| Breast cancer is one of the most frequent malignant tumors of women, which has a strong impact on their physical and psychological health, and what is even more serious is that it can threaten their lives. Its incidence presents the youth oriented tendency and increases year by year. At present, the pathogenesis of breast cancer has not elucidated completely. Several domestic and abroad studies suggest that its occurrence and development is a progressional process in which many factors participate. At present, breast cancer stem cell becomes a hot topic of research.It may initiate the growth of breast cancer and it may be the root cause of its recurrence and far metastasis after neoadjuvant chemotherapy. As a result, exterminating the breast cancer stem cell thoroughly may cure breast cancer completely. However, breast cancer stem cell makes up a tiny proportion of the total breast cancer cell. For the moment, the key is to find methods that can separate and identify the breast cancer stem cell efficiently.Emergencing evidence supported tumor stem cell hypothesis, at present, this hypothesis considered that tumors were drived by tumor stem cell which had several characters such as self-renewal, multilineage differentiation and tumorigenesis. Moreover, it lacked place stability ability, so it could grow unlimitedly. It had multilineage differentiation ability, but it lacked mature differentiation ability. During asymmetrical divisions, breast cancer stem cell can give rise to a stem cell the same as itself and a progenitor cell which can differentiate towards certain direction. It is difficult to distinguish the stem cell from the progenitor cell in practice, so they are collectively called stem-like cell. CD44+/CD24- has been gradually accepted as one marker of breast cancer stem cell. CD44+/CD24-/ESA+ cell expresses observe characteristics of cancer stem cell, it is able to renew in vitro and reform its parental cells, its cell cycle is longer and it's resistant to the effects of chemotherapy, in addition, only a few cells have tumorigenicity. In luminal cell lines, such as MCF7, ESA+is nearly 100%, so CD44+CD24- is enough to enrich cancer stem cell. So far, flow cytometry has been still one of the most important methods selecting and identifying the tumor stem cell. Its principle is that the ability binding the fluorochrorme antibody conjugates of the cells is different and the property discharging the fluorescent dye is different, so the electrical signals they caused are also different and the charge they have are different, therefore they are separated.Oct4 (Octamer binding factor4), also known as Oct3, is a member of the transcription factors family containing Pou domain. It is encoded by Pou5 fl gene and its molecular weight is 18KD. It locates in human chromosome 10p13. At present, it is widely used to identify the undifferentiated human embryonic stem cell. In the human embryonic stem cell cultured in vitro, the expression of Oct4 presents strongly positive, but when the cell differentiates, its expression is down-regulated. Sox2 is a embryonic transcription factor, and locates in human chromosome 3q. Bmi-1, as the polycomb group gene identified first, participates in several biological process such as embryonic development, organogenesis, tumorigenesis and stabilization and differentiation of stem cell. Many studis showed that the expression of Bmi-1 in several human tumors was up-regulated, such as lung cancer, ovarian cancer, acute myelogenous leukemi, nasopharyngeal carcinoma, breast cancer and neuroblastoma, which indicates that Bmi-1 may play an important role in the initiatiation and development of tumors. Bmi-1 also plays a crucial role in the self-renewal and differentiation of stem cell by several routes in vitro and in vivo. In this study, the three genes above were used as breast cancer stem cell related genes.The number of tumor stem-like cells is not constant, but fluctuates within an enormous range, which can be regarded as one of the evidences of mutual transformation between the general cells and stem-like cells. Alteration of tumor microenvironment can give rise to the selectively amplification of tumor stem cell, however, its concrete mechanism has not elucidated. We don't know whether the subpopulation caused by the alteration of different factors in microenvironment is identical. A number of recent studies suggest that the variation of factors in tumor microenvironment caused by radiotherapy, chemotherapy, serum-free, hypoxia and acidic conditions can result in the increase in the phenotype and number of tumor stem-like cell. Chemotherapy can give rise to acute injury of tumor tissue,and the signals that promote cell growth in tumor microenvironment increase, thus promoting the regeneration of tumor tissue, and the tumor stem cell pool increases obviously. Some studies have showed that the proportion of breast cancer stem cell was increased distinctly after chemotherapy, however, whether the characteristic of breast cancer stem cell can be changed by chemotherapy has not reported.In order to primarily test the effect of 5Fu on stem-like cell in MCF7 cell, first, variation tendency of stem-like marks (Oct4, Sox2, Bmi-1) in unsorted MCF7 cell treated with 5Fu was detected,then stem-like cell (CD44+/CD24-cell) in MCF7 cell was sorted and the variation tendency of stem-like cell in stem-like cell group and non-stem-like cell group treated with 5Fu was detected respectively, however, the number of sorted cells was not enough to extract RNA and conduct immunocytochemistry, so only the proportion of CD44+/CD24- cell was detected by FCM. In this study, MCF7 cell was treated with 0.1μg/ml 5Fu, reverse transcription-polymerase chain reaction detected the expression of Oct4, Bmi-1 and Sox2 in untreated cell and six treated generations cell, immunocytochemistry investigates the expression of Oct4 in untreated cell and five treated generations cell. FCM was used to sort CD44+CD24- cells, the two sorted cell groups were treated with 0.1μg/ml 5Fu,finally,the proportion of CD44+CD24- cells in different times (0h,24h,48h,72h, 144h,168h) was detected by FCM.0.1μg/ml 5Fu was used to treat the stem-like cell and the non-stem-like cell, in order to research the effect of chemotherapy drugs on breast cancer stem-like cell, thus providing certain theoretical basis for the research of breast cancer stem-like cell.Materials and methods:1 MCF7 cell was cultured in RPMI-1640 medium with 10% FBS, at 37℃,5%CO2, saturated humidity. Then change the medium, subculture or freeze the cells according to the growth of the cells.2 MCF7 cell was treated with 0.1μg/ml 5Fu, reverse transcription-polymerase chain reaction detected the expression of Oct4, Bmi-1 and Sox2 mRNA in untreated cell and six treated generations cell.3 Immunocytochemistry investigates the expression of Oct4 in untreated cell and five treated generations cell.4 FCM cell was used to sort CD44+CD24- cells. MCF7 cells were divided into two groups after being marked with FITC-CD44/PE-CD24 antibody, they were stem-like cell group (CD44+CD24-) and non-stem-like cell group respectively. The two sorted cell groups were cultured in six-well plate and treated with 0.1μg/ml 5Fu immediately. There were four groups, and they were stem-like cell routinely cultured group, stem-like cell treated with 5Fu group, non-stem-like cell routinely cultured group and non-stem-like cell treated with 5Fu group, in addition, there were three same wells in each group.5 The proportion of CD44+CD24- cells in different times (0h,24h,48h,72h,144h, 168h) was detected by FCM.6 Statistical analyses:The SPSS 17.0 statistical software was used. The mean±standard deviation (X±S) was used to describe the measurement datas; the results were analyzed statistically by ANVOA and t-test. Values of P<0.05 were considered statistically significant.Results:1 The results of RT-PCR:There were significant differences between the expression of Oct4 in untreated GO generation and the other treated groups (F=149.634, P<0.05). The expression of Oct4 was remarkably improved in the third generation and the fifth generation treated celln and was decreased in G1, G2, G4, G6. There was no significant difference between the expression of Bmi-1 in untreated GO generation and the second treated G2 group,but there were significant differences between the expression of Bmi-1 in untreated GO generation and the other treated groups (G1, G3-G6) (F=232.613, P<0.05). The expression of Bmi-1 was also obviously improved in the third generation and the fifth generation treated with 5Fu but was decreased in G1, G4, G6. There was no significant difference between the expression of Sox2 in untreated GO generation and the first treated G1 group, but there were significant differences between the expression of Sox2 in untreated GO generation and the other treated groups (G2-G6) (F=237.808, P<0.05). The expression of Sox2 was also obviously improved in the third generation and the fifth generation treated with 5Fu, but was decreased in G2, G4, G6.2 Immunocytochemistry results:In MCF7 cell, the expression of Oct4 protein located mainly in cell nucleus and it also expressed in cytoplasm. The protein of Oct4 in GO to G5 had a certain degree of regularity. There were significant differences between the expression of Oct4 in untreated GO generation and the other treated groups (G1-G5) (F=2323.082, P<0.05).3 The proportion of CD44+CD24- cell in MCF7 was 24.75%±1.20, however, in order to guarantee the purity of CD44+CD24- cell sorted, the proportion of actual sorting CD44+CD24- cell was 15.35%±0.35. The volume of the stem-like cell (CD44+CD24- cell) was smaller and its shape was closer to round.4 In the stem-like cell routinely cultured group, the proportion of CD44+CD24- cell at 24h,48h,72h,144h,168h declined gradually. Compared to the stem-like cell routinely cultured group, the proportion of CD44+CD24- cell in stem-like cell treated with 5Fu group increased. There were significant differences between the two groups (t=2.977,P=0.041<0.05). In the non-stem-like cell routinely cultured group, the proportion of CD44+CD24- cell at 0h,24h,48h,72h,144h,168h increased gradually. Compared to the non-stem-like cell routinely cultured group, the proportion of CD44+CD24- cell in non-stem-like cell treated with 5Fu group increased. There were significant differences between the two groups (t=5.590, P=0.005<0.05).Conclusions:1 The expressions of Oct4, Bmi-1, Sox2 mRNA and Oct4 protein were positive, which suggested breast cancer stem-like cell might exist in MCF7 cell line. 2 After being treating with 5Fu, the expressions of Oct4, Bmi-1, Sox2 mRNA and Oct4 protein were increased, which prompted that 5Fu might induce the increase in number or proportion of breast cancer stem-like cell.3 In the non-stem-like cell group, breast cancer cell could differentiate reversly to tumor stem-like cell, and 5Fu may play an important role in this process. |
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