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An aptamer specifically targeting osteosarcoma cells and its preparation method and application
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An osteosarcoma cell and aptamer technology, applied in the biological field, can solve the problems of affecting the quality of life, the patient's physical and mental harm, and the failure to make great progress, and achieve the effect of reducing liver toxicity and improving the therapeutic effect.
Active Publication Date: 2020-04-17
BEIJING HELI CONSULTING CO LTD
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This method has a good control effect on the disease, but it does a lot of physical and mental harm to the patient and seriously affects the quality of life after surgery.
Although the prognosis has been greatly improved through neoadjuvant chemotherapy combined with surgery, the 5-year survival rate has risen from about 10% in the late 1970s to about 60% today, but in the past 30 years, osteosarcoma has been treated with surgery3 30-40% recurrence rate after years, despite various efforts, has not been able to achieve significant progress
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Embodiment 1
[0061] Example 1 Screening and preparation of aptamers
[0062] 1. Experimental materials:
[0063] 1.1 ssDNA library, FITC-labeled forward primer and Biotin-labeled reverse primer: ssDNA library:
[0066] 3' primer: 5'-biotin-ACTAAGCCACCGTGTCCA-3'; all provided by Shanghai Sangon Bioengineering Company.
[0067] 1.2 Rat osteoblasts: ROS17 / 2.8, purchased from the Cell Bank of the Chinese Academy of Sciences, the culture medium is DMEM medium with 10% fetal bovine serum.
[0068] 1.3 Rat hepatocytes: BRL-3A, purchased from the Cell Bank of the Chinese Academy of Sciences, the culture medium is DMEM medium with 10% fetal bovine serum.
[0069] Rat peripheral blood cells: PBMCs, isolated directly from peripheral blood of rats.
[0070] 1.4 Culture medium: DMEM medium and fetal bovine serum were purchased from Gbico Company
[0071] 1.5 Flow cytometer: purchased from ...
experiment example 1
[0101] Experimental Example 1 Experiment of Targeting Osteosarcoma Cells with Aptamers and Rekalidin-Aptamer Couplers
[0102] 1.1 Experiment of aptamers targeting osteosarcoma cells
[0103] 1.1.1 Experimental method
[0104] Take the aptamers LC4, LC5, and LC6 prepared in Example 1 of the present invention at a concentration of 200 nM, incubate with osteosarcoma cells, liver cells, and peripheral blood mononuclear cells at 37 degrees for 1 hour, and detect the fluorescence intensity with a flow cytometer , the number of cells was 20,000.
[0105] 1.1.2 Experimental results
[0106] The aptamer loss cytometry data of LC4, LC5, and LC6 of the present invention show that the three sequences can specifically bind to rat osteosarcoma cells, and have no obvious combination with rat hepatocytes and peripheral blood mononuclear cells ( figure 1 ).
[0107] 1.2 LC6-triptolide (hereinafter referred to as "LC6-TP") targeting osteosarcoma experiment
[0108] 1.2.1 Preparation of LC...
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Abstract
The invention relates to the field of biology, and provides an aptamer specifically targeting to osteosarcoma cells as well as a preparation method and applications of the aptamer. The aptamer has the nucleotide sequence shown as the SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The preparation method comprises the following steps: (1) screening aptamers; (2) gathering the aptamers; and (3) sequencing the aptamers. According to the aptamer, the preparation method and the applications, medicines are specifically targeted to the osteosarcoma cells in the body of the patient by adopting the aptamer as a carrier of the osteosarcoma-resisting medicine, so that the osteosarcoma can be effectively treated. The experiment proves that under the same concentration level, triptolide coupled with the aptamer provided by the invention can effectively kill the osteosarcoma cells.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to an aptamer specifically targeting osteosarcoma cells, a preparation method and application thereof. Background technique [0002] Osteosarcoma is the most common type of malignant bone tumor, which develops from mesenchymal cell lines. The rapid growth of the tumor is due to the direct or indirect formation of tumor bone-like tissue and bone tissue through the cartilage stage. After the pathological diagnosis of osteosarcoma, radiotherapy and chemotherapy are required immediately, and amputation is an important method of traditional tumor removal. This method has a good control effect on the disease, but it does great physical and mental harm to the patient and seriously affects the postoperative quality of life. Although the prognosis has been greatly improved through neoadjuvant chemotherapy combined with surgery, the 5-year survival rate has risen from about 10% in the late 1970s...
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