hTERT gene antisense oligodeoxynucleotide, drug composition and application

A technology of antisense oligonucleotide and composition, which is applied in the field of pharmaceutical composition of total soybean progesterone (TFOF), which can solve the problem of loss of function

Inactive Publication Date: 2016-04-06
甘肃省中医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

With the treatment of anti-androgen drugs, prostate cancer cells will transform from androgen...

Method used

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  • hTERT gene antisense oligodeoxynucleotide, drug composition and application
  • hTERT gene antisense oligodeoxynucleotide, drug composition and application
  • hTERT gene antisense oligodeoxynucleotide, drug composition and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Materials and methods

[0027] 1.1.1 Main reagents

[0028] RPMI1640 culture powder is a product of Beijing Tianwei Times Technology Co., Ltd., fetal calf serum is a product of Lanzhou Minhai Biological Products Co., Ltd., trypan blue is a product of Lanzhou Baiao Biological Products Co., Ltd. Products of Wuhan Boster Biological Products Company. Annexin-V-FITC Apoptosis Detection Kit is a product of Beijing Tianwei Times Technology Co., Ltd.

[0029] 1.1.2 Primer synthesis

[0030] According to the sequence analysis of hTERT gene mRNA, the antisense fragment complementary to the start codon was designed to act as a target sequence with a length of 20 bases.

[0031] The sequence of the antisense fragment hTERTASPS-ODN is 5 ， -GGAGCGCGCGGCATCGCGGG-3 ， (SEQ ID NO. 1).

[0032] The sequence of the positive sense fragment SPS-ODN as a control is 5 ， -CCCGCGATGCCGCGCGCTCC-3 ， (SEQ ID NO. 2).

[0033] Each of the above strands undergoes all sulfo-modifi...

Embodiment 2A

[0043] Example 2 Effect of ASPS-ODN on telomerase activity of flutamide-resistant prostate cancer cells LNCaP-flu

[0044] The experiment was divided into ASPS-ODN group, SPS-ODN group and blank control group. Using a 24-well culture plate, each group was inoculated with 3 wells, and each well was inoculated with 1×10 5cell. Telomerase activity was detected by TRAP-PCR-ELISA method, and the operation was performed according to the instructions of the kit. After treatment with ODN at an optimal concentration of 10 μmol / L, collect LNCaP-flu cells treated with oligonucleotides at different periods (24h, 48h, 72h), wash the cells twice with PBS, add 1ml of ice-cold Washbuffer, and place on ice for 5min , (19 x 10 3 centrifuge at 4°C for 30 min); discard the supernatant, add 40 μL of ice-cold Lysisbuffer, place on ice for 5 min, (19×10 3 g4°C for 30 min); after centrifugation, the supernatant was tested for protein concentration to 5-10 μg / mL. Take 5 μL of PCR amplification pr...

Embodiment 3

[0049] Example 3 Trypan blue exclusion method to detect the effect of hTERTASPS-ODN combined with TFOF on the viability of LNCaP-flu cells

[0050] The experiment was divided into 6 groups: ASPS-ODN group, SPS-ODN group, blank control group, TFOF group, TFOF+SPS-ODN group, TFOF+ASPS-ODN group. Using a 24-well culture plate, each group was inoculated with 3 wells, and each well was inoculated with 1×10 5 cell. ODN10μmol / L was added on the first day, 0.1g / LTFOF was added after 24h, and the same amount of culture medium was added to the blank control group. 24, 48, 72, and 96 hours after adding TFOF, the viability of LNCaP-flu cells was detected by trypan blue exclusion method.

[0051] hTERTASPS-ODN acted on LNCaP-flu cells for 24 hours, then added TFOF0.1g / L to act together for 48 hours, the inhibition rate of LNCaP-flu cells was compared with ASPS-ODN group, SPS-ODN group, blank control group, TFOF group, TFOF+SPS-ODN Compared with the group, there is a significant differen...

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Abstract

The invention provides hTERT gene antisense oligodeoxynucleotide, a drug composition and application, belonging to the technical field of gene drugs. The sequence of the antisense oligodeoxynucleotide is 5minute-GGAGCGCGCGGCATCGCGGG-3minute. After hTERT gene total phosphorothioate modified antisense oligodeoxynucleotide (AS PS-ODN) inhibits flutamide tolerance type prostate cancer cell LNCaP-flu telomerase activity, a Tibetan drug, i.e., dry whole grass of O. Falcata Bunge has a sensibilization effect on LNCaP-flu cell apoptosis.

Description

technical field [0001] The present invention provides an antisense oligonucleotide of hTERT gene, a pharmaceutical composition and its application, in particular to an antisense oligonucleotide modified by full sulfuration of hTERT gene, which contains the total corpus luteum of Oxytropis falciparum of Tibetan medicine The pharmaceutical composition of ketone (TFOF) and their application in the preparation of drugs for treating flutamide-resistant prostate cancer belong to the technical field of gene medicine. Background technique [0002] Prostate cancer is one of the most common tumors in the male urinary system. In the United States, its incidence rate is the first among cancer patients, and its mortality rate is the second. Studies have shown that prostate cancer cells are androgen-dependent cells. By inhibiting the production of human androgen, the growth rate of prostate cancer cells slows down, and the phenomenon of inert growth occurs, and even the metastasis of pros...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K36/48A61P35/00A61K31/7088
CPCA61K31/7088A61K36/48C12N15/1137C12N2310/11A61K2300/00C12N2310/33
Inventor 高晓东杨丽霞陈一戎李永新赵永强苗海东巨生贵
Owner 甘肃省中医院
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