CN109055544A - Atherosclerosis molecular marker and its application - Google Patents

Abstract

The present invention relates to a kind of atherosclerosis molecular marker and its applications.The atherosclerosis molecular marker can preferably reflect the development situation of atherosclerosis, to be applied to the Related product of preparation diagnosis or detection atherosclerosis, enrich the diagnosis and detection means of atherosclerosis.And the atherosclerosis molecular marker is able to suppress the adhesive attraction between monocyte THP-1 and vascular endothelial cell HUVECs, and inhibit the expression of the inflammatory factors such as adhesion factor and chemotactic factor (CF), to be effectively suppressed the overactivity of blood vessel endothelium inflammation, therefore it is also used as inhibiting the molecular drug target spot of formation and the development of atherosclerosis, for the pathogenesis of further studying atherosclerotic, explores its protective agents and new empirical theory basis and direction are provided.

Description

Atherosclerosis molecular marker and its application

Technical field

The present invention relates to field of atherosclerosis, more particularly to a kind of atherosclerosis molecular marker and its answer With.

Background technique

Atherosclerosis is a kind of chronic inflammatory diseases, it mainly occurs since low-density lipoprotein (LDL) is metabolized Disorder, the inner membrance deposition of hypodermic layer is caused in the blood vessels.Clinically, atherosclerosis and myocardial infarction, apoplexy, lack These common cardiovascular diseases such as hemorrhagic heart pain and hypertension suffer from close relationship.Currently, by artery The cardiovascular and cerebrovascular disease that atherosis is caused, which has become, endangers one of principal disease of human health.According to the World Health Organization The latest data of WHO counts display, and it is dead that the number for dying of cardiovascular and cerebrovascular disease, which accounts for the 48% of all uninfection total numbers of persons, In twice or more of cancer occurrence numbers.Therefore, the pathogenesis of further studying atherosclerotic, artery congee can be used as by seeking The important symbol object of sample hardening clinical diagnosis has a very important significance.

Summary of the invention

Based on this, it is necessary to which providing one kind can be as the Atherosclerosis of the marker of atherosclerosis clinical diagnosis Chemoattractant molecule marker.

A kind of atherosclerosis molecular marker, the atherosclerosis molecular marker are long-chain non-coding RNA, It has the nucleotide sequence as shown in SEQ ID NO.1.

The present invention also provides a kind of PCR amplification primers, including are used for the above-mentioned Atherosclerosis chemoattractant molecule of specific amplification The upstream primer and downstream primer of marker.

The upstream primer has the nucleotide sequence as shown in SEQ ID NO.2 in one of the embodiments, described Downstream primer has the nucleotide sequence as shown in SEQ ID NO.3.

The present invention also provides a kind of siRNA, sense strand and antisense strand including complementation, and the siRNA The expression of above-mentioned atherosclerosis molecular marker can specifically be inhibited.

The sense strand has the nucleotide sequence as shown in SEQ ID NO.2 in one of the embodiments, described anti- Adopted chain has the nucleotide sequence as shown in SEQ ID NO.3.

The present invention also provides above-mentioned atherosclerosis molecular marker, above-mentioned PCR amplification primer or above-mentioned small interference Application of the RNA in the product of preparation diagnosis or detection atherosclerosis.

The product is detection reagent, kit, genetic chip or Test paper in one of the embodiments,.

The present invention also provides above-mentioned atherosclerosis molecular marker, above-mentioned PCR amplification primer or above-mentioned small interference RNA is preparing the application in the drug for preventing or treating atherosclerosis.

The present invention also provides a kind of for detecting the kit of atherosclerosis, including above-mentioned PCR amplification primer.

It in one of the embodiments, further include that RNA is extracted in reagent, reverse transcription reagents and PCR amplification reagent at least It is a kind of.

2/3rds sequence is by reverse transcription in the gene order of more than 30 hundred million base-pairs of the mankind, and finally only less than 2% nucleic acid sequence is for encoding albumen, and most gene does not express protein, this genoid is referred to as non-coding RNA.It is non- Coding RNA includes short chain non-coding RNA and long-chain non-coding RNA, they may participate in cell differentiation and a on many levels Body developmental regulation, it is closely related with various diseases.Present inventor has found under study for action, long-chain non-coding RNA NKILA The expression quantity of (sequence is as shown in SEQ ID NO.1) in the peripheral blood mononuclear cells of Atheromatosis people is significantly lower than just Expression quantity (P value < 0.05) in the peripheral blood mononuclear cells of ordinary person, can preferably reflect the development situation of atherosclerosis, Therefore it can be used as the clinical diagnosis molecular marker of atherosclerosis, and be applied to preparation diagnosis or detection Atherosclerosis The Related product of change, to enrich the diagnosis and detection means of atherosclerosis.

The generation of atherosclerosis and the exception of vascular endothelial cell have direct association.In atherosclerosis In early days, due to the metabolic disorder of low-density lipoprotein, the inflammatory reaction of vascular endothelial cell can be caused, to promote endothelial cell Secrete the inflammatory factors such as a large amount of adhesion factor, chemotactic factor (CF).The secretion of these inflammatory factors can promote monocyte and endothelium Adhesive attraction between cell, so that monocyte passes through the endangium that endothelial cell gap enters subendothelial layer.Into The low-density lipoprotein induction that monocyte after to inner membrance can be oxidized type becomes macrophage and swallows up lipophagia albumen, thus Foam cells is gradually formed, the formation of plaque is eventually led to.Inventor further investigation revealed that, the artery is athero- Hardening molecular marker is able to suppress the adhesive attraction between monocyte THP-1 and vascular endothelial cell HUVECs, and suppression The expression of the inflammatory factors such as adhesion factor and chemotactic factor (CF) processed, to be effectively suppressed the excessive work of blood vessel endothelium inflammation Change, therefore be also used as inhibiting the molecular drug target spot of formation and the development of atherosclerosis, for further research artery The pathogenesis of atherosis explores its protective agents and provides new empirical theory basis and direction.

Detailed description of the invention

Fig. 1 is the peripheral blood mononuclear cells atherosclerosis molecular marker of Atheromatosis people and normal person Expression quantity comparison；

Fig. 2 is comparison of the siRNA to the interference suppressioning effect of atherosclerosis molecular marker expression quantity；

Fig. 3 is that siRNA reduces the expression of atherosclerosis molecular marker to viscous between monocyte and endothelial cell The influence of attached effect；

Fig. 4 is that the expression of siRNA reduction atherosclerosis molecular marker to adhesion factor VCAM1 and SELE and becomes Change the influence of the expression quantity of factor M CP1 and IL8；

A is the overexpression Efficiency testing of atherosclerosis molecular marker as a result, B is to be overexpressed artery in Fig. 5 in Fig. 5 Influence of the atherosis molecular marker to adhesive attraction between monocyte and endothelial cell；

Fig. 6 is to be overexpressed atherosclerosis molecular marker to adhesion factor VCAM1 and SELE and chemotactic factor (CF) The influence of the expression quantity of MCP1 and IL8.

Specific embodiment

To facilitate the understanding of the present invention, below will to invention is more fully described, and give it is of the invention compared with Good embodiment.But the invention can be realized in many different forms, however it is not limited to embodiment described herein.Phase Instead, purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases Any and all combinations of the listed item of pass.

Atherosclerosis molecular marker and its application are made into one mainly in combination with specific embodiment and attached drawing below Step detailed description.

One, the peripheral blood mononuclear cells atherosclerosis molecule of detection comparison normal person and Atheromatosis people The expression quantity of marker

It is prepared by the separation of 1.1 peripheral blood mononuclear cells

Respectively from the lymph of normal person (10 as a control group) and Atheromatosis people (10 as experimental group) Cell separating liquid (being purchased from the ocean Tianjin Hao biotech firm) separating periphery blood monocytic cell, draws intermediate tunica albuginea layer after centrifugation.So It is washed twice afterwards with 0.9% sodium chloride solution, then cell is resuspended with the X-VIVO culture solution containing 10%PBS, and adjust cell Concentration be 1 × 10⁶A cell/mL.

The extraction of 1.2RNA

In the EP pipe for taking 50 μ L to 1.5mL of peripheral blood mononuclear cells respectively, the Trizol reagent of 500 μ L is added.Use liquid relief Rifle pressure-vaccum is to after no longer sticky, into EP pipe plus 100 μ L chloroforms (1/5th of Trizol volume), sufficiently acutely after concussion (becoming cloudy) stands 5min.12000rpm is centrifuged 10min, three layers is divided after centrifugation, for RNA in top layer, middle layer is albumen, most lower Layer is organic phase.Supernatant to be drawn into new EP pipe, is added isometric isopropanol, concussion mixes, in -20 DEG C of placement 20min, Keep RNA precipitate complete.12000rpm is centrifuged 10min, and supernatant is gone to leave precipitating, and the ethanol washing of 500 μ L 75% is added.Then 12000rpm is centrifuged 5min, removes supernatant, and repeated washing is primary.5~10min is placed at room temperature to dry, and 20 μ L DEPC water are added, 30min is dissolved at room temperature.

1.3 spectrophotometer measurement RNA concentration

Take 2 μ L RNA respectively, 198 μ L DEPC water be added, measured respectively in spectrophotometer after mixing OD230, OD260 and OD280 assesses Nucleic acid quality, and obtains RNA concentration according to following calculation formula.

1.4 digest and remove the residual genomic DNA in RNA

For digesting 5 μ g RNA, 0.4 μ L RNase inhibitor (40U/ μ L), 1 μ L DNA are sequentially added Enzyme, 2 μ 10 × deoxyribonuclease of L buffers and 5 μ g RNA, and DEPC water polishing is added to 20 μ L systems.It is then placed in In PCR instrument, progress following procedure: 37 DEG C, 30min (deoxyribonuclease digests DNA)；68 DEG C, 10min (makes deoxyribose Nuclease-dead).

1.5 reverse transcription

By taking 1 μ g RNA of reverse transcription as an example, 4 μ L RNA (1 μ g), 2 μ L random primers (25 μM) and 6 μ L DEPC water are first added Be configured to 12 μ L systems, PCR instrument be put into after mixing, program is as follows: 70 DEG C, 10min (opens complicated RNA structure)；Then it stands It is placed in 3~5min (RNA template is made to combine upper random primer) on ice.1 μ L dNTP (10mM), 4 are added in the above system 5 × RT Buffer of μ L, 0.5 μ L reverse transcriptase (MLV) and 2.5 μ L DEPC water, are put into PCR instrument, program is such as after mixing Under: 30 DEG C, 10min；42 DEG C, 1h；70 DEG C, 15min.

1.6qRT-PCR detection

RNA sequence (the RefSeq sequence number: NR_131157.1, specific sequence of NKILA is found in ncbi database first Column are as shown in SEQ ID NO.1), the sequence according to NKILA carries out design of primers and is synthesized, upstream and downstream primer (primer-F With primer-R) sequence respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.The cDNA that reverse transcription obtains is matched as template Make following reaction system:

It is then placed in fluorescence quantitative PCR instrument and carries out following amplification program: 95 DEG C, 7min；40 circulations: 95 DEG C, 10s； 60 DEG C, 30s.Carry out melting curve analysis after amplification: 95 DEG C, 15s；60 DEG C, 15s；95 DEG C, 15s.Utilize 2^-ΔΔCTRelatively Sizing technique is compared analysis to the NKILA expression quantity in the peripheral blood mononuclear cells of normal person and Atheromatosis people.

As a result as shown in Figure 1, the expression quantity of NKILA obviously compares just in the peripheral blood mononuclear cells of Atheromatosis people The expression quantity of NKILA wants low (* represents P value ﹤ 0.05 in figure) in the peripheral blood mononuclear cells of ordinary person.

Two, the design of atherosclerosis molecular marker siRNA sequence and its detection of interference effect

RNA interference (RNAi) technology refer to it is being highly conserved during evolution, induced by double-chain small molecule RNA it is homologous The phenomenon that mRNA efficient selective degradation, RNA molecule is by destroying specific mRNA, to inhibit the biology mistake of certain gene expression Journey.It is a kind of gene silencing process of sequence specific post transcriptional level being widely present in animals and plants, is biological gene Group resists a kind of biological protection mechanism of the external hereditary original part invasion of virus etc.Due to using RNAi technology can specificity The expression of specific gene is rejected or closes, which rapidly becomes gene functional research and gene therapy research field is of greatest concern One of research tool, have been widely used for the biological function for exploring gene and the treatment of various diseases.

2.1 design siRNA

According to the RNA complete sequence (as shown in SEQ ID NO.1) of NKILA using siRNA design website (http: // Sidirect2.rnai.jp/), design siRNA sequence and screened, marine growth Sheng Gong company synthesizes in commission, the siRNA Sense strand and antisense strand particular sequence respectively as shown in SEQ ID NO.4 and SEQ ID NO.5.

2.2 detect the interference effect of above-mentioned siRNA using huve cell (HUVECs)

By in HUVECs cell inoculation to six orifice plates, reaches 70% or so to cell density, utilize Lipofectamine 3000 transfection reagents are respectively by control group siRNA (particular sequence is as shown in SEQ ID NO.6 and SEQ ID NO.7) and above-mentioned reality A group siRNA is tested to be transfected into HUVECs.

Steps are as follows for the specific method of transfection siRNA: the serum free medium of 150 μ L being taken to be added to two 1.5mL's respectively In EP pipe, the Lipofectamine 3000 of 6 μ L is taken to be added thereto in an EP pipe, it is dense that 6 μ L are added in another EP pipe The siRNA that degree is 10 μM.The mixture of siRNA culture medium is added to the mixture of 3000 culture medium of Lipofectamine In, vortex oscillation 5 seconds, it is mixed well, is added dropwise in HUVECs cell after the static 10min of room temperature.After 6 hours more Renew fresh complete medium, after 48 hours, carries out reverse transcription using Trizol extracted total RNA as previously described and pass through fluorescence Quantitative PCR carries out the rna expression amount detection of NKILA.

As a result as shown in Fig. 2, the expression quantity of the NKILA in experimental group cell is significantly lower than the NKILA in cellular control unit Expression quantity (* * * represents P value ﹤ 0.001 in figure), illustrate designed siRNA can selectively targeted NKILA, make its expression Amount reduces, therefore the siRNA can be used for further exploring the biological function of NKILA and the treatment of atherosclerosis.

Three, influence of the expression quantity of atherosclerosis molecular marker to blood vessel endothelium inflammation is reduced

Inflammatory reaction is a kind of clinically very common pathological reaction, refers to the living tissue with vascular system to each A kind of basic pathology process based on defense reaction that the stimulation of kind damage factor is occurred.Inflammation often show as it is red, swollen, Heat, pain and dysfunction, also with general reactions such as fever, the changes of tip Leukocyte Counts.The overactivity of inflammation and dynamic Generation, the development of pulse atherosclerosis are closely bound up.

3.1 reduce influence of the expression quantity to adhering between monocyte and endothelial cell

Blood vessel endothelium inflammation can promote some relevant adhesion factors of endothelial cells secrete to promote monokaryon after being activated Adhesive attraction between cell and endothelial cell, so endothelial cell can be reacted by the adhesive attraction between verifying cell Degree of inflammation, specific step is as follows.

Endothelial cell HUVECs is inoculated into 6 orifice plates, overnight, when density reaches 80%, utilizes Lipofectamine Control group siRNA and experimental group siRNA are transfected into HUVECs (for example preceding institute of specific transfection procedure by 3000 transfection reagents respectively It states).Fresh culture is changed within 6 hours after transfection into, after transfection 48 hours, by monocyte THP-1 Tracker Green CMFDA carries out the label of fluorophor, changes fresh culture medium after 1 hour into HUVECs, while being washed with RPMI culture medium THP-1 cell 3 times.THP-1 cell is added separately in control group and the HUVECs of experimental group, it is small to be put into incubator incubation 1 When.It is washed 3 times with the ECM culture medium of culture HUVECs cell, removes the THP-1 cell being not adhered on HUVECs, in fluorescence The THP-1 cell adhered on HUVECs is counted and taken pictures under microscope.

As a result as shown in figure 3, it can be seen from the figure that compared with the control group, after the expression quantity for reducing NKILA, THP-1 Adhesive attraction between HUVECs cell has obtained significant enhancing.

3.2 reduce the influence of inflammatory factor expression amount in expression quantity Human Umbilical Vein Endothelial Cells

By in HUVECs cell inoculation to six orifice plates, reaches 70% or so to cell density, utilize Lipofectamine Control group siRNA and experimental group siRNA are transfected into HUVECs by 3000 transfection reagents respectively.After 48 hours, utilize Trizol extracted total RNA carries out reverse transcription and by quantitative fluorescent PCR to adhesion factor VCAM1 and SELE and chemotactic factor (CF) The mrna expression amount of four kinds of inflammatory factors such as MCP1 and IL8 is detected.

As a result as shown in figure 4, after the expression quantity of NKILA reduces, adhesion factor VCAM1 and SELE and chemotactic factor (CF) Significantly (* * represents P value ﹤ 0.01 to the expression quantity of the inflammatory factors such as MCP1 and IL8 in figure, and * * * represents P value ﹤ in figure for raising 0.001)。

Four, atherosclerosis molecular marker is overexpressed the influence to blood vessel endothelium inflammation

The Lentiviral of 4.1 building atherosclerosis molecular markers

According to the RNA sequence of above-mentioned NKILA, the DNA sequence dna of double-strand is synthesized, and takes BamH I respectively at starting both ends With two restriction enzyme sites of Xba I.By the double digestion in the two sites BamH I and Xba I, the NKILA of synthesis is passed through into DNA T4 ligase is connected on the slow virus empty carrier of pLV CS2.0 (slow virus empty carrier sequence is as shown in SEQ ID NO.8).

Endonuclease reaction system are as follows:

After adding water polishing to 20 μ L, it is placed on 37 DEG C and is incubated for 4 hours.

T4 DNA ligase reaction system are as follows:

10 × buffer, 1 μ L

NKILA segment: pLV carrier (molal quantity) (1~5): 1

1 μ L of T4 ligase

After adding water polishing to 10 μ L, it is placed in 16 DEG C of overnight incubations.

Slow virus packaging is carried out respectively to pLV CS2.0 empty carrier and NKILA over-express vector, the specific steps are as follows: will HEK293T cell inoculation reaches 70% into 6 orifice plates, to cell density, using Lipofectamine 3000 respectively by NKILA It is thin that overexpression plasmid and the empty carrier of control and slow virus the packaging plasmid pSPAX2 and pMD2G of commercialization are transfected into HEK293T In born of the same parents, plasmid is specifically matched are as follows:

pSPAX2 0.5μg

pMD2G 0.5μg

NKILA is overexpressed plasmid or 1 μ g of empty carrier

After 48 hours to be transfected, viral supernatants are collected, infection cell is can be directly used for or freezes in minus 80 degree of ultralow temperature ices Case, in case using.

4.2 detections are overexpressed the influence to adhering between monocyte and endothelial cell.

Huve cell HUVECs is inoculated into six orifice plates, reaches 80% to cell density, discards former culture medium, It takes above-mentioned empty carrier (vec) and NKILA to be overexpressed virus (NKILA-flag) supernatant 1mL respectively, is added in HUVECs, and add The fresh culture of 1mL is eventually adding the polybrene (Polybrene) that 2 μ L concentration are 4 μ g/ μ L.After 8 hours, changed to cell At fresh culture medium.After infecting 48 hours, by the processing of inducer TNF α 1 hour of HUVECs cell inflammatory reaction, together When monocyte THP-1 is carried out to the label of fluorophor with Tracker Green CMFDA, be placed in 37 DEG C and be incubated for 1 hour.With ECM culture medium washs HUVECs 3 times and changes fresh ECM culture medium into, while washing THP-1 cell 3 with RPMI culture medium It is secondary.It is added separately to the THP-1 cell that Tracker Green is marked to have infected empty carrier and NKILA is overexpressed slow virus HUVECs in, be put into incubator be incubated for 1 hour.It is washed 3 times, is removed nonadherent with the ECM culture medium of culture HUVECs cell THP-1 cell on to HUVECs is counted and is clapped to the THP-1 cell adhered on HUVECs under fluorescence microscope According to.

As shown in Figure 5A, compared with the control group of infection empty carrier, the experimental group after infection NKILA overexpression slow virus is thin The expression quantity of NKILA dramatically increases (* * * represents P value ﹤ 0.001 in figure) in born of the same parents.Importantly, as shown in Figure 5 B, NKILA's The adhesive attraction between monocyte THP-1 and endothelial cell HUVECs is significantly suppressed after expression quantity increase.

4.3 detections are overexpressed the influence to inflammatory factor in vascular endothelial cell

HUVECs is inoculated into 6 orifice plates, reaches 80% to cell density, respectively with packaged empty carrier and NKILA It is overexpressed slow-virus infection HUVECs.After infection HUVECs 48 hours, handled 1 hour with the inducer TNF α of inflammatory reaction.With Trizol lytic cell extract total serum IgE, after dnase digestion, take 1 μ g RNA carry out reverse transcription, by the cDNA after reverse transcription into Row quantitative fluorescent PCR, detection infection empty carrier and NKILA are overexpressed the expression quantity of relevant inflammatory factors after slow virus respectively.

As a result as shown in fig. 6, compared with the control group of infection empty carrier, infection NKILA is overexpressed the experiment after slow virus In group cell, the expression quantity of the inflammatory factors such as adhesion factor VCAM1 and SELE and chemotactic factor (CF) MCP1 and IL8 is all significantly reduced (* represents P value ﹤ 0.05 in figure, and * * represents P value ﹤ 0.01 in figure), again demonstrates long-chain non-coding RNA NKILA to intravascular The important inhibiting effect of dermatitis disease.

In summary, long-chain non-coding RNA NKILA (sequence is as shown in SEQ ID NO.1) is in Atheromatosis people Peripheral blood mononuclear cells in expression quantity significantly lower than normal person peripheral blood mononuclear cells in expression quantity (P value < 0.05) it, can preferably reflect the development situation of atherosclerosis, therefore can be used as the clinical diagnosis point of atherosclerosis Sub- marker, and it is applied to the Related product of preparation diagnosis or detection atherosclerosis, to enrich atherosclerosis Diagnosis and detection means.The atherosclerosis molecular marker is able to suppress monocyte THP-1 and vascular endothelial cell Adhesive attraction between HUVECs, and inhibit the expression of the inflammatory factors such as adhesion factor and chemotactic factor (CF), to be able to effectively Ground inhibits the overactivity of blood vessel endothelium inflammation, therefore is also used as inhibiting the molecule of formation and the development of atherosclerosis Drug target is the pathogenesis of further studying atherosclerotic, explores its protective agents and provide new empirical theory base Plinth and direction.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

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gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg 1020

agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat 1080

aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 1140

gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 1200

gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 1260

gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat 1320

ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag 1380

taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat 1440

taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa 1500

gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat 1560

aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt 1620

aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt 1680

atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga 1740

agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta 1800

tcggttaact tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt 1860

agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaattcaa 1920

aattttatcg atgcctcccc gtcaccaccc cccccaaccc gccccgaccg gagctgagag 1980

taattcatac aaaaggactc gcccctgcct tggggaatcc cagggaccgt cgttaaactc 2040

ccactaacgt agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc 2100

atcactgagg tggagaagag catgcgtgag gctccggtgc ccgtcagtgg gcagagcgca 2160

catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga 2220

gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg 2280

agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg 2340

ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 2400

cgggttatgg cccttgcgtg ccttgaatta cttccacgcc cctggctgca gtacgtgatt 2460

cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga 2520

gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga 2580

atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat 2640

ttttgatgat atcctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa 2700

gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc 2760

cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg 2820

tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg 2880

ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt 2940

cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt 3000

gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc 3060

acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg 3120

tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag 3180

actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag 3240

tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt 3300

caggtgtcgt gaaaactacc cctgagctcc ttaaggttaa cgccaccatg gactacaaag 3360

acgatgacga caagtctaga gaattcggat ccaatattcc cgggctcgag ccatggaagc 3420

ttgatatcta actgactgaa ccggtggtac cgatccacgc gtctccggcc tagggataac 3480

agggtaatcc gctagcccct ctccctcccc cccccctaac gttactggcc gaagccgctt 3540

ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg 3600

caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc 3660

ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga 3720

agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc 3780

tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc 3840

acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc 3900

aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga 3960

tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg 4020

ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacac 4080

tagagatcca ccggtcgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg 4140

cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 4200

gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt 4260

cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt 4320

ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc 4380

gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct 4440

ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 4500

ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg 4560

cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg 4620

gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat 4680

gacccgcaag cccggtgcct gagcggccgc gtcgacaatc aacctctgga ttacaaaatt 4740

tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 4800

gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 4860

tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 4920

gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 4980

cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 5040

gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 5100

ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg 5160

cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 5220

ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 5280

atctcccttt gggccgcctc cccgcctgga attcgagctc ggtaccttta agaccaatga 5340

cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 5400

taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag 5460

accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat 5520

aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact 5580

agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc 5640

atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact 5700

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 5760

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 5820

atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 5880

ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 5940

cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac 6000

gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa 6060

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 6120

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 6180

agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 6240

gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 6300

ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 6360

cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 6420

gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 6480

tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 6540

gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 6600

ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg 6660

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 6720

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6780

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6840

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6900

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 6960

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 7020

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 7080

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 7140

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 7200

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 7260

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 7320

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 7380

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 7440

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 7500

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 7560

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7620

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7680

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7740

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7800

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7860

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7920

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 7980

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 8040

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 8100

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 8160

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 8220

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 8280

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 8340

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8400

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 8460

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 8520

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 8580

cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 8640

gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 8700

gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 8760

gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 8820

agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgc 8865

Claims (10)

1. a kind of atherosclerosis molecular marker, which is characterized in that the atherosclerosis molecular marker is long-chain Non-coding RNA has the nucleotide sequence as shown in SEQ ID NO.1.

2. a kind of PCR amplification primer, which is characterized in that including being used for specific amplification Atherosclerosis described in claim 1 The upstream primer and downstream primer of chemoattractant molecule marker.

3. PCR amplification primer according to claim 2, which is characterized in that the upstream primer has such as SEQ ID NO.2 Shown in nucleotide sequence, the downstream primer have the nucleotide sequence as shown in SEQ ID NO.3.

4. a kind of siRNA, which is characterized in that including complementary sense strand and antisense strand, and the siRNA can be special Inhibit the expression of atherosclerosis molecular marker described in claim 1 anisotropicly.

5. siRNA according to claim 4, which is characterized in that the sense strand has as shown in SEQ ID NO.2 Nucleotide sequence, the antisense strand have the nucleotide sequence as shown in SEQ ID NO.3.

6. PCR described in any one of atherosclerosis molecular marker as described in claim 1, claim 2~3 expands Increase siRNA described in any one of primer or claim 4~5 in preparation diagnosis or the product of detection atherosclerosis In application.

7. application according to claim 6, which is characterized in that the product be detection reagent, kit, genetic chip or Test paper.

8. PCR described in any one of atherosclerosis molecular marker as described in claim 1, claim 2~3 expands Increase siRNA described in any one of primer or claim 4~5 in preparation for preventing or treating atherosclerosis Application in drug.

9. a kind of for detecting the kit of atherosclerosis, which is characterized in that expand including PCR described in claim 2 or 3 Increase primer.

10. kit according to claim 9, which is characterized in that further include that RNA extracts reagent, reverse transcription reagents and PCR At least one of amplifing reagent.