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Radiation sensibility tumor-targeted promotor and uses thereof

A technology of chimeric promoters and sequences, applied in the direction of antineoplastic drugs, introduction of foreign genetic material using vectors, drug combinations, etc.

Inactive Publication Date: 2009-08-19
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are no related researches at home and abroad that connect the radiation response element CArG and the telomerase reverse transcriptase promoter hTERT in series, or connect the chimeric promoter in series with the double promoter of the CMV promoter, and combine radiation to improve their regulation of downstream gene expression, but There is reason to believe that the purposeful modification of the promoter can effectively improve its promoter activity and increase the expression of downstream therapeutic genes, which is a promising way to improve the efficacy of gene therapy

Method used

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  • Radiation sensibility tumor-targeted promotor and uses thereof
  • Radiation sensibility tumor-targeted promotor and uses thereof
  • Radiation sensibility tumor-targeted promotor and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of Chimeric Promoter

[0051] 1 Experimental materials:

[0052] 1.1 Plasmid: Plasmid pBasic-hTERT promoter was amplified by Professor Horikawa by PCR to obtain the full length of the hTERT promoter (-3915 to +40) and then cut off by restriction enzymes, containing the hTERT promoter from -385 to +40 (see references: Horikawa, I., Cable, P.L., Mazur, S.J. et al.. Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for anendogenous mechanism of transcriptional repression. Mol Biol Cell, 2002; 13( 8): 2585-2597), the vectors pGL3-Basic and pGL3-Control were purchased from Promega.

[0053] 1.2 Reagents and instruments: plasmid DNA extraction kit (Tiangen Company); gel recovery kit (Omega Company); PCRMix (Tiangen Company); restriction enzymes Mlu I, EcoRI, Pst I, Xho I, BamHI, HindIII , T4 ligase, and Marker (Fermentas). 530 UV-Vis Spectrophotometer (Beckman Coulter Company, US...

Embodiment 2

[0069] Example 2 Study on the Specificity of Chimeric Promoters

[0070] 1 Experimental materials:

[0071] 1.1 Cells: human embryonic lung cells hEL, purchased from China Center for Type Culture Collection.

[0072] 1.2 Plasmid: The chimeric promoter plasmid has been successfully constructed by the applicant; internal reference plasmid pRL-TK (Promega Company) and control plasmids pGL3-Basic, pGL3-Control (purchased from Promega Company).

[0073] 1.3 Reagents: liposome lipofectamine 2000 was purchased from Invitrogen, and the dual fluorescence detection kit was purchased from PROMEGA. RPMI-1640 culture solution containing 10% (V / V) calf serum was purchased from Gibco.

[0074] 1.4 Instrument: TD-20 / 20 Fluorescence Luminometer (Tumer Designs, USA), 530 UV-Vis Spectrophotometer (Beckman Coulter Company, USA).

[0075] 2 Experimental method:

[0076] 2.1 Cell transfection: 1 day before transfection, cells in the exponential growth phase were taken, digested with trypsin,...

Embodiment 3

[0081] Example 3 Effect of Radiation Enhanced Chimeric Promoter Activity in Human Cervical Cancer Hela Cells

[0082] 1 Experimental materials:

[0083] 1.1 Cells: Human cervical cancer Hela cells (China Center for Type Culture Collection), conventional cell culture and passage.

[0084] 1.2 Plasmid: Plasmid p(CArG) n -hTERTp-Luc,p(CArG) 4 -hTERTp-CMVp-Luc has been successfully constructed by the applicant (see Example 1). The internal reference plasmid pRL-TK, the negative control plasmid pGL3-Basic and the positive control plasmid pGL3-Control were purchased from Promega.

[0085] 1.3 Reagents: liposome lipofectamine 2000 was purchased from Invitrogen, and the dual fluorescence detection kit was purchased from PROMEGA. RPMI-1640 culture solution containing 10% (V / V) calf serum was purchased from Gibco.

[0086] 1.4 Instrument: TD-20 / 20 Fluorescence Luminometer (Turner Designs, USA), 530 UV-Vis Spectrophotometer (Beckman Coulter Company, USA).

[0087] 2 Experimental ...

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Abstract

The invention discloses a radiation-sensitive tumor target promoter and application thereof, relates to construction of a chimeric promoter, potentiation of the radiation to activity of the chimeric promoter and synergic action of combination of radiation and gene treatment in killing tumor cells. Normal cells and three different tumor cells are adopted for research, and specificity of the chimeric promoter and the effect of radiation on the activity of the chimeric promoter can be evaluated through expression of a downstream luciferase reporter gene regulated by the chimeric promoter. Synergic killing action when the radiation and the HRP / IAA regulated by the chimeric promoter are combined can be evaluated through inspecting cell proliferation rate and cell apoptosis rate. Results prove that the chimeric promoter has tumor specificity, radiation can enhance the activity of the chimeric promoter; when the radiation is combined with the chimeric promoter, the chimeric promoter has obvious killing effect on a plurality of tumor (cervical carcinoma, liver cancer and lung cancer) cells. Therefore, combined radiotherapy of (CArG)n-hTERTp-HRP / IAA or (CArG)n-hTERTp-CMVp-HRP / IAA can be applied to preparation of medicines for treating or preventing cells of lung cancer, liver cancer and cervical carcinoma.

Description

technical field [0001] The present invention relates to the field of medical technology, more specifically to a radiation-sensitive tumor-targeted promoter, that is, a modified promoter obtained by modifying the promoter of the tumor-targeted telomerase reverse transcriptase gene, and to using the modified The application of the identified promoter to regulate the horseradish peroxidase (HRP) / indole acetic acid (IAA) suicide gene system in drugs for destroying and inhibiting the growth of cervical cancer, lung cancer, and liver cancer cells. Background technique [0002] Looking at the changing pattern of the disease spectrum at home and abroad, malignant tumors have become a major disease that seriously threatens human health. With the advancement of molecular biology, gene therapy has brought hope to defeat tumors. Various countries have carried out a lot of research on gene therapy for malignant tumors and achieved certain results. Some gene therapy programs have been us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63A61K48/00A61P35/00C12N15/113
Inventor 周云峰廖正凯周福祥谢丛华熊杰孙文洁
Owner WUHAN UNIV
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