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Anti-angiogenesis and anti-cancer function of angiogenin combined with nucleotide

An angiogenin and oligonucleotide technology, applied in the direction of angiogenin, angiogenesis factors, medical preparations containing active ingredients, etc., can solve the problem of no effective technology for inhibiting rRNA transcription

Inactive Publication Date: 2004-06-09
上海福缘生化药学研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, so far there is no technique to effectively inhibit angiopoietin-induced rRNA transcription

Method used

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  • Anti-angiogenesis and anti-cancer function of angiogenin combined with nucleotide
  • Anti-angiogenesis and anti-cancer function of angiogenin combined with nucleotide
  • Anti-angiogenesis and anti-cancer function of angiogenin combined with nucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] cell culture

[0051] Human placental vein vascular endothelial cells (HUVE) were cultured in vascular endothelial cell-specific medium (HE-SFM+10% FCS+10ng / ml bFGF) containing 10% fetal bovine serum and 10 ng / ml basic fibroblast growth factor )middle. The culture conditions are constant temperature and humidity at 37°C and 5% carbon dioxide gas phase. The medium was changed every two days, and the culture medium was subcultured at a ratio of 1:3. HUVE cells from passage 3 to passage 15 were used for experiments.

[0052] Human placental venous vascular smooth muscle cells (HuASM), human cervical cancer cells HeLa and human colon adenocarcinoma cells HT-29 were cultured in DMEM containing 10% fetal bovine serum and subcultured at a ratio of 1:4.

[0053] U-937 cells were cultured in RPMI containing 10% fetal bovine serum and subcultured at a ratio of 1:4.

Embodiment 2

[0055] Isolation of angiopoietin-binding nucleotides

[0056] Angiogenin enters the nucleus of vascular endothelial cells and induces the synthesis of total RNA (Xu ZP, TsujiT, Riordan JF, and Hu GF. The nuclear function of angiogenin inendothelial cells is related to rRNA production, Biochem.Biophys.Res.Commun.2002 , 294, 287-292). Since Northern blot showed that angiogenin stimulates the synthesis of ribosomal ribonucleic acid (rRNA), a series of experiments were performed to demonstrate the binding of angiogenin to the transcriptional regulatory region of ribosomal ribonucleic acid gene (rDNA).

[0057] First, a 4.6kb rDNA transcriptional regulatory region fragment was obtained and treated with restriction endonuclease DpnI to obtain 5 smaller fragments. Gel electrophoretic mobility experiments showed that angiopoietin bound to the 3.0 kb fragment, but had no effect on the other 4 fragments (Fig. 1).

[0058] Figure 2 shows that the binding of angiogenin to a 3.0 kb DNA f...

Embodiment 3

[0060] Cloning and sequence analysis of angiopoietin-binding nucleotides

[0061] In this example, in order to further analyze and identify the properties of angiopoietin-binding nucleotides, their sequences were determined.

[0062] The conjugate of the 3.0 kb DNA fragment and angiogenin was treated with deoxyribonuclease (DNase I). In this way, only the nucleotides directly bound to angiogenin will not be enzymatically digested. After these fragments were isolated and cloned, the nucleotide sequences were determined. Figure 3 lists the sequences of five such clones. The commonality of these 5 sequences is that they are all rich in C and T, and contain CT repeats. The sequence of clone No. 12 (CTCTCTCTCTCTCTCTCCCTCTC, SEQ ID NO: 1) was the shortest and was named as angiopoietin binding element (ABE).

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Abstract

The present invention provides a nucleotide-angiogenin conjugated element (ABE) for inhibiting rRNA synthesis induced by angiogenin. The length of the described oligonucleotide is 10-150 bp, and its C+T content is 80%-100% of all the oligonucleotides, and it contains 5-30 continuous CT repetitive units. The experiments show that the specific combination of ABE and angiogenin can inhibit the synthesis of rRNA, and the animal experiments also show that ABE can inhibit the growth of human tumor on naked mouse.

Description

technical field [0001] The present invention relates to the field of medicine. Specifically, the present invention relates to an oligonucleotide specifically binding to angiogenin. The oligonucleotide can inhibit the combination of human angiogenin and the transcriptional regulatory region of the ribosomal ribonucleic acid gene, thereby inhibiting the transcription of the ribosomal ribonucleic acid and the synthesis of ribosomes, protein translation and cell growth. The present invention also relates to a pharmaceutical composition containing the angiopoietin-binding oligonucleotide. Background technique [0002] The dependence of tumor growth and metastasis on angiogenesis has been widely accepted by the scientific community. Tumor tissue can only grow to a size of 1-2 mm in diameter unless it establishes its own vascular network. Tumors of this size do not pose a hazard to the human body. At present, a widely concerned and accepted theory is that long-term use of drugs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/18A61P3/10A61P17/02A61P19/02A61P27/02A61P27/08A61P29/00A61P31/12A61P35/00A61P37/02C07K14/515
Inventor 许正平胡国富
Owner 上海福缘生化药学研发有限公司
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