Expression regulation reagent of NFATc3 and screening kit based on arsenical-sensitive cells of NFATc3

A kit and reagent technology, applied in the field of expression regulation reagents, can solve problems such as reports that the molecular mechanism has not been studied in depth.

Active Publication Date: 2017-02-15
XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the understanding of its mechanism of action is still in its infancy. Studies have found that it mainly induces apoptosis, induces cell differentiation, inhibits nucleic acid synthesis, acts as an inhibitor of vascular endothelial cells, and direct cytotoxicity. No in-depth studies have been reported

Method used

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  • Expression regulation reagent of NFATc3 and screening kit based on arsenical-sensitive cells of NFATc3
  • Expression regulation reagent of NFATc3 and screening kit based on arsenical-sensitive cells of NFATc3
  • Expression regulation reagent of NFATc3 and screening kit based on arsenical-sensitive cells of NFATc3

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Experimental program
Comparison scheme
Effect test

Embodiment 2

[0068] Based on the above experiments of down-regulating the expression of NFATc3, it was found that different gastric cancer cell lines were sensitive to As 4 S 4 There are differences in the sensitivity of reagents.

[0069] For further cytotoxicity experiments on gastric cancer cell lines: AGS, MGC803, MKN28, MKN45, SGC7901, see Figure 14 , the results showed that: at the same dosage concentration, As 4 S 4 Strong inhibitory effect on AGS and MGC803, compared with Figure 8 and Figure 9 The basal expression of NFATc3 can be seen in AGS and MGC803 with the highest basal expression of NFATc3. 4 S 4 sensitivity is the highest.

[0070] Such as Figure 15 As shown, after gene silencing of NFATc3 in the two most sensitive cell lines: AGS and MGC803, the inhibition rate decreased and the IC50 value increased.

[0071] Such as Figure 16 As shown, the sensitivity to arsenic sulfide increased in the drug-resistant strain SGC7901 after overexpressing NFATc3.

[0072] co...

Embodiment 3

[0102] For gastric cancer tissues of different gastric cancer patients, the expression of NFATc3 in gastric cancer tissues and adjacent tissues was detected by immunohistochemical method, so as to judge the effect of different gastric cancer tissues on As 4 S 4 Sensitivity:

[0103] The screening kit for arsenic-sensitive cells provided in this embodiment includes:

[0104] 10% formaldehyde,

[0105] 70%, 80%, 90%, 95%, 95%, 100% ethanol solution,

[0106] A mixed solution of ethanol and xylene (1:1),

[0107] A mixed solution of paraffin and xylene (1:1),

[0108] Xylene,

[0109] citrate buffer,

[0110] Normal non-immune animal serum,

[0111] Antibody,

[0112] endogenous peroxidase,

[0113] Biotinylated secondary antibody,

[0114] streptavidin-peroxidase,

[0115] DAB,

[0116] Gill's hematoxylin.

[0117] The process of using the above kit to detect gastric cancer tissue is as follows:

[0118] Tissue sections

[0119] 1. Cut the tissue and fix it with 1...

Embodiment 4

[0145] RT-PCR was used to detect the relative expression of NFATc3 between gastric cancer and adjacent tumors,

[0146] The steps of PT-PCR (real-time quantitative fluorescent PCR) are as follows:

[0147] (1) Extraction of sample RNA

[0148] Prepare ice in advance, 75% ethanol, put the 1.5ml centrifuge tube in ice to cool for later use, pre-cool the centrifuge in advance, and wear a mask for operation.

[0149] 1. Take an appropriate amount of gastric cancer and paracancerous tissue into a grinding tube, add 1ml of Trizol, digest and grind, transfer to a cooled EP tube, and centrifuge to remove the precipitate.

[0150] 2. Two-phase separation: According to the ratio of Trizol: chloroform = 5: 1, that is, add 0.2 ml of chloroform to every 1 ml of TRIZOL reagent lysed sample, and tightly cap the tube. After shaking the tube vigorously for 15s, let it stand at room temperature for 5min.

[0151] Centrifuge at 12000 rpm for 15 minutes at 3.4°C.

[0152] 4. After centrifugat...

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Abstract

The invention provides an expression regulation reagent of NFATc3 and a screening kit based on the arsenical-sensitive cells of the NFATc3. Compared with existing reagents using gene knockout, RNAi and the like to lower gene expression, As4S4 serving as the expression regulation reagent of the NFATc3 is low in toxicity. An acute toxicity test shows that LD50 calculated by a Bliss method is 19.3+ / -1.1g / kg, the confidential interval of 95% is 18.3-20.4g / kg, and toxic dose recorded in the Pharmacopoeia is exceeded greatly.

Description

technical field [0001] The invention relates to a reagent for regulating the expression of NFATc3, and also relates to a screening kit for arsenic-sensitive cells based on NFATc3. Background technique [0002] Epidemiological studies have shown that gastric cancer is still a tumor with poor prognosis and high degree of malignancy in the world, and almost half of gastric cancer occurs in Asia. In my country, with the improvement of living standards and changes in eating habits, the incidence of gastric cancer has increased. increase year by year. At present, the main treatment for gastric cancer is surgical treatment, but the overall survival rate of each stage of gastric cancer is between 5% and 15%. At least 80% of patients will eventually have local or distant recurrence or metastasis. Therefore, it is necessary to study and find drugs that can effectively inhibit gastric cancer. [0003] Arsenic-containing traditional Chinese medicines have been used in traditional Chin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/574C12Q1/68C12Q1/02A61K33/36A61P35/00C12R1/91
CPCA61K33/36C12Q1/6886C12Q2600/158G01N33/5008G01N33/57446G01N33/68
Inventor 陈思宇张秀丽康婷仝营营丁文评张莲谭圳
Owner XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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