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Applications of Bufalin in preparing preparation capable of realizing targeted inhibition on pancreatic cancer stem cells

A technology of pancreatic cancer stem cells and bufalin, applied in the field of medicine, can solve the problem of low spheroidization rate of SW1990/GEM cell line

Inactive Publication Date: 2017-09-08
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the flow cytometry sorting system is a relatively mature and recognized method for isolating pancreatic CSCs. Among them, the human pancreatic cancer SW1990 / GEM drug-resistant cell line was prepared into a single-cell suspension, and CD44 + CD24 + ESA + After incubation with corresponding fluorescently-labeled antibodies, pancreatic cancer CSCs were sorted by flow cytometry; tumor transplantation experiments in nude mice are an ideal method to identify the proliferation ability of tumor stem cells in vivo. Studies have shown that CD44 + CD24 + ESA + Cells have stronger tumorigenic ability than negative cells, and can differentiate into CD44 + CD24 + ESA + subpopulation of cells, indicating that CD44 in pancreatic cancer + CD24 + ESA + Subpopulation cells have stem cell-like characteristics of self-renewal and differentiation; some studies have shown that the culture of SW1990 / GEM cell line in serum-free suspension culture has a low sphere formation rate through in vitro experiments

Method used

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  • Applications of Bufalin in preparing preparation capable of realizing targeted inhibition on pancreatic cancer stem cells
  • Applications of Bufalin in preparing preparation capable of realizing targeted inhibition on pancreatic cancer stem cells
  • Applications of Bufalin in preparing preparation capable of realizing targeted inhibition on pancreatic cancer stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Enrichment of pancreatic cancer stem cells by serum-free suspension culture

[0031] experimental method:

[0032] 1). Routine culture of human pancreatic cancer Miapaca2 / GEM cells,

[0033] The cells in the logarithmic growth phase were digested with 0.25% trypsin to make a single cell suspension, washed twice with PBS, and inoculated in cells containing basic fibroblast growth factor (Basic Fibroblast Growth Factor, bFGF), epidermal growth factor (Epidermal Growth Factor, EGF) and leukemia inhibitory factor (Leukaemia Inhibitory Factor, LIF) growth factor in serum-free DMEM / F12 medium (CSC medium) culture, 25cm 2 Add 10ml of the above-mentioned medium to the low-adhesion culture flask, and adjust the cell concentration to 1x10 5 / ml.37℃ constant temperature, 5%C0 2 Cultivate in an incubator, shake gently once every 24 hours, and change the medium every 3 days with the half-volume method. After two weeks, you can basically see the cells forming a ball;

...

Embodiment 2

[0045] Example 2 Bufalin inhibits the formation ratio of stem cell spheres in pancreatic cancer drug-resistant cell lines

[0046] Make pancreatic cancer cells 5x10 3 / ml cell suspension, seeded in 96-well plate, 100μl per well. According to the results of the research group’s previous in vitro experiments, the bufolin group was set at 1000nM, 500nM, 100nM, 50, 10nM, 0nM, a total of 4 concentrations, and 5 replicate wells were set for each concentration; the 96-well plate was placed at 37°C, 5% CO 2 Culture in a saturated humidity incubator for 24, 48, and 72 hours respectively, measure the cell proliferation curve according to the CCK8 experiment manual, and take the average value to calculate the intermediate effect concentration of the drug (ie, IC50). Calculate the cell survival rate (%)=(A1—A) / (A0—A)×100%, A1 is the OD value of the Bufalin group, A is the OD value of the blank control group, and A0 is the OD value of the negative control group. Take IC50 as the follow-u...

Embodiment 3

[0049] Example 3 Bufalin significantly inhibits tumor growth in nude mice with Miapaca2 / GEM transplanted tumors

[0050] The pancreatic cancer drug-resistant cell line Miapaca2 / GEM was prepared at a concentration of 1×10 6 0.1 mL of each single cell suspension per mL was inoculated into nude mice (female, 6-8 weeks old, body weight 20±2 g), subcutaneously in the right axilla. On the second day of inoculation, they were randomly divided into 2 groups according to body weight, 6 rats in each group: control group and bufalin group. Bufalin group (1.5mg / kg / d) intraperitoneally injected bufalin 0.2ml, d 1-28 , once a day; control group: intraperitoneal injection of normal saline 0.2ml, d 1-28 , once a day; (the above intraperitoneal injections are fixed at 9:00Am every day). The nude mice were sacrificed by devertebral dissection the next day after the last administration, and the subcutaneous tumors were separated. Measure the maximum diameter and transverse diameter of the tu...

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Abstract

The invention belongs to the technical field of medicines, relates to the novel medicinal application of the traditional Chinese medicinal preparation, namely, Bufalin, and in particular relates to applications of bufalin in preparing a preparation capable of realizing targeted inhibition on pancreatic cancer stem cells. The invention further provides a method for culturing Miapaca2 / GEM by adopting a serum-free suspension culture method, and thus the stem cell spheres with a high proportion are obtained. According to the scheme, from the aspect of tumor stem cells, the experiment shows that in the application of preparing the preparation capable of realizing targeted inhibition on pancreatic cancer stem cells of the traditional Chinese medicinal preparation, namely, Bufalin, the preparation can inhibit the self-renewal of the tumor stem cells, particularly, the preparation can inhibit the proportion of the tumor stem cells in the pancreatic cancer drug resistant cell line Miapaca2 / GEM, and thus the drug tolerance of pancreatic cancer is inverted.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to the new medicinal use of the traditional Chinese medicine preparation bufalin, in particular to the use of bufalin in the preparation of targeted inhibition of pancreatic cancer stem cell preparations, the preparation inhibits the self-renewal of tumor stem cells and reverses pancreatic cancer drug resistance. Background technique [0002] The prior art discloses that pancreatic cancer is the fourth leading cause of death for cancer patients, and the 5-year survival rate is only 5%. Surveys show that most of pancreatic cancer patients have lost the opportunity for surgery when they are discovered, even though some of them have the opportunity to undergo surgery However, most patients still experience local recurrence or metastasis. Therefore, it is necessary to seek new therapeutic strategies to inhibit the occurrence and development of pancreatic cancer. Studies have disclosed ...

Claims

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Application Information

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IPC IPC(8): A61K31/585A61P35/00
Inventor 朱晓燕王海永宁周雨徐立涛孟志强
Owner FUDAN UNIV SHANGHAI CANCER CENT
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