CN112626072A - Meniscal cell-synoviocyte bispecific aptamer and application thereof - Google Patents
Meniscal cell-synoviocyte bispecific aptamer and application thereof Download PDFInfo
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Abstract
The invention provides an aptamer combined with meniscus cells and synovial cells in a targeted mode, wherein the aptamer is a single-stranded DNA molecule and has a nucleotide sequence shown as SEQ ID NO. 1. The bispecific aptamer of the invention also retains the secondary structures of the synoviocyte aptamer and the meniscal cell aptamer, so that the bispecific aptamer can specifically bind to the synoviocyte aptamer and the meniscal cell and can specifically recognize the two cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a single-stranded DNA aptamer capable of being combined with meniscal cells and synoviocytes in a targeted mode and application thereof.
Background
The meniscus is a fibrous cartilage pad located between the femoral condyle and the tibial plateau of the knee joint, can properly extend and displace along with the movement of the knee joint, can conduct load, stabilizes the tibial joint, absorbs impact and shock, reduces the abrasion between the femur and the tibia, and is of great importance to the normal physiological function of the knee joint. Meniscus injury is one of the most common injuries of knee joints, is mostly seen in young and old years, can occur under the action of external force or on the basis of meniscus self-diseases to cause knee joint swelling, pain and movement dysfunction, and the direct excision of the damaged meniscus can lead the stress of articular surface cartilage to concentrate and degenerate to cause osteoarthritis, which has great influence on life.
At present, the main treatment method of meniscus injury is arthroscopic meniscus repair, the meniscus is sewn and repaired through an absorbable suture, the integrity of the meniscus is kept, but the biomechanical property and the function of the repaired meniscus are still different compared with those of the original meniscus, and the meniscus has the risk of poor growth and need to be cut after the operation. Meniscal transplantation is applied to reconstructing a lost meniscus, mainly allogeneic meniscus transplantation is used as a main part, but transplantation rejection reaction to allogeneic cells occurs after part of patients receive the transplantation, and the long-term survival rate of the graft is very low. Tissue engineering techniques are currently more promising in the treatment of reconstructing defective menisci, which utilize seed cells to proliferate in vitro and bind to scaffold materials, stimulating the secretion of extracellular Matrix (ECM) under the action of various cytokines, for replacing damaged menisci. The sources of tissue engineering cells can be roughly divided into stem cells and mature cells, the mesenchymal stem cells are researched and applied more due to wide tissue sources and strong plasticity, but the mesenchymal stem cells are possibly subjected to the problems of reduced proliferation capacity, gene mutation and hypertrophic hyperplasia after in vitro culture, the proliferation and differentiation capacity of the cells is reduced after the in vitro culture monolayer amplification of autologous mature cells, and the sources of the tissue engineering cells most suitable for reconstructing meniscus are still found. The biological structure of the meniscus consists of meniscus cells, collagen I and II and glycosaminoglycan, and experiments prove that the biomechanical strength of the reconstructed meniscus is positively correlated with the deposition amount of collagen, so that the increase of the number of meniscus cells at the damaged part of the meniscus and the promotion of the synthesis of ECM are very important for the repair of the white area of the meniscus. Also, numerous experiments have shown that synovial cells play a major role in meniscal repair as well, since they secrete large amounts of type i collagen-based ECM and can grow synovial tissue at meniscal tear sites. Further experiments demonstrate that the role of synovial cells at the synovial interface of menisci is more critical in repairing endogenous cells of the meniscus. Therefore, there is an urgent need for a therapeutic method that can recruit autologous meniscal and synovial cells locally in a meniscal tear and promote ECM synthesis, so that the damaged meniscus can be anatomically and biostructurally repaired, thereby improving the biological performance of the repaired meniscus tear.
Aptamers are a class of nucleic acids that mediate cell recognition, leading to cell recruitment. Aptamers are single-stranded DNA or RNA nucleic acid sequence fragments that are screened from a specific pool of nucleotides by exponential enrichment system ligand evolution (SELEX) and that bind with high affinity and specificity to a target. The aptamers obtained by this technique are capable of folding to form specific and stable tertiary structures, and are capable of binding target molecules with high affinity and specificity. Compared with the traditional affinity tool, the aptamer has unique advantages, such as low molecular weight, high permeability, strong specificity, capability of being modified for site specificity, reusability, long-term storage, no immunogenicity and the like. The CELL-SELEX technology is a technology for screening aptamers by using living CELLs, the obtained aptamers can be used for target recognition of the living CELLs, the aptamers are applied to tumor diagnosis and treatment at present, and a dual-specificity aptamer crosslinking technology is recently developed on the basis of the CELL-SELEX technology, so that two aptamers with different specificities can be formed into a dual-specificity aptamer to construct a dual-specificity aptamer with dual-specificity recognition capability, and the dual-specificity aptamer can be simultaneously combined with two different CELL targets. In the process of crosslinking, a bispecific aptamer capable of being combined with synovial cells and meniscal cells in a targeted mode is obtained.
Disclosure of Invention
In the invention, the screened synovial cell aptamer and meniscus cell aptamer are crosslinked to obtain a synovial meniscus bispecific aptamer, the cell recruitment capability of the synovial meniscus bispecific aptamer is verified, and the recruitment of meniscus cells and synovial cells and adhesion to a meniscus can be realized in a meniscus repair technology, so that the purpose of recruiting in-vivo cells to repair the meniscus is achieved.
The invention realizes the purpose of the invention by the following technical scheme:
the invention provides an aptamer combined with meniscus cells and synovial cells in a targeted manner, wherein the aptamer is a single-stranded DNA molecule and has a nucleotide sequence shown as SEQ ID NO. 1;
SEQ ID NO:1：
5'-TGGCCCACTATACTGTGCCCATCACAGGGTGTTGCAGTCGTGGGCCACGACCTGGGTTCGCGCACCACAGTACATCACCGTGGGC-3', respectively; and has a secondary structure as shown in fig. 1.
Since the bispecific aptamers of the present invention also retain the secondary structures of the synovial cell aptamers and the meniscal cell aptamers, the bispecific aptamers are capable of specifically binding to the synovial cell aptamers and the meniscal cells and specifically recognizing these two cells.
The invention also provides application of the bispecific aptamer in preparing a biological probe for recognizing synovial cells.
The invention also provides application of the bispecific aptamer in preparing a biological probe for recognizing meniscus cells.
The invention also provides application of the bispecific aptamer in a biological probe for simultaneously recognizing synovial cells and meniscal cells.
The invention also provides application of the bispecific aptamer in preparing a meniscus repair medicament.
The invention has the beneficial effects that: the aptamer constructed by the invention can be used for enriching meniscal cells and synoviocytes at the same time. The dual-specificity aptamer also has good stability and can effectively resist degradation of nuclease. After 12h of incubation with nuclease in the present examples, the band of interest was still detectable.
Drawings
FIG. 1 is a schematic diagram of the process for constructing a bispecific aptamer of the invention;
FIG. 2 is a diagram of the secondary structure of a dual specific aptamer of the invention;
FIG. 3 is a graph showing the results of the anti-nuclease degradation performance of the bispecific aptamers of the present invention;
FIG. 4 is a schematic diagram showing the two-cell (osteoarthritis synovial cell and meniscal cell) binding ability of the bispecific ligand of the present invention;
FIG. 5 is a graph showing the ability of the bispecific aptamers of the present invention to recruit synoviocytes and meniscal cells of osteoarthritis (A is a meniscal cell flow result; B is a synoviocytes flow result; C is a ratio of the number of cell groups of a DNA aptamer library; D is a ratio of the number of cell groups of the bispecific aptamers)
Detailed Description
In order to show technical solutions, purposes and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments.
EXAMPLE 1 construction of bispecific aptamers
1. The specific method for obtaining the aptamer specifically targeting the synovial cells comprises the following steps:
cutting normal synovial tissue in knee joint of patient subjected to arthroscopic meniscus repair operation, extracting human normal synovial cells, screening and synthesizing synovial cell aptamer by biological engineering (Shanghai) corporation, and sequencing. The sequence of the finally obtained aptamer specifically targeting synovial cells is as follows:
5’-GCCCACTATACTGTGCCCATCACAGGGTGTTGCAGTCGTG-3’
2. obtaining the aptamer specifically targeting the meniscal cells by the following specific method:
the method comprises the steps of cutting normal meniscus tissues in the knee joint of a patient subjected to arthroscopic meniscal partial excision, extracting human normal meniscus cells, screening and synthesizing meniscus cell aptamers by biological engineering (Shanghai) GmbH, and sequencing. The sequence of the finally obtained aptamer specifically targeting meniscal cells is as follows:
the sequence of the finally obtained aptamer of the aptamer specifically targeting the meniscal cells is as follows:
5’-CACGACCTGGGTTCGCGCACCACAGTACATCACCGTGGGC-3’
3. the specific method for constructing the dual-specificity aptamer comprises the following steps:
extending the aptamer specifically targeting the synovial cells obtained by screening in the step 1 and the aptamer specifically targeting the meniscal cells obtained in the step 2 by 2 GT nucleotides at the 5 'end of the synovial cell aptamer and 3 CGG nucleotides at the 5' end of the meniscal cell aptamer respectively according to the base complementary pairing principle, so that the two aptamers have a complementary strand with the length of 5 nucleotides; the meniscal-synovial membrane dual-specific aptamers were linked by the base pairing principle of the complementary strands of the two aptamers described above (fig. 1). The secondary structure of the bispecific aptamer is shown in fig. 2, and it can be seen that the bispecific aptamer maintains the secondary structure of both meniscal and synovial aptamers.
Example 2 verification of dual-aptamer stability against nuclease
And (3) preparing a 20 mu M PBS solution from the meniscus cell aptamer, the synovial cell aptamer and the bispecific aptamer, incubating the solution with 10% fetal calf serum for 1, 2, 4, 6, 8, 10 and 12 hours, mixing the solution with a loading buffer solution, loading the mixture into a 1% agarose gel loading hole, carrying out electrophoresis under 100V direct current for 20min, observing the developing condition of an aptamer DNA band under ultraviolet light, and comparing the capacity of the three aptamers on resisting the nucleic acid hydrolase in the fetal calf serum. The results showed that the band brightness of meniscal and synovial aptamers was significantly reduced at 8 hours of hydrolysis, suggesting that the aptamers were hydrolyzed, while the bispecific aptamers still had significant bands after 10 hours of hydrolysis, and it can be considered that the stability of the bispecific aptamers in the nucleic acid hydrolase was slightly better than the former two (FIG. 3).
Example 3 verification of the double cell binding Capacity of bispecific aptamers (immunofluorescence)
Cell preparation: human synovial cells and meniscal cells were isolated and cultured. Human synovial tissue was taken from patients undergoing arthroscopic meniscal repair surgery and was digested by collagenase as the cell source. Synovial cells and meniscal cells were cultured in DMEM medium containing 10% Fetal Calf Serum (FCS) supplemented with 1% penicillin and streptomycin solution in humidified incubator at 37 deg.C in 5% CO2Culturing under the condition. The medium was changed every 3 days and passaged with 70% cell identity.
Cell fluorescent labeling: both cells were washed twice with PBS buffer and then incubated for 30min at 37 ℃ with the addition of a solution of CellTracker green/red fluorescent dye in dimethylsulfoxide, respectively, to label the meniscal cells as green fluorescence and the synovial cells as red fluorescence. Unreacted dye was removed by washing with PBS buffer. Mixing synovium and meniscus cells, then incubating the mixture with an aptamer DNA library or a bispecific aptamer in a binding buffer solution at 4 ℃ for 2h, fixing the cells by paraformaldehyde after incubation, and observing through confocal imaging with the final concentration of 1% (w/v), wherein the observation shows that no meniscus cells and synovial cells are combined after the two cells and DNA library aptamers are incubated, and a meniscus cell-synovial cell group is formed after the addition of the invention, thus confirming that the bispecific aptamer of the invention has bidirectional binding capability of meniscus-synovial cells (figure 4).
Example 4 two-cell recruitment efficiency validation of bispecific aptamers (flow cytometry)
Cell preparation and fluorescent labeling of cells As in example 3, the fluorescent labeling effect of fluorescently labeled meniscal cells and synoviocytes was first detected by flow cytometry(FIG. 5A shows the flow of meniscus and B shows the flow of synovial membrane), after which 1X 106CellTracker Green-labeled meniscal cells and 1X 106The CellTracker red marked synovial cells were incubated with the DNA aptamer library and the bispecific aptamer in PBS buffer solution at 4 ℃ for 1 hour respectively, the aptamer concentration was 5. mu.M, the proportion of double-stained cell clusters was detected by flow cytometry, it was found that the double-stained cell clusters hardly appeared after the DNA aptamer library was added (FIG. 5C shows that the proportion of the number of cell clusters was 0.11%), and the cell forming cell clusters accounting for 79.6% of the total number after the double-aptamer was added (FIG. 5D), which confirmed the superiority of the recruitment ability and efficiency of the present invention.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> grand era memorial Hospital of Zhongshan university
<120> meniscal cell-synovial cell bispecific aptamer and application thereof
<130> 1.12
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 85
<212> DNA
<213> Synthesis
<400> 1
tggcccacta tactgtgccc atcacagggt gttgcagtcg tgggccacga cctgggttcg 60
cgcaccacag tacatcaccg tgggc 85
Claims (6)
1. An aptamer capable of targeting and combining with meniscus cells and synoviocytes, wherein the aptamer is a single-stranded DNA molecule and has a nucleotide sequence shown as SEQ ID NO. 1.
2. The aptamer according to claim 1, wherein the secondary conformation of the nucleotide sequence shown in SEQ ID NO. 1 is:
3. use of the aptamer of claim 1 for the preparation of a biological probe that recognizes synovial cells.
4. Use of an aptamer according to claim 1 for the preparation of a biological probe for identifying meniscal cells.
5. Use of the aptamer of claim 1 in a biological probe for simultaneous recognition of synovial cells and meniscal cells.
6. Use of an aptamer according to claim 1 for the preparation of a meniscus repair drug.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105985692A (en) * | 2015-02-04 | 2016-10-05 | 天长市高新技术创业服务中心 | Antibacterial environment-friendly coating material |
| CN107794267A (en) * | 2017-03-14 | 2018-03-13 | 湖南大学 | A kind of targeting PD1 PDL1 bispecific aptamer and its derivative |
| CN110283826A (en) * | 2019-07-03 | 2019-09-27 | 福州大学 | A kind of bispecific aptamer, derivative, preparation method and applications |
| CN112094846A (en) * | 2020-05-20 | 2020-12-18 | 中山大学孙逸仙纪念医院 | Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof |
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- 2021-01-12 CN CN202110037269.7A patent/CN112626072B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105985692A (en) * | 2015-02-04 | 2016-10-05 | 天长市高新技术创业服务中心 | Antibacterial environment-friendly coating material |
| CN107794267A (en) * | 2017-03-14 | 2018-03-13 | 湖南大学 | A kind of targeting PD1 PDL1 bispecific aptamer and its derivative |
| CN110283826A (en) * | 2019-07-03 | 2019-09-27 | 福州大学 | A kind of bispecific aptamer, derivative, preparation method and applications |
| CN112094846A (en) * | 2020-05-20 | 2020-12-18 | 中山大学孙逸仙纪念医院 | Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof |
Non-Patent Citations (1)
| Title |
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| NIRIT等: "DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis", 《NATURE COMMUNICATIONS》 * |
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