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Method for regulating expression, quantity and activity of heat shock protein 70 and application thereof

A heat shock protein, active technology, applied in peptide/protein components, medical preparations containing active ingredients, pharmaceutical formulations, etc., can solve the problems of limited stability and solubility, limited clinical application, low bioavailability, etc. Inhibition of solid tumor growth, promotion of activation, and enhancement of sensitivity

Active Publication Date: 2012-12-12
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the drugs geldanamycin and 17-AAG targeting HSP90 as therapeutic targets have entered clinical research. However, although geldanamycin and 17-AAG have good anti-tumor activity, they still have limitations, mainly in the stability and solubility. Large, can be metabolized by cytochrome P450, low bioavailability, and geldanamycin has severe liver toxicity, which limits its clinical application

Method used

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  • Method for regulating expression, quantity and activity of heat shock protein 70 and application thereof
  • Method for regulating expression, quantity and activity of heat shock protein 70 and application thereof
  • Method for regulating expression, quantity and activity of heat shock protein 70 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Using RNA interference technology to down-regulate the expression of HSP70 in tumor cells or tissues, combined with the anti-tumor drug TRAIL to induce tumor cell apoptosis, and treat the mouse A549 solid tumor model as an example:

[0073] 1. Construction of HSP70 interference plasmid:

[0074] Genscript's software siRNA target finder was used to select the appropriate target sequence from the HSP70 sequence, and then Genscript's software siRNA Construct Builder was used to construct a DNA fragment for interference that could be connected to the vector pRNAT-U6.1 / Neo. The DNA fragment includes a sense strand (sense strand, which is the same as the target sequence), a hairpin structure, an antisense strand (antisense strand, which is complementary to the sense strand), and a poly T signal at the 3' end that can terminate transcription; in addition, in order to facilitate It was connected to the vector pRNAT-U6.1 / Neo, and restriction sites for BamH I (G↓GATCC) and Hind I...

Embodiment 2

[0121] Using RNA interference technology to down-regulate the expression of HSP27 in tumor cells or tissues, combined with the anti-tumor drug TRAIL to induce tumor cell apoptosis, and treat the mouse A549 solid tumor model as an example:

[0122] 1. Construction of HSP27 interference plasmid:

[0123] The design and construction method of the HSP27 interference plasmid is the same as that described in Example 1. The fragment for HSP27 RNA interference designed by Gensicript software is as follows: 5'-GGATCCCGTCTCATCGGATTTTGCAGTTGATATCCGCTGCAAAATCCGATGAGACTTTTTTCCAAAAGC-3', and the two fragments synthesized by Shenergy Gambling Biotechnology Co., Ltd. are: 5'-GATCCCGTCTCATCGGATTTTGCAGTTGATATCCGCTGCAAAATCCGATGAGACTTTTTTCCAAA-3' -AGCTTTTGGAAAAAAGTCTCATCGGATTTTGCAGCGGATATCAACTGCAAAATCCGATGAGACGG-3'.

[0124] 2. Detection of the interference effect of HSP27 interference plasmid and its ability to induce A549 cell apoptosis at the cellular level when combined with TRAIL:

[0125]...

Embodiment 3

[0154] Detection of the ability of HSP70 interference combined with TRAIL to induce apoptosis of various non-small cell lung cancer cells NSCLC (A549, SW1573 and H460) at the cellular level (cell apoptosis detected by flow cytometry):

[0155] Transfect the HSP70 interference plasmid into A549, SW1573 and H460 cells with GenEscort Reagent transfection reagent, add TRAIL to induce cell apoptosis 24 hours later, harvest the treated cells after 18 hours, resuspend in 100 μl sample buffer, and keep aside While oscillating, add 1ml of pre-cooled 70% ethanol dropwise, and fix overnight at 4°C. Then centrifuge at 3000rpm for 5 minutes, suck off the supernatant, add 1ml PI staining solution, mix well, place in the dark at room temperature for more than 30 minutes, and detect within 24 hours. Data were collected and analyzed using CellQuest software.

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for regulating expression, quantity and activity of a heat shock protein 70 and application thereof. The method provided by the invention can also regulate tumor tissue, activity of a transcription factor NF-kB, expression and activity of a death receptor DR4 and DR5, phosphorylation and activity of JNK and c-Jun, expression and activity of a p53 protein, expression and proapoptotic activity of proapoptotic molecules Bax, Bid, t-Bid, capsase 3, capsase 8 and capsase 9, and expression and activity of c-FLIP and Bcl-2, and promote apoptosis of tumor tissues and cells. The invention also relates to application of the above method to preparation of drugs cooperatively applied with cell apoptosis induced drug.

Description

1. Technical field: [0001] The invention belongs to the field of biological technology, and in particular relates to a method for regulating the expression, quantity and activity of heat shock protein 70 and its application. 2. Background technology: [0002] According to statistics from the Ministry of Health of China, the total number of new tumor patients in China is about 2.127 million each year, of which, there are about 1.06 million new patients with malignant tumors every year; There are about 1.485 million cancer patients. Taking the morbidity and mortality of lung cancer as an example, about 160,000 people die of lung cancer in the United States every year, and 75% of them are non-small cell lung cancer (NSCLC). In major cities in my country, the incidence of lung cancer has occupied the first place in the incidence of various malignant tumors. Nearly 800,000 people die of lung cancer in my country every year, and non-small cell lung cancer patients account for ab...

Claims

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Application Information

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IPC IPC(8): C12N15/85A61K45/00A61K45/06A61K48/00A61K38/19A61P35/00
Inventor 华子春庄红芹
Owner NANJING UNIV
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