Meniscus cell-synovial cell bispecific aptamer and use thereof
A technology of synoviocytes and aptamers, which is applied in the direction of pharmaceutical formulas, organic active ingredients, and medical preparations containing active ingredients, etc., can solve problems such as hypertrophy and hyperplasia, decreased proliferation ability, and decreased ability of cell proliferation and differentiation, and achieve good Stability, effect against degradation
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Embodiment 1
[0022] Example 1 Construction of bispecific aptamers
[0023] 1. To obtain aptamers specifically targeting synoviocytes, the specific method is as follows:
[0024] The normal synovial tissues in the knee joints of patients undergoing arthroscopic meniscus repair were excised, and human normal synovial cells were extracted, which were sent to Sangon Bioengineering (Shanghai) Co., Ltd. to screen and synthesize synovial cell aptamers and sequence them. The sequence of the finally obtained aptamer specifically targeting synoviocytes is as follows:
[0025] 5'-GCCCACTATACTGTGCCCATCACAGGGTGTTGCAGTCGTG-3'
[0026] 2. To obtain aptamers specifically targeting meniscus cells, the specific method is as follows:
[0027] The normal meniscus tissues in the knee joints of patients undergoing arthroscopic partial meniscectomy were excised, human normal meniscus cells were extracted, and sent to Sangon Bioengineering (Shanghai) Co., Ltd. to screen and synthesize meniscus cell aptamers and...
Embodiment 2
[0032] Example 2 Bispecific aptamer anti-nucleolytic stability verification
[0033] Prepare the meniscus cell aptamer, synovial cell aptamer and bispecific aptamer into 20 μM PBS solution, and incubate with 10% fetal bovine serum for 1, 2, 4, 6, 8, 10, 12 hours , mixed with the loading buffer and added to the sample hole of 1% agarose gel, electrophoresis under 100V direct current for 20 minutes, and then observed the development of the aptamer DNA band under ultraviolet light, and compared the three aptamers against fetuses. Nucleolytic enzyme capacity in bovine serum. The results showed that the meniscal aptamer and synovial aptamer bands were significantly lighter after 8 hours of hydrolysis, suggesting that the aptamers were hydrolyzed, while the bispecific aptamers still had obvious bands after 10 hours of hydrolysis, which can be considered The stability of bispecific aptamers in nucleolytic enzymes was slightly better than that of the former two ( image 3 ).
Embodiment 3
[0034] Example 3 Verification of the double-cell binding ability of the bispecific aptamer (immunofluorescence method)
[0035] Cell preparation: Isolate and culture human synoviocytes and meniscus cells. Human synovial tissue was obtained from patients undergoing arthroscopic meniscal repair surgery and digested with collagenase as a cell source. Synoviocytes and meniscus cells were cultured in DMEM medium containing 10% fetal calf serum (FCS), supplemented with 1% penicillin and streptomycin solution in DMEM, in a humidified incubator at 37°C, in 5% CO 2 cultured under conditions. The medium was changed every 3 days and the cells were passaged at 70% consistency.
[0036] Fluorescence labeling of cells: Wash the two kinds of cells twice with PBS buffer, then add CellTracker green / red fluorescent dye in dimethyl sulfoxide solution and incubate at 37°C for 30 minutes, so that meniscus cells can be labeled with green fluorescence, and synoviocytes can be labeled with green fluo...
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