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Method for establishing hepatocellular carcinoma cis-platinum drug-resisting cell strain

A technology for drug-resistant cells and hepatocellular carcinoma, applied in the field of biomedicine, can solve the problems of poor prognosis of liver cancer patients, and achieve the effect of simple and easy method, high scientific research and production application value

Inactive Publication Date: 2016-01-27
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Primary liver cancer is one of the most common malignant tumors in the world. Its incidence rate ranks sixth among malignant tumors, and its mortality rate ranks third. Most patients with liver cancer are already in the middle and late stages when they are diagnosed, and they frequently recur after surgery. metastases, leading to poorer prognosis in HCC patients

Method used

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  • Method for establishing hepatocellular carcinoma cis-platinum drug-resisting cell strain
  • Method for establishing hepatocellular carcinoma cis-platinum drug-resisting cell strain
  • Method for establishing hepatocellular carcinoma cis-platinum drug-resisting cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Method for Inducing and Establishing Liver Cancer Drug-resistant Strain LM3 / CDDP Cells

[0035] (1) Take LM3 cells and use DMEM culture medium after recovery, at 37°C, 5% CO 2 Cultivated in an incubator;

[0036] (2) In order to find the most suitable stimulating concentration of cisplatin, LM3 cells in the logarithmic growth phase were planted in 24-well plates, and 0.1 μg / L, 0.25 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L , 5μg / L, 10μg / L cisplatin, timely subculture or change medium. Each concentration of chemotherapeutic drugs was added correspondingly at each subculture or medium change. The optimal concentration is the maximum concentration at which the cells can survive and grow slowly after the chemotherapeutic drug is added. The long-term growth referred to in this example refers to the growth time of more than 3 months.

[0037] The experimental results show that: when the concentration of cisplatin is 0.5 μg / mL, LM3 cells can survive but grow very slowly; when the con...

Embodiment 2

[0040] Morphological Observation of Drug-resistant Liver Cancer LM3 / CDDP Cells

[0041] Use an inverted phase-contrast microscope to observe the morphology of living cells: take LM3 cells and LM3 / CDDP cells in the logarithmic growth phase, wash and replace with normal saline, and observe the morphology of living cells under an inverted microscope with a magnification of 200 times. Such as figure 2 As shown in (a), it can be observed by light microscopy that the LM3 cells are basically the same size, polypodia-like polygonal, and some are round. And as figure 2 As shown in (b), LM3 / CDDP cells can be observed by light microscopy in different sizes, including round and polygonal shapes, and the polygonal shapes are different.

Embodiment 3

[0043] Determination of Growth Curve of Liver Cancer Drug-resistant Strain LM3 / CDDP Cells

[0044] Take LM3 and LM3 / CDDP cells in the logarithmic growth phase, digest and count with 0.5% trypsin, inoculate into 96-well plate at a density of 5000 cells per well and set up 3 duplicate wells, add 200 μL of medium to each well , placed in a 37°C, 5% CO2 incubator for culture. Aspirate the medium at each time point of 0h, 24h, 48h, 72h and 96h respectively, add 100μLCCK8 working solution to each well, incubate in the incubator for 1 hour, put it into a microplate reader to detect with a wavelength of 570nm, and read the The OD value and the average value were calculated, and the growth curve was drawn with the time as the abscissa and the average OD value as the ordinate.

[0045] The result is as image 3 shown. The growth curves of LM3 and LM3 / CDDP, the results are as follows image 3 shown. After several days of continuous measurement of the growth curves of the two cells, ...

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Abstract

The invention provides a method for establishing a hepatocellular carcinoma cis-platinum drug-resisting cell strain. The method is characterized by comprising the following steps: step 1, taking human hepatoma carcinoma LM3 cells, thawing the cells, and culturing the cells by adopting DMEM culture solution in a culture box; step 2, taking the LM3 cells in the logarithmic phase, planting the LM3 cells in the logarithmic phase in a 24 well-plate, respectively adding 0.1mu mol / L cis-platinum, 0.25 mu mol / L cis-platinum, 0.5 mu mol / L cis-platinum, 1 mu mol / L cis-platinum, 2 mu mol / L cis-platinum, 5 mu mol / L cis-platinum, and 10 mu mol / L cis-platinum into the 24 well-plate, and carrying out passage or liquid change timely, wherein after the addition of cis-platinum, the maximum concentration for keeping the long-term growth of the cells is taken as the optimum concentration; and step 3, searching for the optimum concentration as 0.5 mu mol / L, and exciting the cells for the term of 4 months with the optimum concentration, and thus obtaining the hepatocellular carcinoma cis-platinum drug-resisting cell strain. The obtained hepatocellular carcinoma cis-platinum drug-resisting cell strain can be used for searching for the tumour drug-resisting marker and screening and evaluating novel anti-tumor drugs, etc.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a method for establishing a cisplatin-resistant cell line of hepatocellular carcinoma. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in the world. Its incidence rate ranks sixth among malignant tumors, and its mortality rate ranks third. Most patients with liver cancer are already in the middle and advanced stages when they are diagnosed, and they frequently recur after surgery. Metastasis leads to a poor prognosis for HCC patients. Chemotherapy is one of the important methods for the treatment of liver cancer. However, tumor multidrug resistance (MDR) often occurs and is difficult to solve in chemotherapy, that is, tumors are simultaneously resistant to multiple chemical drugs with different structures and mechanisms of action. It is the leading cause of chemotherapy failure. Therefore, overcoming tumor multidrug resistance and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02C12R1/91
Inventor 李恒宇程卓宁北芳盛湲李晓峰刘娜吴凯男秦科宇阎娜
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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