Application of NTNG1 protein in preparation of reagent or kit for liver cancer diagnosis

A technology of diagnostic reagents and kits, which is applied in the field of cell biology and can solve the problems that the role of NTNG1 development has not been reported.

Inactive Publication Date: 2020-10-30
叶甲舟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no report on the role of NTNG1 in the occurrence and development of human primary liver cancer.

Method used

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  • Application of NTNG1 protein in preparation of reagent or kit for liver cancer diagnosis
  • Application of NTNG1 protein in preparation of reagent or kit for liver cancer diagnosis
  • Application of NTNG1 protein in preparation of reagent or kit for liver cancer diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0095] 1.CCK-8 experiment (cytotoxicity detection)

[0096] The specific steps of the CCK-8 experiment were performed according to the manufacturer's standard protocol. Cells were diluted in serum-free medium and seeded in 96-well cell culture plates at a density of 2000 cells / well. The plate was incubated at 37°C in 5% CO 2 incubator. To measure the growth rate of the cells, replace 100 μL of the spent medium with an equal volume of fresh medium containing 10% CCK-8. Cells were then further incubated at 37°C for 3 hours. Finally, a microplate reader (5082Grodig, Tecan, Austria) was used to measure the value of cell absorbance at 450 nm. The experiment was repeated 3 times and the results were recorded.

[0097] 2. Plate colony formation experiment

[0098] Cells in the logarithmic growth phase were taken from the cells in good growth state, digested with trypsin, resuspended with medium, inoculated into six-well plates with 500 cells per well, and cultured normally for ...

Embodiment 3

[0107] Flow Cytometry

[0108] Collect the HepG2 and SMMC7721 cells of each group transfected for 48 hours, digest with EDTA-free trypsin, wash twice with PBS, add 500 μL of binding buffer to resuspend the cells, and then add 5 μL of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), protected from light, incubated at room temperature for 15 min, and then detected the apoptosis rate of cells in each group on a flow cytometer (BD, USA). Then the cells were collected and fixed with 70% ethanol at 4°C for 2 hours, treated with PI (50 μg / mL) and RNase A I (100 μg / mL), incubated at 37°C in the dark for 30 minutes, and placed in a flow cytometer The changes of DNA content in cells were analyzed in each period. Experiments were repeated three times.

[0109] Conclusion: NTNG1 inhibits apoptosis and regulates cell cycle progression in human hepatocellular carcinoma cells

[0110] The uncontrolled proliferation of tumor cells caused by apoptosis and cycle regulat...

Embodiment 4

[0115] 1 Transwell cell experiment

[0116] The Transwell chamber was placed in a 24-well plate prepared with artificial basement membrane (Matrigel) matrigel in advance, placed in an environment of 37°C for 30 minutes, and a medium containing 10% fetal bovine serum was added to the lower chamber of the Transwell chamber. Add the upper chamber of Transwell to resuspend the cells of each group with serum-free DMEM high-glucose medium, 5% CO 2 , Cultivate for 48 hours at 37°C with saturated humidity. After 48 hours, the culture solution in the lower chamber was removed, the small chamber was fixed in 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet solution for 10 minutes, dehydrated with ethanol, and the polycarbonate film was placed on a glass slide, and the high power lens ( ×200) to count the number of invasive cells. The experiment was repeated three times.

[0117] 2 Cell scratch experiment

[0118] The HepG2 and SMMC7721 cell lines that knocked dow...

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Abstract

The invention belongs to the technical field of cell biology, and particularly discloses application of NTNG1 protein in preparation of a reagent or a kit for liver cancer diagnosis. The NTNG1 proteinshows higher expression quantity in a liver cancer cell line than that in normal liver cells, which indicates that the expression level of the NTNG1 gene is obviously related to hepatocellular carcinoma; the expression quantity of NTNG1 mRNA in cancer tissues is higher than that of NTNG1 mRNA in para-carcinoma tissues, which indicates that the NTNG1 gene and its expression product can be used asa molecular marker of hepatocellular carcinoma and provide effective information for diagnosis and targeted therapy of liver cancer. Results of clinical tissue samples and cell experiments show that NTNG1 plays a role of oncogene in the development process of liver cancer. Meanwhile, NTNG1 is suggested to be a promising biomarker of liver cancer and a potential treatment target.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to the use of NTNG1 protein in the preparation of diagnostic reagents or kits for liver cancer. Background technique [0002] Hepatocellular carcinoma (HCC, hereinafter referred to as liver cancer) is the sixth most prevalent cancer worldwide and the third most common cause of cancer-related death. According to cancer statistics in 2018, there are 841,000 new cases of HCC every year worldwide, and about 55% of HCC patients are from China. In recent years, liver cancer is also one of the main causes of cancer death in my country, and its mortality rate ranks second. Due to the lack of effective clinical diagnostic targets, and the high degree of malignancy, strong invasiveness, and rapid progression of liver cancer, most patients have reached the middle and late stages when they are diagnosed. Even if they receive standard systemic treatment, the median OS often does not exceed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886G01N33/574A61K45/00A61P1/16A61P35/00
CPCC12Q1/6886G01N33/57438G01N33/57484G01N33/74A61K45/00A61P1/16A61P35/00C12Q2600/158G01N2333/475
Inventor 叶甲舟林燕梁嵘马良余红平
Owner 叶甲舟
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