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Aptamer specifically targeting to osteosarcoma cells as well as preparation method and applications of aptamer

A technology of osteosarcoma cells and aptamers, applied in the biological field, can solve the problems of affecting the quality of life, causing great physical and mental harm to patients, and failing to make great progress, so as to reduce liver toxicity and improve the therapeutic effect

Active Publication Date: 2017-01-04
BEIJING HELI CONSULTING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Aptamer specifically targeting to osteosarcoma cells as well as preparation method and applications of aptamer
  • Aptamer specifically targeting to osteosarcoma cells as well as preparation method and applications of aptamer
  • Aptamer specifically targeting to osteosarcoma cells as well as preparation method and applications of aptamer

Examples

Experimental program
Comparison scheme
Effect test

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:

[0064] 5’-ATCCAGAGTGACGCAGCA-55nt-TGGACACGGTGGCTTAGT-3’

[0065] 5'primer: 5'-FITC-ATCCAGAGTGACGCAGCA-3';

[0066] 3'primer: 5'-biotin-ACTAAGCCACCGTGTCCA-3'; all were provided by Shanghai Shenggong Bioengineering Company.

[0067] 1.2 Rat osteoblasts: ROS17 / 2.8, purchased from the Cell Bank of the Chinese Academy of Sciences, and the culture medium is DMEM medium with 10% fetal bovine serum.

[0068] 1.3 Rat liver cells: BRL-3A, purchased from the Cell Bank of Chinese Academy of Sciences, and the culture medium is DMEM medium with 10% fetal bovine serum.

[0069] Rat peripheral blood cells: PBMCs, isolated directly from rat peripheral blood.

[0070] 1.4 Culture medium: DMEM medium and fetal bovine serum were purchased from Gbico

[0071] 1.5 Flow cytometer: purchased from BD Immunocytometry Syst...

experiment example 1

[0101] Experimental example 1 Targeting osteosarcoma cells by aptamer and triptolide-aptamer conjugate

[0102] 1.1 Experiments 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. After incubating with osteosarcoma cells, liver cells and peripheral blood mononuclear cells for 1 hour at 37 degrees, the fluorescence intensity is measured by a cytometer , The cell number is 20,000.

[0105] 1.1.2 Experimental results

[0106] The aptamer loss cytometer data of LC4, LC5, and LC6 of the present invention shows that the three sequences can specifically bind to rat osteosarcoma cells, and at the same time do not significantly bind to 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 LC6-triptolide (herein...

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PUM

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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, and a preparation method and application thereof. Background technique [0002] Osteosarcoma is the most common type of bone malignant tumor, which develops from the mesenchymal cell line. The rapid growth of tumor is due to the formation of tumor bone-like tissue and bone tissue directly or indirectly through the cartilage stage. Radiochemical treatment is required immediately after the pathological diagnosis of osteosarcoma, and amputation is an important method of traditional tumor tissue removal. This method has a better control effect on the disease, but it does great physical and mental harm to the patient, and seriously affects the quality of life after surgery. Although neoadjuvant chemotherapy combined with surgical treatment has greatly improved the prognosis, the 5-year survival rate has risen from about 10% in the late 197...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68C12Q1/04A61K47/48A61P35/00
Inventor 吕诚梁超张保亭刘振丽
Owner BEIJING HELI CONSULTING CO LTD
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