Establishing method of osteosarcoma EPI (Epirubicin) drug-resistant cell line

A technology of epirubicin and drug-resistant cells, applied in the medical field, can solve the problems of long time-consuming, unstable tolerance, passive treatment of osteosarcoma patients, etc., and achieve the effect of short construction period and stable drug resistance

Pending Publication Date: 2018-11-16
ZHONGSHAN HOSPITAL FUDAN UNIV
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Problems solved by technology

But just like all drugs, once used, drug resistance is unavoidable, even for epirubicin with low toxicity, and the treatment of osteosarcoma patients will therefore be passive, and metastasis and recurrence will be inevitable. on schedule
[0004] At present, the mechanism of epirubicin resistance in osteosarcoma is still unclear, and reports on how to reverse epirubicin resistance in osteosarcoma are nowhere to be found. One of the main reasons for this research gap is that Scarcity of rubicin-resistant cell lines
The establishment of drug-resistant cell lines mainly includes the method of gradually increasing the drug concentration induction method (or called the method of gradually increasing the drug concentration) and the method of high-dose shock induction. To 12 months, the latter requires a short cycle, but the cells are prone to death during the shock process, and the tolerance is unstable, making the establishment of drug-resistant cell lines a long-term high-risk and high-cost situation

Method used

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  • Establishing method of osteosarcoma EPI (Epirubicin) drug-resistant cell line
  • Establishing method of osteosarcoma EPI (Epirubicin) drug-resistant cell line
  • Establishing method of osteosarcoma EPI (Epirubicin) drug-resistant cell line

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Embodiment 1

[0038] Example 1 Construction of epirubicin-resistant cell lines for osteosarcoma

[0039] Human 143b cells in the logarithmic growth phase were inoculated in DMEM medium containing 10% FBS at 37°C and 5% CO 2 cultured in a saturated humidity incubator. Add an appropriate amount of epirubicin to the culture medium for induction, the initial concentration is 5nM / L, when the cells return to normal growth, increase the concentration by 5nM / L, when the cells can grow normally and pass on at a concentration of 20nM / L, use 40nM / L EPI shock culture for 10h, immediately replace the medium with an EPI concentration of 20nM / L and culture for about a week, when the cells grow stably and pass passage, shock again until the cells can grow in 40nM / L EPI, and then culture at 10nM / L The concentration of L is increasing. When the cells can grow and pass normally at a concentration of 80nM / L, shock induction with 120nM / L EPI for 10h, immediately replace the medium with an EPI concentration of ...

Embodiment 2

[0040] Morphological characteristics comparison of embodiment 2 human 143b cells and 143b-EPI cells

[0041] The morphology of 143b cells before and after epirubicin induction was observed with an inverted phase-contrast microscope, and photographed and recorded. The result is as figure 1 As shown, 143b is mainly short-spindle-shaped with sharp edges and corners, while 143b-EPI is mainly long-spindle-shaped, and some cells are obviously elongated, suggesting that the morphology of cells has changed significantly after induction.

Embodiment 3

[0042] The growth comparison of embodiment 3 people's 143b cell and 143b-EPI cell

[0043] Take the 143b in the logarithmic growth phase and the drug-resistant 143b-EPI cells respectively, trypsinize and resuspend the cells in DMEM complete culture medium, and adjust the density to 3×10 4 cells / mL, inoculated in 96-well plate, 100 μL per well. Set up 5 replicate wells in each group and place at 37°C, 5% CO 2 Cultivate overnight in a saturated humidity incubator, add an appropriate amount of EPI to the two types of cells the next day, the final concentration is 20nM / L, and culture them in the incubator for 5 days. curve. like figure 2 As shown, the two kinds of cells grew slowly in the first two days, and on the third day, the growth of 143b-EPI accelerated and entered the exponential phase, while the growth of the parental cell 143b was relatively stable, and the exponential phase was not obvious. In general, under the condition of 20nM / L EPI, drug-resistant cells 143b-EP...

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Abstract

The invention discloses an establishing method of an osteosarcoma EPI (Epirubicin) drug-resistant cell line. The establishing method comprises the following step: by taking human 143b cells as parentcells and EPI as an induction drug, jointly implementing a drug gradual increment induction method and a large dosage impact induction method with in a drug concentration range of 3 to 120 nM/L untilobtaining a drug-resistant cell line which can be in normal growth and passage under the drug concentration of at least 120 nM/L. The establishing method disclosed by the invention has the characteristics of short establishing period and stable drug resistance of the drug-resistant cell line, and the established drug-resistant cell line can be used for research on an osteosarcoma EPI drug-resistant mechanism and research on reversing drug resistance.

Description

technical field [0001] The invention belongs to the medical field, and in particular relates to a method for establishing an epirubicin drug-resistant cell line of osteosarcoma. Background technique [0002] Osteosarcoma is a common primary bone malignancy, more common in adolescents, with high malignancy and early distant metastasis. The 5-year survival rate after simple amputation is less than 20%. With the emergence of neoadjuvant chemotherapy, the 5-year survival rate of patients can reach 50% to 80%, and about 60% of patients with limb osteosarcoma can save limbs, but for patients with distant metastasis, the curative effect is not ideal , the 5-year survival rate is only 20 to 30%. Molecular targeted therapy is an important direction in cancer treatment, which can effectively prolong the survival period of cancer patients and improve the quality of life of patients. However, targeted therapy for osteosarcoma is still in the research stage, and there is a lack of large...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693G01N33/5011
Inventor 陈子贤曹渊武张亮王晓峰万盛成姜晓幸
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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