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Objective: use of chemotherapy drugs enrichment mouse breast cancer cell line 4T1 tumor stem cells (cancer stem cell, CSC), to study the resistance mechanisms of cancer stem cells, to further explore the regulatory mechanism of breast cancer stem cells, development target breast cancer stem cells the molecular preparations to lay the foundation. Method: a chemotherapy drug enrichment breast cancer stem cells in animal model. Collect the logarithmic growth phase 4T1 cells inoculated in BALB / c mice right scapular subcutaneous mouse breast cancer model. The mice were randomly divided into four groups, respectively, for the control group and 5 - fluorouracil (5-Fu) low, medium and high concentration treatments. Subject to the mouse tumor control group was given saline ,5-Fu treatment group were given low, medium and high concentrations (0.05mg/ml, 0.1mg/ml, 0.2mg/ml) 5-Fu intraperitoneal injection daily time, shared seven days, seven days later, changed once a week, eagerly mice were killed after four weeks, the tumor tissue is divided into three parts, a fixed in neutral formaldehyde detected by immunohistochemistry, one for RNA extraction, a system The proportion of cell suspension for flow cytometry the the proportion of of CD44 CD24-/low cells and Hoechest33342 staining detection of side population (side population, SP) cells. Take of CD44 CD24-/low cells the highest proportion of 5-Fu treated mouse tumor cell suspension was prepared, the establishment of the second generation of CSC-enriched mouse xenograft model, tumor-bearing mice with the same concentration of 5-Fu chemotherapy produced four generations, the medication the same way as before, so passaged mouse xenograft model. By flow cytometry (flow cytometry, FCM) detection of the proportion of of CD44 CD24-/low cells in the generation of the control group and 5-Fu treated mice tumor xenograft model. 3 to adopt Hoechst33342 staining proportion of SP cells detected in each generation of the control group and 5-Fu treated mice xenograft model tumor. 4 immunohistochemical method to detect the generation of the control group and 5-Fu treated mouse xenograft model of tumor tissue, CD55 and ALDH1 protein expression. 5 observed in serum-free culture each generation of the control group and 5-Fu treated mice transplanted tumor tissue microsphere formation, count the number of microspheres and calculation of the efficiency of the formation of the microspheres. Microspheres 6 observed the third generation of 5-Fu treated mice transplanted tumor cells in a serum-free culture environment induced differentiation. 7 animals cause tumor assay the third generation of the control group and 5-Fu treatment of mice transplanted tumor cells tumorigenic ability. 8 HE staining, light microscope to observe the morphological changes of the tumor tissue of the mouse xenograft model. 9 by reverse transcription - polymerase chain reaction (reverse transcription PCR, RT-PCR) semi-quantitative and quantitative real-time PCR (Real-time PCR) to detect the third generation of the control group and 5-Fu treated mice transplanted tumor tissue MDR1 , BCRP, ALDH1 and β-catenin mRNA expression. Results: 1 chemotherapy drug 5-Fu induced under 4T1 cell lines passaged in mice to establish mouse xenograft model enrichment of breast cancer stem cells from 5-Fu treatment group, two, three, separating the resulting cells in the mouse model of tumor tissue of four generations were named as 4T1-1st, the 4T1-2nd, 4T1-3rd, of 4T1-4th cells, and separating the resulting cells of the tumor tissue of mice in the control group is named for the parental 4T1 cells. 2 FCM test results showed that the CD44 CD24-low percentage of cells in the first generation of control mice transplanted tumor tissue to 11.5 ± 0.9%, low, high concentrations of 5-Fu treatment rates were 40.1 ± 3.4%, 49.8 ± 1.2%, 45.6 ± 1.6%, significantly higher than the control group (P lt; 0.05). , The high concentration group was significantly higher than the low-dose group (P lt; 0.05), the high concentration group, no significant difference (P gt; 0.05). CD44 CD24-low mice transplanted tumor tissue cells in the concentration of 5-Fu treatment is higher than the other two groups in the concentration (0.1mg/ml) 5-Fu, so I chose to follow-up experiments. 5-Fu treatment group, two, three, four generations of mice transplanted tumor tissue of CD44 CD24-/low cells proportion were 49.8 ± 1.26%, 56.8 ± 1.76%, 66.4 ± 1.5%, 69.0 ± 1.6%, significantly higher than those in the control group (P lt; 0.01). With the increase of the number of passages, CD44 CD24-/low proportion of cells gradually increased, not obvious (P gt addition to the difference between the third and fourth generations; 0.05) outside the other generations difference was statistically significant (P lt ; 0.05). The 3 Hoechst33342 staining showed ,5-Fu treated first, second, third and fourth generation of mice transplanted tumor tissue SP cells were 25.0 ± 1.21%, 42.6 ± 2.8%, 58.4 ± 2.2%, 61.3 ± 2.6% , significantly higher than the 9.7 ± 1.4% (P lt; 0.01). SP cell ratio gradually increased with the increase of the number of passages, not obvious (P gt addition to the difference between the third and fourth generations; 0.05) outside, the difference was statistically significant (P lt; 0.05) between generations. 4 Immunohistochemical results showed that the control group of mice transplanted tumor tissue ALDH1 expression is negative ,5-Fu treated, two, three, four generations of mice transplanted tumor tissue ALDH1 expression weakly positive to positive, with the passage the ALDH1 expression level of the increase in the number of 5-Fu treatment group gradually increased. 5-Fu treatment group, two, three, four generations of mice transplanted tumor tissue CD55 strongly expressed in cell number was 7.8 ± 1.6%, 10.1 ± 2.0%, 15.6 ± 1.4%, 17.3 ± 1.9%, significantly higher in the control group, 0.6 ± 0.3% (P lt; 0.01), and gradually increased with the increase of the number of passages 5-Fu treated CD55 expression levels. Serum-free culture of mouse xenograft tumor cells results show 4T1-3rd cells microsphere formation efficiency of 5.9 ± 0.4%, parental 4T1 cell micro-sphere formation efficiency of 0.5 ± 0.2%, the former is about 12 times that of the latter (P lt After not; 0.01); 4T1-3rd cells to form the first generation of microspheres passages form the same proportion of the second, three generations of microspheres and can spread to the 5th generation microspheres formed by the parental 4T1 cells spread to the three generations and then form a ball of cells began adhesion, and differentiation. The 4T1-4th cell mammosphere formation efficiency of 6.1 ± 0.3%, with between 4T1-3rd cells no significant differences (P gt; 0.05). 6 has a serum-free culture microspheres induced differentiation results show 4T1-3rd microsphere cells in serum medium gradually adherent differentiation, and can be continuous passage. 7 animals tumorigenic experimental results show that the cells of 4T1-3rd tumorigenic ability was significantly higher than the parental 4T1 cells. 8 after HE staining observed results show that, all tumor-bearing mice transplanted tumor tissue are breast tissue inoculated with 4T1-3rd cells and parental 4T1 cells in mice tumor was no significant difference in morphology. 9 semi-quantitative RT-PCR test results show that the third generation of 5-Fu treated mice transplanted tumor tissue of MDR1, BCRP elevated ALDH1mRNA expression levels, a statistically significant difference compared with the control group (P lt; 0.01) β-catenin mRNA expression levels between the two groups did not show significant differences (P gt; 0.05). Rt-PCR test results showed that the expression of MDR1, BCRP and ALDH1mRNA ,5-Fu treated mice transplanted tumor tissue than the control group were raised 4.35,6.14,3.78 times, both compared to a statistically significant difference (P lt ; 0.01); expression of β-catenin mRNA increases 1.75 times, both compared to a statistically significant difference (P lt; 0.05). Conclusion: The use of the chemotherapy drug 5-Fu enrichment of breast cancer stem cells to establish a tumor-bearing mouse model, highly malignant mouse breast cancer cells in mice passaged 4T1-3rd, 4T1-3rd cells rich set of high-purity the CSC, prompt application of chemotherapy drugs is an effective method of screening breast cancer stem cells. 2 third-generation 5-Fu treated mice transplanted tumor tissue high expression of MDR1 and BCRP, raised produce resistance to chemotherapeutic drugs, escape chemotherapy drugs attack breast cancer stem cells may be the expression of MDR1 and BCRP. The expression levels of β-catenin in the third generation of 5-Fu treated mice transplanted tumor tissue, suggesting that the Wnt signal transduction pathway activation may be involved in the regulation of breast cancer stem cells, but whether the relationship with the breast cancer stem cell drug needs further research is clear. 4 5-Fu treated mice transplanted tumor tissue, high expression of CD55 and ALDH1, prompted CD55 and ALDH1 available as the new tumor markers in breast cancer stem cells.
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