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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

Pending Publication Date: 2022-06-03
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports of nucleic acid aptamers targeting tumor-associated fibroblasts

Method used

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  • Nucleic acid aptamer for targeting tumor-associated fibroblasts and application of nucleic acid aptamer
  • Nucleic acid aptamer for targeting tumor-associated fibroblasts and application of nucleic acid aptamer
  • Nucleic acid aptamer for targeting tumor-associated fibroblasts and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention belongs to the technical field of biomedicine, and particularly discloses a nucleic acid aptamer for targeting tumor-associated fibroblasts and application of the nucleic acid aptamer. The sequence of the nucleic acid aptamer is 5 '-AGCGTGGAGGATAATTAGGCATCGTTCCGCCTAGGAAATTATTCAATCTACGC-3', and the sequence of the nucleic acid aptamer is shown in the description. The nucleic acid aptamer has high specificity and high affinity to tumor-associated fibroblasts, can be used as a molecular probe for imaging of tumor-associated fibroblasts, can be used as a drug carrier to participate in targeted transportation and fixed-point release of drugs in a targeted drug delivery system, and can be used for imaging of tumor-associated fibroblasts, tumor-associated fibroblasts, tumor-associated fibroblasts, tumor-associated fibroblasts, tumor-associated fibroblasts, tumor-associated fibroblasts and tumor-associated fibroblasts. The method can be used for identification and functional research of tumor-associated fibroblast specific target molecules. The nucleic acid aptamer can keep good activity at the temperature of 4 DEG C and 37 DEG C, and has the advantages of being easy and convenient to operate, low in cost, short in period, high in accuracy and the like in research and detection of tumor-related fibroblasts.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a nucleic acid aptamer targeting tumor-related fibroblasts and applications thereof. Background technique [0002] Cancer-associated fibroblasts (CAFs) are a special type of stromal cells in the tumor microenvironment. Studies have shown that CAFs can maintain the hallmark features of tumors, such as the continuous activation of proliferative signals, induction of angiogenesis, resistance to Cell death and changes in cellular energy metabolism are of great significance. Previous studies have suggested that CAFs are highly heterogeneous and lack surface markers for effective clustering, which leads to difficulties in identification and seriously hinders the research on the function of different subsets of tumor fibroblasts and the development of targeted therapies. Therefore, it is very necessary to further explore and search for new surface-related molecular mark...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/574G01N33/569G01N33/58
CPCC12N15/115G01N33/574G01N33/56966G01N33/582C12N2310/16Y02A50/30
Inventor 方瑾李婉明巴微
Owner 中国医科大学
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