Construction method of dormant or recurrent cell strain induced by tumor endocrine drug

A tumor cell and construction method technology, which can be applied to tumors/cancer cells, animal cells, and cells modified by introducing foreign genetic material, etc., can solve the problems of dormancy, complex drug resistance mechanism, unfavorable tumors, etc. Conducive to stable labeling and in vivo tracking, with strong specificity

Pending Publication Date: 2021-11-05
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are no cell lines for dormancy and relapse that simulate clinical in vivo construction, only some cell lines with drug resistance, and these drug-resistant cell lines produce multidrug resistance, resulting in complex drug resistance mechanisms, which are not conducive to Study on tumor dormancy, drug resistance, recurrence and metastasis caused by a single drug
[0004] With the deepening of tumor research, there are many limitations in the application of traditional methods (such as caliper measurement of tumor volume, tumor tissue slices, etc.) in tumor research
For example, mice need to be killed to take out tumor tissues before tissue section observation. Therefore, at different time points or in different experimental groups, a batch of experimental mice need to be killed to obtain sufficient statistical data, which not only greatly increases the cost of the experiment, but also It is difficult to eliminate errors due to individual differences in mice, it is impossible to obtain reliable repeatability data, and the accuracy of experiments cannot be guaranteed when making slices
In addition, micrometastases are also difficult to detect by traditional means

Method used

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  • Construction method of dormant or recurrent cell strain induced by tumor endocrine drug
  • Construction method of dormant or recurrent cell strain induced by tumor endocrine drug
  • Construction method of dormant or recurrent cell strain induced by tumor endocrine drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of Human Estrogen Receptor Positive Breast Cancer MCF7-FD / MCF7-FR Cells

[0046] The construction process of human estrogen receptor positive breast cancer MCF7-FD / MCF7-FR cells is as follows: figure 1 .

[0047] (1) Screen MCF7 cells stably expressing luciferase gene

[0048] An expression vector containing the luciferase gene was constructed for future use. The construction of the vector refers to the instructions in the kit.

[0049] MCF7 cells were seeded in 6-well plates, and transient transfection was performed when the confluence of the cells reached 80%. Each well of a 6-well plate was transfected with 2ug of plasmid DNA and 6ul of liposomes, at 37°C, 5% CO 2 Incubate for 24h. After 72 hours of transfection of MCF7 cells, the supernatant was discarded, and RPMI 1640 medium containing 10% FCS and 800 μg / ml G418 was added, and cultured for about 3 weeks. named Luc-MCF7).

[0050] Detection of bioluminescence in cell lines stably expr...

experiment example 1

[0057] Experimental example 1: Morphological observation of human estrogen receptor positive breast cancer MCF7-FD / MCF7-FR cells

[0058] Use an inverted phase-contrast microscope to observe the morphology of living cells: Take dormant MCF7-FD cells and relapse-like MCF7-FR cells in the logarithmic growth phase, and observe the morphology of living cells under an inverted microscope at 200 times magnification, as shown in figure 2 shown.

[0059] It can be observed by light microscopy that the size and shape of MCF7-FD cells are relatively consistent, like paving stones, showing a more mesenchymal morphology, and a larger proportion of cells with less polarity and more roundness.

[0060] Through the light microscope, it can be observed that the MCF7-FR cells are of different sizes, with fusiform and polygonal shapes, and many protrusions, and the polygonal shapes are different, showing a more epithelial-like shape with stronger polarity, which can be seen clearly tentacles....

experiment example 2

[0061] Experimental Example 2: Determination of resistance index of human estrogen receptor positive breast cancer MCF7-FD / MCF7-FR cells to fulvestrant

[0062] Take the dormancy-like cells MCF7-FD, relapse-like cells MCF7-FR and parental cells obtained in Example 1, culture them in RPMI1640 medium containing 2% fetal bovine serum, cultivate them to the logarithmic growth phase, and digest them with 0.25% iodine And count, adjust the cell concentration to 2×10 with 1640 culture medium 4 / ml. At the same time, set up no-drug control wells in which only cells were inoculated and zero-adjustment wells in which only culture medium was added. Inoculate 0.1ml of single-cell suspension in each well of a 96-well plate, each well containing 2000 cells, and culture overnight in an incubator. Discard the supernatant in each well, wash once with PBS, and then add 0.1 ml of cell culture medium containing fulvestrant, the concentration gradient of fulvestrant is shown in Table 1, and the ...

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Abstract

The invention discloses a construction method of a dormant or recurrent cell strain induced by a tumor endocrine therapeutic drug. The construction method comprises the following steps: transfecting a carrier for expressing a luciferase gene into tumor cells through a biological means, screening the tumor cells for stably expressing the luciferase gene, and carrying out a nude mouse in-situ xenotransplantation tumor formation experiment, and after tumor formation of nude mice, injecting the tumor endocrine therapeutic drug for treatment; and after the treatment is finished, taking out non-proliferative tumor blocks and proliferative-recovered tumor blocks, and respectively carrying out primary culture to obtain a dormant cell strain and a recurrent cell strain. The invention further discloses a dormant cell strain MCF7-FD and a recurrent cell strain MCF7-FR of human estrogen receptor positive breast cancer induced by fulvestrant. The construction method simulates clinical administration, innovatively constructs a dormant or recurrent cell model in vivo, and has high specificity. Meanwhile, luciferase is also beneficial to stable marking and in-vivo tracing, and especially has higher sensitivity to tiny metastasis.

Description

technical field [0001] The invention belongs to the field of tumor biology, and in particular relates to a method for constructing dormant or recurrent cell lines induced by tumor endocrine therapy drugs. Background technique [0002] Breast cancer is currently the gynecological malignancy with the highest morbidity and mortality, and it will replace lung cancer as the world's largest cancer for the first time in 2020, accounting for 11.7% of all new cancer patients, of which more than 50% of breast cancer patients Estrogen receptor positive, the treatment of this type of breast cancer has also become the focus of attention. Due to the lack of specificity, traditional chemotherapy and radiotherapy often bring great toxic and side effects to patients while achieving curative effect, and the clinical outcome is not good. Therefore, in order to optimize the curative effect and minimize the toxicity, it is of great clinical significance to carry out selective "individualized" t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A01K67/027A61K49/00C12Q1/02
CPCC12N5/0693A01K67/0271A61K49/0008G01N33/5011C12N2510/00C12N2501/06A01K2207/12A01K2227/105A01K2267/0393A01K2267/0331C12N2503/02G01N2500/10
Inventor 陈淑罗贝·彼特维杰·潘迪张熙木兰黄鹏
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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