| Objectives:To establish a rapid and high efficient method of primary culture of BCAFs by modifying the common primary culture methods of human breast cancer-associated fibroblasts?BCAFs?.Methods:Fresh cancer tissue specimens from 20 patients with invasive breast cancer were collected and each specimen is about 1cm3 in size.Each specimen was randomly divided into two groups of the same size and quality?modified group and traditional group?.The modified group was digested by modified enzyme digestion?type II collagenase+hyaluronidase digestion for 10 min?,and the traditional group was digested by traditional enzymatic digestion?type I collagenase digestion for 10 h?.The total number of cells obtained by digestion between two groups were compared by cell counting;The absorbance value?OD value?at 570 nm was measured by MTT colorimetry,and the activity of adherent cells was compared after 48h of culture by OD value;By selecting?-SMA and Vimentin proteins as marker proteins of BCAFs,The purity of BCAFs obtained by the two digestion methods was compared by cell immunofluorescence assay;The primary cell characteristics of BCAFs at each stage were observed under inverted microscope.Finally,SPSS 16.0 statistical software was used to analyze the experimental data.Results:In two breast cancer tissue masses of the same size and mass,The total number of cells obtained in the modified group was about?8.26±0.46?*105,while that in the traditional group was about?4.61±0.40?*105.The number of cells isolated by the modified enzyme digestion method was significantly higher than that by the traditional enzyme digestion method[?8.26±0.46?*105,N=20 VS?4.61±0.40?*105,N=20;P<0.05];The OD value of the modified group was?0.31±0.03?and that of the traditional group was?0.26±0.02?.The activity of adherent cells in the modified group at 48 hours was higher than that of the traditional group?0.31±0.03,N=20 VS 0.26±0.02,N=20;P<0.05?;The purity of BCAFs in the modified group was about?77.85±3.65?%and that in the traditional group was about?70.85±2.23?%.The purity of BCAFs obtained in the modified group was higher than that in the traditional group?77.85±3.65?,N=20 VS 70.85±2.23),N=20,P<0.05).Under the inverted microscope,it was found that after 3 days of BCAFs primary cell culture,the cells adhered to the wall and extended like paving stones.After 7-12 days of culture,the cells can be passaged,most of them are single nuclei with more granules in the cytoplasm.After passage,the cells were spindle-shaped or spindle-shaped,with different lengths,transparent cytoplasm,irregular cell arrangement and sometimes spiral growth.Conclusions:In this study,a rapid and efficient primary culture method of BCAFs was established by digesting breast cancer tissue with modified enzymatic digestion method?type II collagenase+hyaluronidase digestion for 10 minutes?.To establish a basis for further study on the function of BCAFs in breast cancer.Objectives:The breast cancer cell line MCF-7 cells were used as the research object to study the effects of primary human BCAFs cells and human normal fibroblasts?NFs?primary cells on paclitaxel sensitivity of MCF-7 cells in vitro.Methods:Take 5 pairs of breast cancer patients' tissue blocks for primary culture of BCAFs and NFs.The primary cells of BCAFs and NFs identified by cell immunofluorescence were co-cultured with human breast cancer cell line MCF-7 for 48 h,,and used as experimental group?MCF-7BCAFsgroup,MCF-7NFsgroup?..MCF-7 was cultured alone as a control group.1.5,2.5,3.5,4.5,5.5 ?g/ml paclitaxel was applied to each group of MCF-7 cells for 24 hours.The OD values of each group were measured by CCK-8 method,and the IC50 values of each group were calculated.Finally,the experimental data was analyzed by SPSS 16.0 statistical software.Results:The IC50 of the MCF-7BCAFs group was higher than that of the blank control group [IC50:?4.49±0.22?,n=5 VS?3.83±0.12?,n=5;P<0.05],and the IC50 of the MCF-7BCAFs group was also Higher than MCF-7NFs group?IC50:?4.49±0.22?,n=5 VS?3.92±0.15?,n=5;P<0.05?.Half of MCF-7 cells were killed in the same time,and higher concentrations of paclitaxel were required in the MCF-7BCAFs group.Breast cancer MCF-7,which was co-cultured with BCAFs,was less sensitive to paclitaxel.The IC50 of MCF-7NFs group was not significantly different from the blank control group [IC50:?3.92±0.15?,n=5 VS?3.83±0.12?,n=5,P=0.488,P>0.05],IC50 was not significant difference,There was no significant difference in the concentration of paclitaxel required between the MCF-7NFs group and the blank control group when half of MCF-7 cells were killed in the same time.Conclusions:This study used breast cancer MCF-7 cell as the research object.By using BCAFs and NFs in a non-contact co-culture manner with MCF-7,it was found that BCAFs can significantly reduce the sensitivity of paclitaxel in breast cancer cells,while NFs have no significant effect on the sensitivity of breast cancer to paclitaxel.This study provides a new idea and research background for further study of breast cancer resistance to paclitaxel. |
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