CN105985962B - The aptamer of the selectively targeted inflammatory synovial cell of rheumatoid arthrosis and its application - Google Patents
The aptamer of the selectively targeted inflammatory synovial cell of rheumatoid arthrosis and its application Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, provide the aptamer of selectively targeted inflammatory synovial cell in human rheumatoid joint a kind of, and the aptamer has the nucleotide sequence of SEQ ID NO.1；Invention further provides the aptamers in the application of the bioprobe of preparation identification inflammatory synovial cell and in the application of preparation targeting resisting rheumatoid arthritis drug or pharmaceutical carrier.Aptamer of the present invention is highly selective to having for inflammatory synovial cell, and eliminates or weaken in conjunction with liver cell；Bioprobe containing aptamer of the present invention can recognize external people's inflammatory synovial cell, such as the inflammatory synovial cell in patient RA synovial tissue slice, provide foundation for clinic RA diagnosis and targeted therapy.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of selectively targeted inflammatory synovial membrane of human body rheumatoid arthrosis
The single-strand DNA aptamer of cell and its application.
Background technique
Rheumatoid arthritis (Rheumatoid Arthritis, RA) is a kind of to become special with multi-joint synovial membrane inflammation venereal disease
The chronic generalized autoimmune disease of sign, is known as " first of disabling disease ", not dead cancer is known as by world medical circle,
One of the four big persistent ailments of human health are threatened as 21 century.RA disease incidence in adult is higher, in every 100,000 adult population about
There is the disease incidence of 20-40%.The RA patient of studies have shown that 10% may occur in which serious dysfunction in morbidity 2 years, about
50% patient disability after illness 10 years.Currently, China RA patient year per capita disease expense up to 13506 yuan of people
Coin accounts for the 51% of current year per-capita gross domestic product (GDP), all causes serious financial burden to patient and society.
RA there is no specific treatment method so far.Traditional antirheumatic includes non-steroidal anti-inflammatory drugs, hormone, antirheumatic drug
Etc. curative effects it is not high, and there are toxic side effect and drug resistances.Novel biological agent can be effectively improved the RA state of an illness, become RA in recent years and control
The new milestone treated.Tumor necrosis factor (TNF) inhibitor has become the Consensus biopharmaceuticals for RA.However, connecing
By tnf inhibitor treat patient there are the risk of the complication such as severe infections, limit clinical application.Therefore, exploitation has
Efficiently, less toxic anti-RA alternative medicine is particularly necessary.
Oligonucleotide aptamer is the cluster small molecule DNA with target substance specific binding screened by SELEX process
Or RNA segment is studied more noticeable as the promising substitution molecule of antibody molecule.SELEX(systematic
Evolution of ligands by exponential enrichment) technology is that one kind that the nineties grow up is new
In-vitro screening technology, it uses the random oligonucleotide library of large capacity, and the outer PCR amplification of combination, with index richness
It is suitable to obtain high affinity, the oligonucleotides of high specificity by multi-turns screen for the oligonucleotides of collection and target molecule specific bond
Gamete (aptamers) successfully applies to the screening of many target molecules, including metal ion, organic dyestuff, protein, medicine
Object, amino acid and various cell factors etc., or even can be used for complete cell, virus, spore and prion etc..This method
Have the characteristics that it is easy, quick, economical, with other combinatorial chemical libraries such as random peptide library, antibody library and phage display text
Library is compared, and the aptamer filtered out from oligonucleotide library has higher affinity and specificity, and nucleic acid is adapted in recent years
Son is constantly applied to basic research, drug development, clinical application etc., has a good application prospect.Specific bond in
The nucleic acid aptamer of sick cell is conducive to the diagnosing and treating of disease.Compared with antibody, aptamer has the following advantages: chemistry is steady
Qualitative height can be resistant to 80 DEG C of high temperature, can store under the intolerable various solvents of protein drug and mal-condition；Nothing is exempted from
Epidemic focus, can repetitively administered, safety be better than monoclonal antibody；Differences between batches are small, and quality is stablized, and are conducive to industrialized production；
It is easy to carry out various chemical modifications, convenient for extending its application range；Relative molecular mass is small, it is readily permeable enter organization internal.Base
In above-mentioned advantage, aptamer can substitute antibody in terms of many biologic applications.Aptamer occurs during the last ten years, being applied to enzyme
It is external real to join oligonucleotides experiment, flow cytometer, aptamer chip (aptamer chip), protein quantification and protein purification etc.
It tests, while having the function of that interaction or inhibitory enzyme activity of growth factor and receptor etc. can be inhibited in vivo, can also carry out body
Interior imaging etc..Aptamer can be used as diagnosing and treating reagent, can recognize when for diagnosing and combines certain target molecules, for treating
When can be used as targeting conveying, function blocking substance or directly block disease process.CN 200610015738.0 discloses one group
With human breast carcinoma tissue high specific and the nucleic acid aptamer of high-affinity and the preparation method and application thereof；
CN201210224174.7 discloses a kind of nucleic acid aptamer molecular beacon probe for platelet derived growth factor detection；
CN201410468490.8 discloses a kind of cardiac muscle troponin I nucleic acid aptamer and its application, kit；CN
201010295767.3 disclosing nucleic acid aptamer and its screening technique and the application of a kind of specific recognition Shigella；
CN201210240284.2 disclose it is a kind of can specific bond tubercle bacillus antigen nucleic acid aptamer and its application.
However up to the present, it not yet finds successfully to be used to develop Diagnosis of Rheumatoid Arthritis examination for SELEX screening technique
The report of agent and therapeutic agent.
Summary of the invention
For the above technical situation, the present invention provides a species specificity, high-affinity combination human rheumatoid joint are scorching
Property synovial cell's film surface single-strand DNA aptamer, the aptamer have following SEQ ID NO.1 shown in nucleotides sequence
Column: SEQ ID NO.1:5 '-GCAAC CAGGG GAACC GGGGA ATTGGAACGC TTTCC TCCCG TTGGC-3 '；Institute
It states aptamer and shows secondary structure with Fig. 4.
The present invention also provides a kind of screening technique of aptamer that selectively targeted inflammatory synovial membrane is thin, the method packets
It includes:
(1) synthesis of the building of ssDNA pool and primer
The random library that length is 81nt is constructed, both ends are fixed sequence program, and centre is 45 nucleotide random sequences, storage capacity
Amount is about IO15-IO16, upstream and downstream primer is designed according to ssDNA pool both ends fixed sequence program.
The sequence in the library ssDNA: 5 '-ATCCAGAGTGACGCAGCA- (45N)-TGGACA CGG TGG CTT AGT-
3 ', wherein N represents A, T, C, tetra- randomized bases of G；
Upstream primer P1:5 ' FITC-ATC CAG AGT GAC GCA GCA-3 '；
Downstream primer P2:5 ' Biotin-ACTAAG CCA CCG TGT CCA-3 '；
(2) SELEX is screened
The culture of target cell (human desmocyte synovial cell) and negative selection cell (liver cell)；
Target cell, negative selection cell and the library ssDNA are incubated for；
The amplification of ssDNA in conjunction with target cell；
Streptavidin paramagnetic particle method prepares the library ssDNA；
It repeats the above process, carries out 8-12 wheel screening altogether；
(3) each wheel of flow cytometer monitoring of DNA aptamer screens enrichment condition
It prepares FITC and marks ssDNA；
The processing of target cell；
Target cell and FITC label ssDNA are incubated for；
Flow cytometry, the case where enrichment is compared by the size of fluorescence intensity level.
(4) clone and sequencing
The ssDNA obtained through multi-turns screen PCR amplification dsDNA, recovery purifying connect carrier T, screen through blue hickie, with
Machine selects multiple clones and carries out sequencing.
(5) structural analysis of DNA aptamer I and II and prediction and homologous sequence analysis.
As one of embodiment of the present invention, the screening technique further comprises:
(6) identification of DNA aptamer specificity
The affinity dissociation that Flow cytometry DNA aptamer and target cell (human desmocyte sample synovial cell) combine is normal
Number Kd；
DNA aptamer specificity identifies the DNA aptamer screened by Flow cytometry and human desmocyte
The affinity of sample synovial cell, liver cell and people's Kupffer cell are to identify its specificity.
The identification of DNA aptamer selectivity in vitro
Pass through fluorescence microscope imaging and streaming fluorescence intensity change, choosing of the preliminary analysis ssDNA aptamer to target cell
Selecting property.
As one of embodiment of the present invention, the human desmocyte sample synovial cell in the screening technique includes but is not limited to
MH7A cell.
As one of embodiment of the present invention, the liver cell in the screening technique includes but is not limited to L-02 cell.
As one of embodiment of the present invention, the adaptation of the selectively targeted inflammatory synovial cell in human rheumatoid joint
Son screening technique include:
(1) random single-stranded DNA banks and primer are synthesized
Random library:
5’-ATCCAGAGTGACGCAGCA-(45N)-TGGACACGGTGGCTTAGT-3’
5 ' primers: 5 '-FITC-ATCCAGAGTGACGCAGCA-3 '
3 ' primers: 5 '-biotin-ACTAAGCCACCGTGTCCA-3 '
(2) SELEX is screened
Human desmocyte sample synovial cell MH7A (target cell) and human liver cells L-02 (negative selection cell) cell culture and sieve
Select pre-treatment.The good cell of upgrowth situation is handled using the cell dissociation buffer of no enzyme, by 2 × 105A cell inoculation is in P100
It is cultivated in culture dish, when the fusion rate of cell reaches 95% screening that can be used to library.
Target cell, negative selection cell and the library ssDNA are incubated for and screening
Collect about 1 × 106A MH7A cell, with Washing Buffer (DPBS+CaCl2+MgCl2) washing cell 2 times,
Binding Buffer(DPBS+CaCl2+MgCl2+ tRNA) washing cell it is primary；
By the target cell after above-mentioned library 8nmol and washing in 37 DEG C of incubation 1h, cell 2 is washed with Washing Buffer
It is secondary, ultrapure water 500ul, 95 DEG C of water-bath 10min are added into cell precipitation,
In 4 DEG C, 13600g is centrifuged 10min, collects supernatant, screens the ssDNA of specific bond in supernatant containing the first round, on
Clear liquid can be used for PCR amplification, the sub-library of preparation next round screening as template.- 80 DEG C freeze it is spare.
Single-stranded DNA banks PCR amplification after first round screening
Single-stranded DNA banks are expanded as double-stranded DNA library, amplification condition by upstream and downstream primer are as follows: in 95 DEG C of initial denaturations
3min, then 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 30s are carried out, totally 12 recycle, 72 DEG C of extension 3min；
Using double-stranded DNA PCR product as template, carries out second of amplification cycles number and grope, amplification condition are as follows:
95 DEG C of initial denaturation 3min, then carry out 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 30s, expand 6 respectively, 10,14,18,
22,26,30 circulations, 72 DEG C of extension 3min；
3% gel electrophoresis of pcr amplification product row is identified, best amplification cycles number 5 is selected；
It is 5 (Fig. 1) that remaining first time PCR product, which is continued amplification cycles number, according to above-mentioned PCR condition；
Streptavidin MagneSphere method separates single stranded DNA preparation the second wheel screening sub-library；
By following condition massive amplification double-stranded DNA in case preparing single-stranded；
Preparation ssDNA is separated using streptavidin magnetic bead method；
Streptavidin magnetic bead is washed 3 times with 1 × PBS, PCR product is dissolved in ddH2In 0, it is incubated at room temperature with magnetic bead
1h, through a chain with biotin labeling in conjunction with Streptavidin MagneSphere；1 × PBS is washed 3 times, and 0.25mol/L is added
NaOH is denaturalized 5min at room temperature, and another chain in conjunction with target cell is by separate out；Upper magnetic frame, room temperature 3min are drawn
Supernatant is transferred in EP pipe, and the dense HCl that certain volume is added is neutralized, and nucleic acid quantification instrument surveys ssDNA concentration, for the next round of screening.
4th wheel of screening introduces negative selection cell and carries out abatement cell-SELEX screening；
By the 4th wheel screening the library ssDNA be immediately placed on after 95 DEG C of denaturation 5min be fully cooled on ice it is spare；
Take about 1 × 107Negative sieve cell L-02 is washed 2 times with Washing buffer, screens text after ice bath denaturation is added
Library, 37 DEG C of incubation 1h, careful collection combine rear supernatant, obtain the library ssDNA, that is, subtractive library in conjunction with subtractive cell line.
Continue the screening that target cell specific bond ssDNA aptamer is carried out according to aforesaid operations, and every wheel is followed while being born
Screening；
(3) fluorescence microscope identification and streaming monitoring
After FTIC-ssDNA after about 10 wheel screenings is neutralized with hydrochloric acid HCl, detectable concentration is used for streaming or fluorescence microscopy
Mirror monitors each wheel enrichment condition；
Streaming monitors the target cell processing of enrichment condition；
The EDTA liquid without pancreatin is used to digest target cell, in case damaging cells film surface component, influences nucleic acid aptamer
And the combination of target cell；Collect MH7A cell, wash 2 times with PBS, with Binding Buffer resuspension cell and be adjusted to 1 ×
106/ml；
Target cell and FITC label ssDNA are incubated for
It filters out library ssDNA pool and target cell MH7A to wash cell 3 times in 37 DEG C of incubations 30min, PBS, addition
Cell precipitation, up flow type detection is resuspended in 450ulI × PBS.
Fluorescence microscope monitors aptamer situation in conjunction with target cell
The subtractive cell line for being inoculated with proper density and target cell are in the burnt capsule of copolymerization, adherent overnight growth；It is light with 1 × PBS
Light rinsing 2 times, Binding buffer washed once；Be added Bindingbuffer of the 200ul containing FITC-ssDNApool, 37
DEG C be incubated for 30min；1 × PBS is rinsed cell 3 times；Fluorescence microscopy combines situation under the microscope.
(4) carrier cloning and sequencing
The PCR4-TOPO vector of following (6ul): the 1ul (10ng) of reaction system；The salt solution of 1ul；At most
The ssDNA pool of 4ul (30ng) is mixed gently, and reacts at room temperature 30min；On ice or -20 DEG C freeze；
It takes 10ul connection product to be added in host's bacterium competence cell of 100ul ice bath, mixes gently, ice bath 30min, 42
DEG C water-bath heat shock 90sec, immediately ice bath 2min；The not antibiotic room temperature SOC culture medium of 700ul, 37 DEG C of slowly vibrations are added
Shake 45min；It takes appropriate volume bacterium solution to be coated on LB/Amp/IPTG/X-gal plate, is inverted overnight incubation in 37 DEG C of incubators.
Picking white monoclonal every other day carries out bacterium colony PCR identification and is sequenced.
(5) oligonucleotide aptamer primary sequence and secondary structure analysis
Utilize 4.5 software of RNA structure, DNAMAN software and MEME online software.
Http:// meme.sdsc.edu/meme/cgi-bin/meme.cgi carries out having such as SEQ ID NO.1
Shown in nucleotide sequence single-strand DNA aptamer: SEQ ID NO.1:5 ' GCAAC CAGGG GAACC GGGGA ATTGG
AACGC TTTCC TCCCG TTGGC3'；There is the aptamer Fig. 4 to show secondary structure.
As one of embodiment of the present invention, the method further includes:
(6) identification of DNA aptamer specificity (referring to Fig. 3)
The DNA aptamer and human desmocyte sample synovial cell MH7A, people liver screened by Flow cytometry are thin
The binding force of born of the same parents L-02 and people's Kupffer cell is to identify its specificity.
The single-stranded oligonucleotide aptamer to be detected of synthesis FITC label is prepared respectively；
Above-mentioned survey cell to be checked is washed with 1 × PBS, cell is resuspended with Binding buffer and adjust separately as 1 ×
106/ml。
Take about 400pmol FITC-ssDNA respectively with above-mentioned cell incubation about 30min；Centrifugation carefully removes supernatant, uses PBS
Washing 2 times, adds 450ulPBS that cell is softly resuspended；
Flow cytomery compares the combination situation of aptamer by fluorescence intensity level.
The present invention further provides the functionalization biomolecule that a kind of above-mentioned single-strand DNA aptamer couples fluorescent molecule formation
Probe can identify the inflammatory synovial cell in histotomy；
The present invention further additionally provides a kind of above-mentioned single-strand DNA aptamer or derivatives thereof and is preparing rheumatoid arthrosis
Application in scorching targeting diagnosis reagent.
Invention further provides a kind of above-mentioned single-strand DNA aptamer or derivatives thereof in preparation targeting
Application in resisting rheumatoid disease arthritis drug or targeting delivery vehicles.
The present invention is respectively to screen target and abatement target cell with human desmocyte sample synovial cell MH7A and human liver cells L-02,
Using abatement cell-SELEX technology, filtering out can specific recognition human desmocyte sample synovial cell MH7A and nonrecognition human liver cell
The single-strand DNA aptamer of L-02, while remaining highly selective to synovial cell, eliminate or to weaken it possible with liver cell
In conjunction with.
With fluorescein or the functionalization probe biomolecule of quantum dot-labeled aptamer of the present invention, it can recognize that external people is scorching
Property synovial cell.The functionalization probe biomolecule that fluorescein or quantum dot couple aptamer of the present invention can recognize that patient RA is sliding
Inflammatory synovial cell in membrane tissue slice, this provides foundation for clinic RA diagnosis and targeted therapy.
Detailed description of the invention
Fig. 1: the amplified production electrophoresis result of aptamer ssDNA in conjunction with MH7A target cell；
Fig. 2: each wheel of flow cytometer monitoring of DNA aptamer screens enrichment condition；
Fig. 3: fluorescent marker aptamer selectivity in vitro research；
Fig. 4: the secondary structure map of aptamer.
Specific embodiment
Following embodiment does not limit effective range of the invention for the present invention is further explained in any manner.
Embodiment 1
Cut down the nucleic acid aptamer of cell-SELEX screening specific bond human desmocyte sample synovial cell MH7A
(1) random single-stranded DNA banks and primer are synthesized
Random library:
5’-ATCCAGAGTGACGCAGCA-45nt-TGGACACGGTGGCTTAGT-3’
5 ' primers: 5 '-FITC-ATCCAGAGTGACGCAGCA-3 '
3 ' primers: 5 '-biotin-ACTAAGCCACCGTGTCCA-3 '
(2) SELEX is screened
2.1 human desmocyte sample synovial cell MH7A (target cell) and with human liver cells L-02 (negative selection cell) cell culture
And screening pre-treatment.
The good cell of upgrowth situation is handled using the cell dissociation buffer of no enzyme, by 2 × 105A cell inoculation is trained in P100
It supports and is cultivated in ware, when the fusion rate of cell reaches 95% screening that can be used to library.
2.2 target cells, negative selection cell and the library ssDNA are incubated for and screening
Collect about 1 × 106A MH7A cell washs cell 2 with Washing Buffer (DPBS+CaCl2+MgCl2)
Secondary, it is primary that Binding Buffer (DPBS+CaCl2+MgCl2+tRNA) washs cell；
By the target cell after above-mentioned library 8nmol and washing in 37 DEG C of incubation 1h, cell 2 is washed with Washing Buffer
It is secondary, ultrapure water 500ul, 95 DEG C of water-bath 10min are added into cell precipitation,
In 4 DEG C, 13600g is centrifuged 10min, collects supernatant, screens the ssDNA of specific bond in supernatant containing the first round, on
Clear liquid can be used for PCR amplification, the sub-library of preparation next round screening as template.- 80 DEG C freeze it is spare.
Single-stranded DNA banks PCR amplification after first round screening.
Single-stranded DNA banks are expanded as double-stranded DNA library, amplification condition by upstream and downstream primer are as follows:
In 95 DEG C of initial denaturation 3min, then 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 30s are carried out, totally 12 circulations, 72 DEG C of extensions
3min；
Using double-stranded DNA PCR product as template, carries out second of amplification cycles number and grope, amplification condition are as follows:
95 DEG C of initial denaturation 3min, then carry out 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 30s, expand 6 respectively, 10,14,18,
22,26,30 circulations, 72 DEG C of extension 3min；
3% gel electrophoresis of pcr amplification product row is identified, best amplification cycles number 5 is selected；
It is 5 (Fig. 1) that remaining first time PCR product, which is continued amplification cycles number, according to above-mentioned PCR condition；
Streptavidin MagneSphere method separates single stranded DNA preparation the second wheel screening sub-library；
By following condition massive amplification double-stranded DNA in case preparing single-stranded；
Preparation ssDNA is separated using streptavidin magnetic bead method；
Streptavidin magnetic bead is washed 3 times with 1 × PBS, PCR product is dissolved in ddH2In 0, it is incubated at room temperature with magnetic bead
1h, through a chain with biotin labeling in conjunction with Streptavidin MagneSphere；
1 × PBS is washed 3 times, and 0.25mol/L NaOH is added and is denaturalized 5min at room temperature, and another in conjunction with target cell
Chain is by separate out；Upper magnetic frame, room temperature 3min draw supernatant and are transferred in EP pipe, and the dense HCl that certain volume is added is neutralized, core
Sour quantitative instrument surveys ssDNA concentration, for the next round of screening.
4th wheel of screening introduces negative selection cell and carries out abatement cell-SELEX screening
By the 4th wheel screening the library ssDNA be immediately placed on after 95 DEG C of denaturation 5min be fully cooled on ice it is spare；
Take about 1 × 107Negative sieve cell L-02 is washed 2 times with Washing buffer, screens text after ice bath denaturation is added
Library, 37 DEG C of incubation 1h, careful collection combine rear supernatant, obtain the library ssDNA, that is, subtractive library in conjunction with subtractive cell line.
Continue the screening that target cell specific bond ssDNA aptamer is carried out according to aforesaid operations, and every wheel is followed while being born
Screening.
(3) fluorescence microscope identification and streaming monitoring
After FTIC-ssDNA after about 10 wheel screenings is neutralized with hydrochloric acid HCl, detectable concentration is used for streaming or fluorescence microscopy
Mirror monitors each wheel enrichment condition target cell that I.7.2 streaming monitors enrichment condition and handles；
The EDTA liquid without pancreatin is used to digest target cell, in case damaging cells film surface component, influences nucleic acid aptamer
And the combination of target cell.Collect MH7A cell, wash 2 times with PBS, with Binding Buffer resuspension cell and be adjusted to 1 ×
106/ml；
Target cell and FITC label ssDNA are incubated for
It filters out library ssDNA pool and target cell MH7A to wash cell 3 times in 37 DEG C of incubations 30min, PBS, addition
Cell precipitation, up flow type detection is resuspended in 1 × PBS of 450ul.
Fluorescence microscope monitors aptamer situation in conjunction with target cell (referring to Fig. 1)
The subtractive cell line for being inoculated with proper density and target cell are in the burnt capsule of copolymerization, adherent overnight growth；It is light with 1 × PBS
Light rinsing 2 times, Binding buffer washed once；The Binding buffer of 200ul pool containing FITC-ssDNA is added,
37 DEG C of incubation 30min；1 × PBS is rinsed cell 3 times；Fluorescence microscopy combines situation under the microscope.
(4) carrier cloning and sequencing
Reaction system is following (6ul):
PCR4-TOPO vector 1ul(10ng)；
Salt solution (salting liquid) 1ul；
SsDNA pool is up to 4ul (30ng)；
It mixes gently, reacts at room temperature 30min (can amplify in right amount according to above-mentioned reaction system)；On ice or -20 DEG C freeze；
It takes 10ul connection product to be added in host's bacterium competence cell of 100ul ice bath, mixes gently, ice bath 30min,
42 DEG C water-bath heat shock 90 seconds, ice bath 2min immediately；The not antibiotic room temperature SOC culture medium of 700ul, 37 DEG C of slowly vibrations are added
Shake 45min；It takes appropriate volume bacterium solution to be coated on LB/Amp/IPTG/X-gal plate, is inverted overnight incubation in 37 DEG C of incubators.
Picking white monoclonal every other day carries out bacterium colony PCR identification and is sequenced.
(5) oligonucleotide aptamer primary sequence and secondary structure analysis
Utilize 4.5 software of RNA structure, DNAMAN software and MEME online software.
Http:// meme.sdsc.edu/meme/cgi-bin/meme.cgi carries out having such as SEQ ID NO.1
Shown in nucleotide sequence single-strand DNA aptamer: SEQ ID NO.1:5 ' GCAAC CAGGG GAACC GGGGA ATTGG
AACGC TTTCC TCCCG TTGGC3'；There is the aptamer Fig. 4 to show secondary structure.(referring to fig. 4)
(6) identification (referring to figure 2-3) of DNA aptamer specificity
The DNA aptamer and human desmocyte sample synovial cell MH7A, people liver screened by Flow cytometry are thin
Born of the same parents L-02 and people Kupffer cell binding force are to identify its specificity.
The single-stranded oligonucleotide aptamer to be detected of synthesis FITC label is prepared respectively；
Above-mentioned survey cell to be checked is washed with 1 × PBS, cell is resuspended with Binding buffer and adjust separately as 1 ×
106/ml。
Take about 400pmol FITC-ssDNA respectively with above-mentioned cell incubation about 30min；Centrifugation carefully removes supernatant, uses PBS
Washing 2 times, adds 450ulPBS that cell is softly resuspended；
Flow cytomery compares the combination situation of aptamer by fluorescence intensity level.
Claims (4)
1. a kind of aptamer of the selectively targeted inflammatory synovial cell in human rheumatoid joint, which is characterized in that the aptamer
It for the nucleotide sequence as shown in SEQ ID NO.1, and is secondary structure shown in Fig. 4.
2. the application that aptamer described in claim 1 is used to prepare the bioprobe of identification inflammatory synovial cell.
3. the application that aptamer described in claim 1 is used to prepare targeting resisting rheumatoid arthritis drug.
4. application of the aptamer described in claim 1 as the pharmaceutical carrier of targeting inflammatory synovial cell.
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| CN109837281B (en) * | 2019-03-19 | 2020-08-28 | 温州医科大学附属第一医院 | Aptamer specifically binding to S100P protein and screening, identification and application thereof |
| CN110684774B (en) * | 2019-11-07 | 2021-11-19 | 郑州大学 | Aptamer specifically binding to DEK protein and application thereof |
| CN112094846B (en) * | 2020-05-20 | 2022-02-18 | 中山大学孙逸仙纪念医院 | Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof |
| CN114404613B (en) * | 2021-12-15 | 2023-10-13 | 南京大学 | Construction method and application of bone joint synovial fibroblast targeting aptamer nanoparticle |
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