Nucleic acid aptamer for targeting tumor-associated fibroblasts and application of nucleic acid aptamer
A nucleic acid aptamer and fibroblast technology, applied in the field of biomedicine, to achieve good reproducibility, broad application prospects, and stable chemical properties
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Embodiment 1
[0029] Example 1 Screening of nucleic acid aptamers.
[0030] (1) Preparation of random screening library:
Entrust Sangon Bioengineering Co., Ltd. to synthesize a random single-stranded DNA library (5'-AAGGAGCAGCGTGGAGGATANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTAGGGTGTGTCGTCGTGGT-3'), where N represents any of the bases A, T, C, and G; take one tube of 10OD random single-stranded DNA Library, add binding buffer, vortex to dissolve, cover the centrifuge tube cap, water bath at 95°C for 5 minutes, quickly add to ice for 8 minutes, and set aside.
[0031] (2) Isolation and preparation of tumor-associated fibroblasts and paracancerous fibroblasts: Take fresh tissue or paracancerous tissue from clinical colorectal cancer from the same patient, and use PBS (containing double antibody, gentamicin and amphotericin) Rinse until the tissue is clear, add 3-5 times the tissue volume of collagenase (2 mg / mL), and place it in a tissue processor for processing. The tissue was resuspend...
Embodiment 2
[0038] Example 2 Observation of the expression of fibroblast markers on tumor-associated fibroblasts by fluorescence microscopy.
[0039] The isolated and cultured tumor-associated fibroblasts were taken and inoculated on a coverslip. After 24 h, the cells were washed twice with PBS; fixed with 4% paraformaldehyde at room temperature for 25 min, and washed with PBS three times; added with 0.1% Triton X-100 at room temperature for 10 min , washed three times with PBS; added 5% FBS to block at room temperature for 60 min, aspirated and discarded FBS, added primary antibodies (α-SMA, FAP, PDGFRβ) and incubated overnight at 4°C. Wash three times with PBS, add fluorescent secondary antibody for 45 min at room temperature; wash three times with PBS, add DAPI for 30 min at room temperature; wash three times with PBS, ddH 2 O Rinse once, cover slides, and air dry naturally. like figure 2 As shown, strong fluorescent signals were observed on the isolated tumor-associated fibroblasts...
Embodiment 3
[0040] Example 3 Flow cytometry was used to detect the binding of the nucleic acid aptamer to tumor-associated fibroblasts and paracancerous fibroblasts.
[0041] The above tumor-associated fibroblasts or paracancerous fibroblasts were taken, digested with non-enzymatic digestion solution and pipetted into a single cell suspension, centrifuged at 1000 rpm for 5 min, and then the supernatant was removed, and the cells were washed twice with 4°C pre-cooled washing buffer . The nucleic acid aptamers labeled with FAM were incubated with tumor-associated fibroblasts or paracancerous fibroblasts on a shaker at 4 °C for 30 min, centrifuged at 1000 rpm for 5 min at room temperature, and then the supernatant was removed, and the washing buffer was pre-cooled at 4 °C. Wash cells twice. Finally, 300 µL of PBS was added for flow cytometry to measure the fluorescence intensity of cells. like image 3 As shown, the fluorescence intensity on tumor-associated fibroblasts is significantly h...
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