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Hepatocellular carcinoma lenvatinib drug-resistance cell strain and preparation method and application thereof

A human liver cancer cell line, lenvatinib technology, applied in the field of biomedicine, to achieve the effect of high scientific research and production application value

Active Publication Date: 2019-10-08
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lenvatinib was officially approved as a first-line drug for the treatment of advanced liver cancer in 2018. Although it is more suitable for the use of oriental populations with hepatitis background than sorafenib, doctors and patients will also face the thorny problem of tumor drug resistance , but there is no liver cancer cell line and drug-resistant animal model resistant to lenvatinib, and the method of constructing tumor drug-resistant cell lines needs to be improved urgently

Method used

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  • Hepatocellular carcinoma lenvatinib drug-resistance cell strain and preparation method and application thereof
  • Hepatocellular carcinoma lenvatinib drug-resistance cell strain and preparation method and application thereof
  • Hepatocellular carcinoma lenvatinib drug-resistance cell strain and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of hepatocellular carcinoma lenvatinib drug-resistant cell line

[0032] Get human liver cancer HuH-7 cells with DMEM medium (penicillin 100 units / mL, streptomycin 100 ug / mL) containing 10% fetal bovine serum, at 37 ℃, 5% CO 2 cultured in an incubator; in order to determine the IC50 value of HuH-7 cells, the logarithmic phase cells were planted in a 96-well plate, and the concentration gradients of lenvatinib were set to 0, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25 μmol / L, keep the blank control (only add 100ul DMEM medium containing 10% fetal bovine serum), set 3 duplicate wells for each gradient, treat with lenvatinib for 48 hours, use CCK-8 kit The IC50 value of the cells was measured, and the IC50 value was 3.13 μmol / L, so the initial administration concentration was 3 μmol / L.

[0033] The cells were treated with lenvatinib at a concentration of 3 μmol / L, and the medium was changed every 48 hours. When the cells were full, they were subcult...

Embodiment 2

[0034] Embodiment 2: the IC50 value of liver cancer cell line HuH-7 and the IC50 value of drug-resistant strain ML30

[0035]HuH-7 and ML30 cells in the logarithmic growth phase were digested with 0.5% trypsin (Gibco) and counted, and seeded into 96-well plates at a density of 5000 cells per well in DMEM containing 10% fetal bovine serum for 6 hours After the cells adhered to the wall, the medium was removed, and the medium (DMEM containing 10% fetal bovine serum) containing different concentrations of lenvatinib was added, and the concentration gradient was 0, 1, 10, 20, 30, 40, 50 μmol / L (more than 50 μmol / L lenvatinib can not be dissolved in the culture medium, which will affect the cell state and absorbance measurement), leave a blank control (only add DMEM medium containing 10% fetal bovine serum), set 3 duplicate holes for each concentration, Add 100 μL of medium to each well, place at 37°C, 5% CO 2 Incubate for 48 hours in the incubator.

[0036] Add 10 μL of CCK-8 wo...

Embodiment 3

[0044] Embodiment 3: the cell shape of HuH-7 and ML30 cell under light microscope

[0045] Observe the morphology of live cells with an inverted phase-contrast microscope: HuH-7 cells and ML30 drug-resistant cells in the logarithmic growth phase were taken respectively, and after changing the liquid with PBS (pH=7.2-7.4), the morphology of living cells was observed under an inverted microscope. It can be observed by light microscopy that the size of HuH-7 cells is basically the same. figure 1 A, 1B shown); while ML30 drug-resistant cells vary in size and shape, and more cells fuse into cell colonies (such as figure 1 C, shown in 1D).

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Abstract

The invention discloses a hepatocellular carcinoma lenvatinib drug-resistance cell strain. The hepatocellular carcinoma lenvatinib drug-resistance cell strain is characterized in that the cell strainis named human hepatocellular carcinoma lenvatinib drug-resistance cell strain ML30, and the preservation number is (C2019127). A preparation method of the hepatocellular carcinoma lenvatinib drug-resistance cell strain comprises the following steps that 1, after resuscitation of human hepatocellular carcinoma cell strain HuH-7 cells, culturing is carried out in a 5% CO2 incubator at 37 DEG C by aDMEM medium containing fetal bovine serum of 10%; and 2, the human hepatocellular carcinoma cell strain HuH-7 cells in the logarithmic phase are taken, lenvatinib is added in a culture dish, the initial concentration is 3[mu]mol / L, and timely passage or liquid exchange is carried out; after each passage, the concentration of 0.5[mu]mol / L is increased, continuous culture is carried out for more than six months; until the human hepatocellular carcinoma cell strain HuH-7 cells which can grow rapidly in the range of being greater than 30[mu]mol / L are selected and used as the drug-resistance cellstrain, and the drug-resistance cell strain is named as the human hepatocellular carcinoma lenvatinib drug-resistance cell strain ML30. The hepatocellular carcinoma lenvatinib drug-resistance cell strain has important application prospects in studying the mechanism of liver cancer drug-resistance, reversing the drug-resistance of liver cancer cells, guiding the patient to use drugs and establishing a drug-resistance animal model.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a hepatocellular carcinoma lenvatinib drug-resistant cell line and a preparation method and application thereof. Background technique [0002] Primary liver cancer is a common malignant tumor of the digestive system. There are more than 850,000 new cases and 700,000 deaths each year worldwide, half of which occur in China, and the incidence rate is increasing year by year. It has become the first malignant tumor in my country. Two leading causes of death. Among all primary liver cancers, hepatocellular carcinoma (HCC) is the most common type, accounting for about 90%, and has the characteristics of easy recurrence, metastasis, high mortality, and poor prognosis. Clinically, most patients with liver cancer are already in the middle or late stage when they are clearly diagnosed, thus losing the opportunity for surgical treatment, and drugs have become the main force in the trea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02A01K67/027
CPCC12N5/0693G01N33/5011A01K67/0271C12N2501/06C12N2503/02G01N2500/10A01K2207/12A01K2227/105A01K2267/03
Inventor 胡倍源钦伦秀覃伟董琼珠
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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