CN108531483A - A kind of and the relevant circular rna of nucleus pulposus and application - Google Patents
A kind of and the relevant circular rna of nucleus pulposus and application Download PDFInfo
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Abstract
The present invention relates to a kind of and relevant circular rnas of nucleus pulposus comprising such as SEQ ID NO:Sequence shown in 1.The circular rna can be used for preparing the medicament of early diagnosis reconstruction of nucleus gelatinosus tissue regression；It can be used for preparing the medicament of identification nucleus pulposus cell regression aging；It can be used for the auxiliary diagnosis medicament of reconstruction of nucleus gelatinosus tissue sample；It can be used for the bio-guide healing potion of nucleus pulposus regression.
Description
Technical field
The present invention relates to biomedicine technical fields, and are more particularly related to a kind of relevant with nucleus pulposus
Circular rna and its application.
Background technology
Current China aging degree getting worse, has become the most country of elderly population in the world, with aging
What is accompanied is then that age related chronic disease incidence and morbidity quantity constantly increase.Degenerative disc disease is one
The common age related chronic disease of class, can cause the symptoms such as lumbago, can seriously cause patient's disability even residual
Disease brings white elephant to family and society.Many factors can lead to the generation of Degenerative disc disease, common special
Point be cell quantity reduce and hypofunction, Extracellular Matrix Content decline and it is out of proportion etc..And nucleus pulposus is as interverbebral disc
Important component, its own aging play very crucial effect during intervertebral disc degeneration.
In recent years, circular rna (circular RNA, circRNA) is the research hotspot in the fields RNA, its own ring-type knot
The distinctive stability of structure make its have as biomarker inherent advantage, this for intervertebral disc degeneration early diagnosis and
Molecule diagnosis provides new possibility.It is found that circPVT1 in nucleus pulposus and cell by sequencing technologies, and confirms to move back
Become circPVT1 expression quantity in nucleus pulposus and aging nucleus pulposus cell to reduce, indication circRNA and nucleus pulposus regression and nucleus pulposus
Cell ageing has close relationship.CircRNA has the function of various biological, is risen in the expression regulation after genetic transcription
Important function, thus it there are relevances below with disease：First, the variation of circRNA may be the cause of disease, this is because
The inhibiting factor and promotive factor of disease all may be the target site of circRNA, when disorderly table first has occurred in circRNA itself
It reaches, final result can all lead to the variation of downstream series of genes expression and the whole disorder of certain accesses, and then induce
Disease occurs；Secondly, the variation of circRNA is also likely to be disease as a result, this is because when disease occurs, and can cause to dye
The loss of body segment, the mutation of gene or the violent amplification of chromosome segment, if circRNA is placed exactly in this varied sections
It is interior, then highly significant variation will occur for its expression quantity.
Therefore, theoretically circRNA can be used as a new class of disease markers, its specific variations certainty and disease
It is associated to generate development.CircRNA is also used as potential drug target simultaneously, is raised in lysis by inhibiting
CircRNA or be overexpressed lower circRNA, it would be possible to greatly alleviate disease occurrence and development.
In conclusion work out a kind of circular rna can be used for provide such use, be those skilled in the art urgently
The technical problem solved.
Invention content
The present invention is in view of the above-mentioned problems, be designed to provide a kind of and the relevant circular rna of nucleus pulposus and its application.
In order to achieve the above objectives, the present invention adopts the following technical scheme that：
On the one hand, the invention discloses a kind of and relevant circular rnas of nucleus pulposus, including such as SEQ ID NO:1 institute
The sequence shown.
Second aspect, the embodiment of the invention also discloses above-mentioned circular rnas to be used to prepare early diagnosis nucleus pulposus group
Knit the application of the medicament of regression；It is used to prepare the application of the medicament of identification nucleus pulposus cell regression aging；For nucleus pulposus group
Knit the application of the auxiliary diagnosis medicament of sample；The application of bio-guide healing potion for nucleus pulposus regression.
The beneficial effects of the invention are as follows：
Circular rna according to the present invention is used to prepare the medicament with reconstruction of nucleus gelatinosus tissue relevant disease, can be used as new
Disease markers, also can be used as potential drug target, can greatly alleviate the hair tonic and development of disease, to human body vertebra
The diagnosing and treating of disc disease is of great significance.
Description of the drawings
Fig. 1 is the expression of circPVT1 in normal disc tissue and degeneration intervertebral disc tissue in one embodiment of the invention
Level view；
Fig. 2 is that cyclic annular rna expression level illustrates in the 2nd, 4,6 generation cell of nucleus pulposus in one embodiment of the invention；
Fig. 3 is normal cell and the intracellular circPVT1 interfered by circPVT1siRNA in one embodiment of the invention
Expression illustrates；
Fig. 4 is related to the intracellular aging interfered by circPVT1siRNA point of normal cell in one embodiment of the invention
The gene expression amount of sub- p53, p21, p16 illustrate；
Fig. 5, Fig. 6 are the influence diagrams for interfering circPVT1 expression to nucleus pulposus cell aging.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
Embodiment 1
RT-PCR verifies the circPVT1 differential expressions of regression nucleus pulposus and normal nucleus pulposus.
1, sample collection
2, people's normal disc tissue is derived from wound and causes vertebral burst fracture patient.5, people's degeneration intervertebral disc tissue,
It is derived from intervertebral disk retrogression disease surgery patient, average age 55 years old is severe regression according to Gr1es (5) standards of grading.
Patient is obtained when sample collection and its family members agree to.
2, nucleus pulposus extracts
Normal nucleus pulposus：Normal disc tissue is won in operation, it is seen that the fibrous ring and center jelly of periphery white
The nucleus pulposus of sample.Interverbebral disc is impregnated with the physiological saline containing dual anti-(a green streptomysin) 10 minutes, with curet gently by nucleus pulposus
Tissue is detached from interverbebral disc, and dual anti-physiological saline soaking flushing is until without apparent bloodstain, about 3-4 times.
Regression nucleus pulposus：Intervertebral spinal fusion mirror perform the operation from side approach, cannot completely take out interverbebral disc.Detach fibrous ring group
When knitting with nucleus pulposus, distinguished by naked eyes, feel etc..Nucleus pulposus is in gelatin translucent, and fibrous ring is white
Flexible shape.Nucleus pulposus is easy to shred with eye scissors, immalleable.
3, RNA is extracted
The vessel such as pestle and homogenizer will be ground and do roasting 4h at 200 DEG C, remove RNA enzyme, it is cooling；It is added in liquid nitrogen and is pre-chilled, by group
It knits and is taken out rapidly from liquid nitrogen, is crushed into powder；Tissue is put into the homogenizer for being previously added TRIzol reagents with curet, is homogenized
Several minutes；Liquid after homogenate is transferred in the centrifuge tube of no RNA enzyme, after chloroform is added, 4 DEG C of centrifugation layerings；By upper strata aqueous phase
It is transferred in a centrifuge tube without RNA enzyme, after chloroform is added, 4 DEG C of centrifugation layerings；Upper strata aqueous phase is transferred to a centrifugation without RNA enzyme
Guan Zhong adds isopropanol, 4 DEG C of centrifugation RNA；Precipitation is washed with 75% ethyl alcohol 2 times；It is heavy with the deionized water dissolving of no RNA enzyme
It forms sediment.The RNA of extracting carries out Quality Identification (using spectrophotometric determination RNA concentration, purity and integrality).Quality Identification is good
It is for use that RNA is stored in -80 DEG C of refrigerators.
4, reverse transcription
Using the reverse transcription reagent box (DRR047) of TAKARA companies, 1 μ g RNA are inverted.Reaction system and reaction
Condition is carried out with reference to kit specification.
5, RT-PCR reacts
Using cDNA as template, SYBR (R) Premix of PCR primer and Takara for target circRNA is used
ExTaqTM quantitative fluorescent PCR systems are expanded on stratagen MX3000P quantitative PCR apparatus, and measure the Ct of sample
Value.The formula that gained Ct values are obtained by substituting into bioassay standard curve, is obtained using the computational methods of absolute quantitation in sample
Target circRNA content.GAPDH genes are standardized correction as interior shine to circRNA qPCR testing results.
It is as follows for the primer of circPVT1：
Forward primer is 5 '-CGACTCTTCCTGGTGAAGCATCTGAT-3 ',
Reverse primer is 3 '-TACTTGAACGAAGCTCCATGCAGC-5 '
It is as follows for the primer of GAPDH：
Forward primer：5’-CTCTCTGCTCCTCCTGTTCG-3’
Reverse primer：5’-TTAAAAGCAGCCCTGGTGAC-3’
6, the results are shown in Figure 1, compared with normal disc tissue, the expression water of circPVT1 in degeneration intervertebral disc tissue
It is flat to significantly reduce.
Embodiment 2
RT-PCR verifies the circPVT1 differential expressions in normal nucleus pulposus cell and continuous passage renaturation aging nucleus pulposus cell.
1, cell extraction and culture
Normal disc nucleus pulposus is put into the 100ml beakers for filling 10m12%II Collagenase Types, and magnetic stirring apparatus stirs
It mixes about 60 minutes.After tissue is completely dissolved, 1000r/min is centrifuged 10 minutes, supernatant is sucked out, with containing 10% fetal calf serum
DMEM culture mediums lml gently dispels cell, is drawn in 50ml culture bottles, and the DMEM culture medium 6-8ml of 10% fetal calf serum are added,
It is statically placed in 37 DEG C, saturated humidity, cultivates 3 days in 5%CO2 incubators.Inverted microscope observes cell adherent growth situation after 3 days,
Liquid is changed every other day.
2. building nucleus pulposus cell continuous passage replicative senescence model
Extraction gained nucleus pulposus cell is placed in and is statically placed in 37 DEG C, saturated humidity, cultivates in 5%CO2 incubators.When cell grow to
When cell bottle 70%-80%, primary rear trypsin digestion cell is cleaned with sterile PBS, gained cell will be digested with 1：3 mixings are distinguished
It is inoculated in three new culture bottles, is denoted as second generation cell.It is and so on passaged to the 6th generation cell, collects the 2nd, 4,6 generation cells
Carry out subsequent experimental.
2, RNA is extracted
2,4,6 generation cell of gained is put into and is previously added in TRIzol reagents, is cracked on ice several minutes；By the liquid after cracking
Body is transferred in the centrifuge tube of no RNA enzyme, after chloroform is added, 4 DEG C of centrifugation layerings；Upper strata aqueous phase is transferred to a centrifugation without RNA enzyme
Guan Zhong, after chloroform is added, 4 DEG C of centrifugation layerings；Upper strata aqueous phase is transferred in a centrifuge tube without RNA enzyme, adds isopropanol, 4 DEG C from
The heart precipitates RNA；Precipitation is washed with 75% ethyl alcohol 2 times；It is precipitated with the deionized water dissolving of no RNA enzyme.The RNA of extracting carries out quality
Identification (measures RNA concentration, purity and integrality) with Agilent 2100.The good RNA of Quality Identification is stored in -80 DEG C of refrigerators and waits for
With.
3, reverse transcription
Using the reverse transcription reagent box (DRR047) of TAKARA companies, 1 μ g RNA are inverted.Reaction system and reaction
Condition is carried out with reference to kit specification.
4, RT-PCR reacts
Using cDNA as template, SYBR (R) Premix of PCR primer and Takara for target circRNA is used
ExTaqTM quantitative fluorescent PCR systems are expanded on stratagen MX3000P quantitative PCR apparatus, and measure the Ct of sample
Value.The formula that gained Ct values are obtained by substituting into bioassay standard curve, is obtained using the computational methods of absolute quantitation in sample
Target circRNA content.GAPDH genes are standardized correction as interior shine to circRNA qPCR testing results.
It is as follows for the primer of circPVT1：
Forward primer is 5 '-CGACTCTTCCTGGTGAAGCATCTGAT-3 ',
Reverse primer is 3 '-TACTTGAACGAAGCTCCATGCAGC-5 '
It is as follows for the primer of GAPDH：
Forward primer：5’-CTCTCTGCTCCTCCTGTTCG-3’
Reverse primer：5’-TTAAAAGCAGCCCTGGTGAC-3’
5, result
As shown in Fig. 2, the 2nd, 4,6 generation cell of nucleus pulposus, the expression of circRNA continuously decreases in cell.
Embodiment 3
RT-PCR verification interference circPVT1 expresses the influence to nucleus pulposus cell aging
1, siRNA of the design synthesis for circPVT1
Sequence is：CUGUCAGCUGCAUGGAGCUUCGU(SEQ ID NO:1).
2, cell extraction and culture
Normal disc nucleus pulposus is put into the 100ml beakers for filling 10m12%II Collagenase Types, and magnetic stirring apparatus stirs
It mixes about 60 minutes.After tissue is completely dissolved, 1000r/min is centrifuged 10 minutes, supernatant is sucked out, with containing 10% fetal calf serum
DMEM culture mediums lml gently dispels cell, is drawn in 50ml culture bottles, and the DMEM culture medium 6-8ml of 10% fetal calf serum are added,
It is statically placed in 37 DEG C, saturated humidity, cultivates 3 days in 5%CO2 incubators.Inverted microscope observes cell adherent growth situation after 3 days,
Liquid is changed every other day.
3, cell transfecting
Nucleus pulposus cell is divided into 2 groups, respectively control group, circPVT1 siRNA interference groups, nucleus pulposus cell is used and is turned
Transfection reagent LipofectamineTM 2000 is transfected, and transfection method is with reference to specification.It collects each group cell and uses within 4 days after transfection
In subsequent experimental.
4, RT-PCR is tested
Cell total rna extracts and PCR step is the same as embodiment 2.
5, result
Such as Fig. 3, shown in Fig. 4, wherein Fig. 3 indicates compared with the control group, circPVT1 siRNA interference groups it is intracellular
The level of circPVT1 is significantly lowered, and shows that circPVT1 is interfered to express successfully；Fig. 4 indicate compared with the control group, circPVT1
Intracellular aging relevant molecule p53, p21, p16 gene expression amount of siRNA interference groups significantly increases.
Embodiment 4
SA- β-gal dyeing detection verification interference circPVT1 expresses the influence to nucleus pulposus cell aging.
1, the aging situation of SA- β-gal dyeing detection nucleus pulposus cell.SA- β-gal are a kind of lifes for identifying senile cell
Object marker.Illustrate to detect according to cell ageing assay kit (Cell Signaling Technology companies), observe
And the case where having the percentage of cells of blue under counting microscope in cytoplasm, judging cell ageing.Every group sets 3 multiple holes, often
Secondary experiment is repeated 3 times.
2, cell culture and transfection are the same as embodiment 3.
3, cell transfecting carries out SA- β-gal dyeing after 4 days.
4, result
Such as Fig. 5, shown in Fig. 6, cellular control unit group blue cell average percentage is 5%, circPVT1 interference group cells
Group blue cell average percentage is 27%, and difference has statistical significance (P<0.05), the above results show to interfere
CircPVT1 expresses the aging course that can accelerate nucleus pulposus cell, and the enlightenment for giving us is can be by increasing circPVT1 tables
The mode that reaches inhibits cell ageing.
The foregoing is merely presently preferred embodiments of the present invention, practical range not for the purpose of limiting the invention；If do not taken off
It from the spirit and scope of the present invention, modifies or equivalently replaces the present invention, should all cover in the claims in the present invention
In protection domain.
Sequence table
<110>The second affiliated hospital of army medical university of ground force of the Chinese People's Liberation Army
<120>A kind of and the relevant circular rna of nucleus pulposus and application
<130> 18P99118-CN
<141> 2018-04-09
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cugucagcug cauggagcuu cgu 23
<210> 2
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cgactcttcc tggtgaagca tctgat 26
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tacttgaacg aagctccatg cagc 24
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctctctgctc ctcctgttcg 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ttaaaagcag ccctggtgac 20
Claims (5)
1. a kind of and relevant circular rna of nucleus pulposus, which is characterized in that including such as SEQ ID NO:Sequence shown in 1.
2. circular rna described in claim 1 is used to prepare the medicament of early diagnosis reconstruction of nucleus gelatinosus tissue regression.
3. circular rna described in claim 1 is used to prepare the medicament of identification nucleus pulposus cell regression aging.
4. circular rna described in claim 1 is used for the auxiliary diagnosis medicament of reconstruction of nucleus gelatinosus tissue sample.
5. circular rna described in claim 1 is used for the bio-guide healing potion of nucleus pulposus regression.
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Cited By (2)
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| CN110916842A (en) * | 2019-11-28 | 2020-03-27 | 中国人民解放军陆军军医大学 | Rat cartilage degeneration induction fiber ring and nucleus pulposus degeneration model and application |
| CN111057764A (en) * | 2019-12-25 | 2020-04-24 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of CircRNA PVT1 and peptide fragment in tumor growth prediction, metastasis prediction, prognosis evaluation and treatment |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110916842A (en) * | 2019-11-28 | 2020-03-27 | 中国人民解放军陆军军医大学 | Rat cartilage degeneration induction fiber ring and nucleus pulposus degeneration model and application |
| CN111057764A (en) * | 2019-12-25 | 2020-04-24 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of CircRNA PVT1 and peptide fragment in tumor growth prediction, metastasis prediction, prognosis evaluation and treatment |
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