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Application of oligonucleotide YW002 CpG ODN in preventing and treating periodontitis bone absorption

An oligonucleotide and periodontitis technology, applied in the field of molecular drugs, can solve the problems of unreported bone regeneration research, and achieve the effects of good experimental repeatability, low price and low risk

Inactive Publication Date: 2018-05-11
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, CpG ODN has been widely used in the research and development and preliminary clinical trials of anti-infection, anti-tumor, and vaccine adjuvants, but the research on bone regeneration mediated by CpG ODN has not been reported yet.

Method used

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  • Application of oligonucleotide YW002 CpG ODN in preventing and treating periodontitis bone absorption
  • Application of oligonucleotide YW002 CpG ODN in preventing and treating periodontitis bone absorption
  • Application of oligonucleotide YW002 CpG ODN in preventing and treating periodontitis bone absorption

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Mouse osteoblasts (MC3T3-E1, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were seeded on six-well plates and cell slides at 300,000 / well, and placed in a 37°C cell culture incubator containing 5% (volume ratio) carbon dioxide nourish. After overnight cell attachment, replace the high-glucose modified medium (containing 4.0mML glutamic acid, 4500mg / L glucose, without sodium pyruvate), and add Cy5-labeled YW002 CpG ODN (5 'end-labeled cyanine derivatives, which can be synthesized by TAKARA company), the final concentration is 2 μg / mL. After co-cultivation for 1 hour, the culture medium was discarded, washed twice with phosphate buffered saline, and the cells in the six-well plate were observed under a fluorescent microscope, photographed and used for flow cytometry detection; the cells on the cell slide were treated with 4% ( volume ratio) paraformaldehyde (prepared by dissolving paraformaldehyde in phosphate buffered saline solution) for 10 ...

Embodiment 2

[0021] (1) MC3T3-E1 cells were seeded in a six-well plate at 300,000 / well, cultured in a 37°C cell incubator with 5% (volume ratio) carbon dioxide for 24 hours, and then replaced with an osteogenic induction medium (β-sodium glycerophosphate 10mmol / L and ascorbic acid 50mg / L) high-sugar modified medium, while adding 2006 CpG ODN and YW002 CpG ODN. After co-culture with CpG ODN for 72 hours, total ribonucleic acid (RNA) extraction, reverse transcription into complementary deoxyribonucleic acid (cDNA), and real-time fluorescent polymerase chain reaction to generate deoxyribonucleic acid (DNA) were performed to detect the expression of osteogenic differentiation genes . Using actin (β-actin) as an internal reference gene, calculate 2 -ΔΔCt value.

[0022] (2) The procedure is the same as in Example 2 (1). Collect MC3T3 cells after 72 hours of CpG ODN action, extract the total protein of the tissue, measure the protein concentration, adjust the protein to the same concentration ...

Embodiment 3

[0025] (1) MC3T3-E1 cells were seeded in 96-well plates at 5,000 / well, cultured in a 37°C cell incubator with 5% (volume ratio) carbon dioxide for 24 hours, and then replaced with an osteogenic induction medium (β-sodium glycerophosphate 10mmol / L and ascorbic acid 50mg / L) high glucose modified medium, while adding 2006 CpG ODN and YW002 CpG ODN. After co-cultivating with CpG ODN for 24h, 48h, and 72h (detecting the short-term and long-term expression of ALP activity in MC3T3 cells under the action of CpG ODN), the cells were washed twice with phosphate buffered saline. Cells were lysed with 1% (volume ratio) Tritox cell lysate, and kept in a water bath at 37°C for 30 minutes. Determination of protein concentration (Catalog No. P0010 can be purchased from Biyuntian Reagent Company) and alkaline phosphatase (AKP) activity (Cat. No. A059-2 can be purchased from Nanjing Jiancheng Reagent Company), and the absorbance value is detected with a microplate reader. According to the for...

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Abstract

The invention relates to application of oligonucleotide YW002 CpG ODN containing a CG sequence motif in preventing and treating periodontitis bone absorption, and belongs to the technical field of molecular medicines. The YW002 CpG ODN can high-efficiently and quickly enter cells and does not need a carrier; on a gene level, a protein level and an osteoblast secretion function level, an osteogenicdifferentiation capacity and a function of osteoblast are remarkably improved by the YW002 CpG ODN; in addition, the YW002 CpG ODN not only inhibits macrophage proliferation and promotes apoptosis, but also remarkably inhibits gene and protein expressions in an osteoclast differentiation process, and influences the maturity of the osteoclast at the same time. The YW002 CpG ODN has a remarkably superior influence on osteogenesis and osteolysis compared with 2006 CpG ODN, and the YW002 CpG ODN can realize double conditions favorable to osteanagenesis for promoting osteogenesis and inhibiting osteolysis.

Description

technical field [0001] The invention belongs to the technical field of molecular medicine, and specifically relates to the application of an oligonucleotide YW002 CpGODN containing a CG motif in the prevention and treatment of periodontal inflammatory bone resorption. The YW002 CpG ODN has the function of regulating osteoblasts and osteoclasts. It can promote the osteogenic differentiation of pre-osteoblasts and inhibit the activation of pre-osteoclasts. Background technique [0002] Inflammation and bone resorption are the two main symptoms of periodontitis. Studies have shown that periodontitis is a natural immune response to inflammation in the body caused by bacterial infection. In the prevention and treatment of clinical periodontal disease, a drug that can control inflammation, resist bone resorption and obtain bone regeneration is very rare, and it is also a hot spot and difficult problem in periodontal bone regeneration research. During the treatment of periodontiti...

Claims

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Application Information

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IPC IPC(8): A61K31/711A61P1/02
CPCA61K31/711
Inventor 申玉芹于文雯边立新徐晓薇
Owner JILIN UNIV
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