effective acting time of miR-744, maybe can promote miR-744.The target of described promotor is not limited to miR-744 itself, also comprises the upstream and downstream of miR-744, such as: the genome sequence of coding miR-744, and the target gene of miR-744, the albumen of regulation and control miR-744 or gene.
Further, miR-744 promotor 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-744 promotor is the expression vector of miR-744 itself or miR-744 sequence.
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 miR-744 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 miR-744.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-744 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-744 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-744 and intervertebral disk retrogression lesions treatment.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention miR-744 is to intervertebral disk retrogression pathology relevant, by detecting the expression of experimenter miR-744, 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-744, 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-744 in intervertebral disk retrogression pathological tissues;
The disturbed condition that Fig. 2 display utilizes QPCR detection anti-miR-744 to express miR-744;
Fig. 3 shows miR-744 and expresses the impact of breeding nucleus pulposus cell.
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 1Solexa order-checking screening is relevant to intervertebral disk retrogression pathology
1, sample collection
People's normal disc tissue 5 example, takes from wound and causes vertebral burst fracture patient.Get disc tissue 5 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 extracts: will grind the vessel such as pestle and homogenizer at 200 DEG C of dry roasting 4h, and remove RNA enzyme, cooling; Add precooling in liquid nitrogen, tissue is taken out rapidly from liquid nitrogen, be crushed into powder; With curet, tissue is put into the homogenizer adding TRIzol reagent in advance, homogenate number minute; Liquid after homogenate is proceeded in the centrifuge tube without RNA enzyme, after adding chloroform, 4 DEG C of centrifugal layerings; Upper strata aqueous phase is proceeded in a centrifuge tube without RNA enzyme, after adding chloroform, 4 DEG C of centrifugal layerings; Upper strata aqueous phase is proceeded in a centrifuge tube without RNA enzyme, add Virahol, 4 DEG C of centrifugation RNA; 2 times are precipitated by 75% washing with alcohol; Precipitate with the deionized water dissolving without RNA enzyme.The RNA of extracting carries out quality evalution (measuring RNA concentration, purity and integrity by Agilent 2100).It is stand-by that the RNA that quality evalution is good leaves-80 DEG C of refrigerators in.
4, reverse transcription: utilize the Reverse Transcription box (OneStepPrimeScriptmiRNAcDNASynthesisKit) of TaKaRa company to build the cDNA library of two kinds of tissues, reverse transcription is carried out in the explanation according to test kit.
5, Solexa method is utilized by the sample after reverse transcription to check order (Solexa order-checking is completed by Hua Da genome company).
6, result:
Analyze Solexa method sequencing result, miR-744 there are differences expression in normal control tissue and intervertebral disk retrogression pathology, significantly reduces in the level of intervertebral disk retrogression pathological tissues miR-744.
Embodiment 2QPCR verifies the miR-744 of differential expression
1, sample collection: the detected result of embodiment 1 selects miR-744 to carry out large sample QPCR checking.According to the sample collection way selection normal control tissue in embodiment 1 and each 40 examples of intervertebral disk retrogression pathological tissues.
2, RNA extracts: will grind the vessel such as pestle and homogenizer at 200 DEG C of dry roasting 4h, and remove RNA enzyme, cooling; Add precooling in liquid nitrogen, tissue is taken out rapidly from liquid nitrogen, be crushed into powder; With curet, tissue is put into the homogenizer adding TRIzol reagent in advance, homogenate number minute; Liquid after homogenate is proceeded in the centrifuge tube without RNA enzyme, after adding chloroform, 4 DEG C of centrifugal layerings; Upper strata aqueous phase is proceeded in a centrifuge tube without RNA enzyme, after adding chloroform, 4 DEG C of centrifugal layerings; Upper strata aqueous phase is proceeded in a centrifuge tube without RNA enzyme, add Virahol, 4 DEG C of centrifugation RNA; 2 times are precipitated by 75% washing with alcohol; Precipitate with the deionized water dissolving without RNA enzyme.The RNA of extracting carries out quality evalution (measuring RNA concentration, purity and integrity by Agilent 2100).It is stand-by that the RNA that quality evalution is good leaves-80 DEG C of refrigerators in.
3, reverse transcription:
Adopt the Reverse Transcription box (DRR047) of TAKARA company, reverse to 1 μ gRNA, this test kit adds the step removing genomic dna compared with classical inverse transcript reagent box, can ensure the purity of RNA and the specificity of amplification to the full extent.Reaction system and reaction conditions reference reagent box specification sheets carry out.
4, QPCR reaction:
Be template with cDNA, use SYBR (R) the PremixExTaqTM quantitative fluorescent PCR system of PCR primer for target miRNA and Takara, stratagenMX3000P quantitative PCR apparatus increases, and records the Ct value of sample.By the formula that gained Ct value draws by substituting into bioassay standard curve, the method for calculation of absolute quantitation are adopted to draw the content of the target miRNA in sample.U6 gene carries out stdn correction as interior photograph to miRNAqPCR detected result.
Primer for miR-744 is as follows:
Forward primer is 5 '-TGCGGGGCTAGGGCTAACAGCA-3 ' (SEQIDNO.1),
Reverse primer is general reverse primer (purchased from Beijing Quanto Biotechnology Co., Ltd.).
Primer for U6 is as follows:
Forward primer: 5 '-CTCGCTTCGGCAGCACA-3 ' (SEQIDNO.2),
Reverse primer: 5 '-ACGCTTCACGAATTTGCGT-3 ' (SEQIDNO.3).
5, result
As shown in Figure 1, compared with normal control tissue, in intervertebral disk retrogression pathological tissues, the expression level of miR-744 significantly reduces, consistent with Solexa sequencing result.
Embodiment 3 disturbs miR-744 to express
1, design and synthesis is for the antisense oligonucleotide (anti-miR-744) of miR-744
The sequence information of miR-744 is found, according to the sequence information of miR-744 by the precious biotinylated biomolecule Technology Co., Ltd. design and synthesis anti-miR-744 in Dalian and random controls sequence (anti-miR-NC) in miRNA database (http://www.sanger.ac.uk).
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
Nucleus pulposus cell is divided into 2 groups, be respectively control group (anti-miR-NC group), miR-744 interference group (anti-miR-744 group), used by nucleus pulposus cell transfection reagent LipofectamineTM2000 to carry out transfection, transfection method is with reference to specification sheets.The working concentration of antisense oligonucleotide is 5 μMs.After transfection, 48h collects each group of cell for subsequent experimental.
4, QPCR experiment
Cell total rna extraction and PCR step are with embodiment 2.
5, result
As shown in Figure 2, compared with anti-miR-NC group, in the cell of anti-miR-744 group, the level of miR-744 is significantly lowered, and shows that interference miR-744 expresses successfully.
Embodiment 4 disturbs miR-744 to express the impact of breeding nucleus pulposus cell
1, cell transfecting: transfection is carried out to nucleus pulposus cell according to the method for embodiment 3.
2, transfection adds 3H-TdR (1 μ Ci/ hole) after 24 hours, then cultivates 24 hours, collecting cell, adds liquid scintillation solution, and β calculating instrument detects cpm value.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, employing SPSS13.0 statistical software carries out statistical study, difference between interference miR-744 expression group and control group adopts t to check, and thinks to have statistical significance as P<0.05.
4, result
Result such as Fig. 3 shows, and compared with anti-miR-NC group, the cell proliferation of anti-miR-744 group is slack-off, and difference has statistical significance (P<0.05).
Embodiment 5 disturbs the impact of miR-744 expression on 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 anti-miR-NC group is (9.25+0.14) %, the apoptosis rate of anti-miR-744 is (45.21+1.94) %, above-mentioned difference has statistical significance (P<0.05), the above results shows, reducing miR-744 expression then can cause apoptosis to aggravate, and the enlightenment giving us is that the mode can expressed by increasing miR-744 carry out inhibited apoptosis.
Embodiment 5 disturbs the impact of miR-744 expression on 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 anti-miR-NC groups of cells blue cell average percentage is 12%, transfection anti-miR-744 groups of cells blue cell average percentage is 41%, difference has statistical significance (P<0.05), the above results shows that reducing miR-744 expresses the aging course can accelerating nucleus pulposus cell, and the enlightenment giving us is that the mode can expressed by increasing miR-744 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-744.
2. application according to claim 1, is characterized in that, described Microrna is ripe miR-744.
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 promotor of the Microrna described in claim 1.
9. application according to claim 8, it is characterized in that, described promotor can promote the expression of Microrna according to claim 1 or can promote the stability of Microrna according to claim 1 or can promote the activity of Microrna according to claim 1 or can promote effective acting time of Microrna according to claim 1.
10. application according to claim 8 or claim 9, it is characterized in that, described promotor is selected from following group: albumen, oligonucleotide, micromolecular compound, oligonucleotide expression vector.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2758928A1 (en) * | 2009-04-29 | 2010-11-04 | Academisch Medisch Centrum Bij De Universiteit Van Amsterdam | Means and methods for counteracting, preventing and/or determining heart failure, or a risk of heart failure |
| CN102421917A (en) * | 2009-04-29 | 2012-04-18 | 阿姆斯特丹大学学术医学中心 | Means and methods for counteracting, preventing and/or determining heart failure, or a risk of heart failure |
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| WANG H Q等: ""Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3"", 《JOURNAL OF PATHOLOGY》 * |
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