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Liver cancer diagnostic marker, kit and application of liver cancer diagnostic marker

A technology of diagnostic markers and kits, applied in the field of biomedical diagnosis, to achieve high detection sensitivity, improve early diagnosis and prognosis of HCC, and induce cell apoptosis

Inactive Publication Date: 2020-08-14
BEIJING TONGREN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although previous studies have found that a variety of biomarkers can be used for the diagnosis of liver cancer, none of them is significantly better than AFP. Therefore, the combined detection of AFP and other markers may be able to improve the early diagnosis and prognosis of HCC.

Method used

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  • Liver cancer diagnostic marker, kit and application of liver cancer diagnostic marker
  • Liver cancer diagnostic marker, kit and application of liver cancer diagnostic marker
  • Liver cancer diagnostic marker, kit and application of liver cancer diagnostic marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] DcR3 expression in liver cancer cell lines

[0030] Cell culture: The culture medium for culturing human liver cancer cells is RPM I-640 (containing 10% calf serum, antibiotic concentration is 100μg / ml streptomycin and 100U / L penicillin). Cell culture in CO 2 The concentration is 5% in a cell culture incubator at 37°C. Collect the liver cancer cells in the logarithmic growth phase into a centrifuge tube, then add 1-2 times the aspirated amount of PBS to mix thoroughly, and centrifuge at 1500 rpm at room temperature for 5 min. After centrifugation, the supernatant was discarded, and 1ml PBS was added to resuspend the cell clumps in the centrifuge tube, and transferred to a 1.5ml EP tube, and centrifuged at 1500rpm at room temperature for 5min. Remove the remaining PBS and dispense according to the amount of clumps in the centrifuge tube. They are used for mRNA and protein detection respectively.

[0031] Extraction of total cell protein: 1) Aspirate the medium in the cultu...

Embodiment 2

[0035] Effect of DcR3 knockdown on apoptosis of human hepatocellular carcinoma cells

[0036] Human liver cancer HepG2 cells were purchased from the cell bank of Cancer Hospital of Peking Union Medical College. Using RNA interference target lentivirus Lv-DcR3-EGFP-shRNA, blank control lentivirus Lv-NC-EGFP-shRNA successfully transfected DcR3 siRNA knockdown HepG2 cells (KD) and DcR3 blank plasmid control HepG2 cells (negative control, NC), Take wild-type HepG2 cells (Wildtype, WT) as normal control cells.

[0037] Three groups of WT cells, NC cells, and KD cells were cultured to the logarithmic phase. The cells were trypsinized and collected by centrifugation. The serum-free medium was washed 3 times, counted and prepared into a cell suspension; 5×105 cells / well Inoculate into a 6-well plate; 24h later, change to a culture medium containing 10ng / ml FasL, use DMSO as a control, and continue to incubate for 48h; aspirate the culture medium, wash with PBS, add an appropriate amount o...

Embodiment 3

[0040] Influence of cell cycle

[0041] The two groups of WT cells and KD cells were cultured to the logarithmic phase. The cells were trypsinized and collected by centrifugation. The serum-free medium was washed 3 times, counted and prepared into a cell suspension; the WT cells and KD cells were divided into 5×10 5 Cells / well were inoculated into a 6-well plate; cultured for 24h and replaced with 10ng / ml FasL culture medium, with DMSO as control; after 48h, aspirate the cell culture medium, wash once with PBS, add appropriate amount of trypsin cell digestion solution to digest the cells , Gently pipette to blow down the adherent cells, transfer the cells to a centrifuge tube, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, shake the suspended cells and add 0.9% NaCl to wash the cells once; discard the supernatant and shake the centrifuge tube Suspend the cells. While shaking, add 1ml of 75% ethanol dropwise to fix the cells overnight at -20°C; remo...

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Abstract

The invention determines that DcR3 protein can be used as a marker for detecting liver cancer, and provides application of the DcR3 protein which is used for preparing a reagent or a kit for detectingliver cancer molecules. The reagent is a specific reagent corresponding to the DcR3 protein, and certain immune cells suitable for targeted knockdown of expression of the DcR3 can be used to inhibitliver tumors and include CAR-T cells, CIK cells and / or NK cells; and similarly, the reagent can specifically inhibit compound or antibody of the DcR3 and can be used as an inhibitor for inhibiting liver tumors. The kit is used for liver cancer detection, is high in sensitivity, strong in specificity, rapid, simple and convenient, and will become an important means for early diagnosis and prognosisprediction of liver cancer. In addition, precise medical treatment can be realized by targeted knock-down of expression of the DcR3 protein.

Description

Technical field [0001] The invention relates to the technical field of biomedical diagnosis, in particular to the application of a liver cancer diagnostic marker, a kit and the liver cancer diagnostic marker. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the common malignant tumors in my country. Because of its insidious onset, early diagnosis is difficult, it often progresses to the middle and late stage at the time of treatment, and it is often accompanied by intrahepatic metastasis, and the treatment effect is poor. The reason is the lack of effective early diagnosis methods, so the early diagnosis of HCC has always been a research hotspot. At present, alpha fetoprotein (AFP) has been used for early diagnosis of HCC, but its sensitivity and specificity are not high. Studies have confirmed that the accuracy of using AFP alone to diagnose HCC is only 60%, especially for early HCC, its diagnostic sensitivity is greatly reduced; at the same time, patients...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886A61K45/00A61P35/00
CPCC12Q1/6886A61K45/00A61P35/00C12Q2600/158C12Q2600/112C12Q2600/118
Inventor 许英晨张东欣伍冀湘梁超杰张立军栗光明
Owner BEIJING TONGREN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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