| [Background]Breast cancer is highest incidence and mortality in women just below those of lung cancer, malignant tumor, which cause serious damage to the women’s health around the world each year more than 1.3 million women newly diagnosed with breast cancer, more than 50 women died of breast cancer. With the rapid change of life way and ecological environment deteriorated sharply, breast cancer incidence in our country present obvious rising trend, up to 3～4% of new cases every year, become the one of the fastest rising incidence of malignant tumors in our country, and has a younger trend, the incidence of female breast cancer patients aged about ten years earlier than the west. Therefore, clear the pathogenesis of breast cancer, and control the incidence of breast cancer, to improve the effect of treatment of breast cancer, to reduce breast cancer mortality has become a major issue to be solved. Surgery is generally accepted radical treatment for breast cancer, but surgery alone cannot effectively improve the prognosis of patients with breast cancer, cancer recurrence and metastasis are the main factor that leads to poor prognosis. Surgery is generally accepted radical treatment for breast cancer, but surgery alone cannot effectively improve the prognosis of patients with breast cancer, cancer recurrence and metastasis are the main factor that leads to poor prognosis.Radiation and chemotherapy can improve local control and improve the long-term survival rate, is an important auxiliary means to clinical treatment of breast cancer.But long-term radiation and chemotherapy has a serious adverse reaction, and greatly improve the tumor radiation and chemotherapy resistance effect, so need to explore more effective means of adjuvant therapy. Cell apoptosis is refers to the body to maintain a stable internal environment, under the influence of some physical factors, caused by factors in vivo and in vitro cell independent of ordered death, the process is also known as programmed cell death.This is an orderly process, was not above pathological conditions on the damage phenomenon.Cell apoptosis and cell necrosis, apoptosis is not a passive process, but the active process, it involves a series of activation, expression and regulation of genes.In the evolution of the organism, the stability of internal environment and plays an important role in the development of multiple systems.Apoptosis plays an important role in the body to maintain its own dynamic balance.Apoptosis occurred most is redundant in an organism or cell aging cells, it is possible that occurs when tumor cells or cells infected by the virus or bacteria.In many cases, apoptosis is for signals from other cells or follow in the environment of a reaction. Tumor occurrence and development is excessive proliferation and cell apoptosis pathways blocked the result of joint action.Cell apoptosis and its role in tumorigenesis and treatment more and more attention, at the same time, based on the mechanism of promoting tumor cell apoptosis of tumor biological treatment has important progress. The cell cycle is the life generation to the next generation passed the most basic and most important in the process of continuous process.The process starts from cells dividing a complete, at the end of the next cell division, including DNA synthesis and cell division are two important stages of the cell cycle, contains G1 phase and S phase and G2 and M.In the evolution of species, in order to ensure that the cell cycle strictly follow an orderly alternately and the phase sequence of the change of physiological processes, somatic cell life development and established a series of regulatory mechanism. When stimulated by certain factors inside and outside the body and the regulation of cell cycle of the extraordinary molecular network members, the disorder of cell cycle, leading to the hyperplasia of cells not harmonious, DNA damage and other abnormal situation, to prevent abnormal cells apoptosis, leading to the onset of cancer.Thus, the cell cycle of disorder is closely related to the occurrence of tumor. As a result, the cell cycle of tumor occurrence and evolution, is of great significance to the clinical diagnosis and treatment. Chinese medicine Safflower (Safflower) is also called the red and blue flowers, the grass Safflower, compositae plants, is a traditional Chinese medicinal materials in our country, its temperature, taste, with promoting blood circulation to remove blood stasis and analgesic effects.Red flower in most areas are distributed in our country, the largest in xinjiang, sichuan, etc. Safflower, modern pharmacology study proves, contain a variety of biological active ingredients, such as Safflower polysaccharide, flavonoids, flavone, Safflower yellow pigment, Organic acid and Safflower glycosides, a lot of studies have shown that safflower polysaccharide has many pharmacological activities, such as antioxidation, antitumor, anticoagulant, lower blood pressure and immunity, etc.Because the red flower polysaccharide has rich resources, side effects, high security, small and easy to the advantages of the industrial production, has been widely concerned.In addition, safflower polysaccharide with the chemotherapy drug combination, can strengthen its effectiveness and reduce the side effects of auxiliary functions. Prophase research proves, safflower polysaccharide by regulating apoptosis and cell cycle, realize to gastric cancer, liver cancer and other malignant tumor cells in vitro inhibitory effect, but its effect on breast cancer cells and its mechanism is not yet clear.[Objectives]This stady to evaluate the safflower polysaccharide extraction method, and in human breast cancer MCF-7 cell as the research object, explore the safflower polysaccharide in vitro on breast cancer MCF-7 cell apoptosis and the effect of safflower polysaccharide on the basis of the research on human breast cancer cell apoptosis related factors, the influence of reference for clinical treatment of breast cancer to explore new ideas and methods, for the clinical application of Chinese medicine polysaccharides also provide reliable experimental basis.[Methods]Part Ⅰ:Selected of traditional Chinese medicine Safflower as raw material and with boiling water decoction, used the rotary evaporation apparatus to enrichment the solution, water extract-alcohol precipitation method were used to extract crude polysaccharide, after repeated freezing and thawing to remove impurities, Sevage method remove protein, decoloration, washing and finally obtained refined safflower polysaccharide. The sulfate-phenol method were used to determinat the polysaccharide content in the refined safflower polysaccharide. Safflower polysaccharide 20 mg into 100 mL volumetric flask, distilled water to be diluted to scale line, stirred to fully dissolve. With a pipette gauged safflower polysaccharide solution 1.0 mL, and to join the 1.0 mL dual steaming water,1.0 mL of 6% benzoic acid and 5.0 mL of concentrated sulfuric. Measured the absorbance at 490 nm wavelength and used the regression equations to measure the content of polysaccharide. Part Ⅱ:The cell line MCF-7 was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% of FBS (fetal bovine serum), and incubated at 37℃, with 5% CO2 and constant humidity. When cell had a confluence of about 80%, intervention treatment was given. The MCF-7 cells were divided into a control group and a treatment group of different concentrations of safflower polysaccharides (0.06mg/mL,0.12mg/mL,0.25mg/mL,0.5mg/mL, 1.00mg/mL,2.00 mg/mL). At 12,24,48,72 hours after intervention, the changes of cell number, morphology and state were observed by phase contrast microscope. The inhibitions of cell proliferation were detected by MTT assay. The change of cell cycle phase and cell apoptosis was analyzed by flow cytometry. Part Ⅲ:The cell line MCF-7 was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% of FBS (fetal bovine serum), and incubated at 37℃, with 5% CO2 and constant humidity. When cell had a confluence of about 80%, intervention treatment was given. The MCF-7 cells were divided into a control group and a treatment group of different concentrations of safflower polysaccharides (0.06mg/mL,0.12mg/mL,0.25mg/mL, 0.5mg/mL, 1.00mg/mL,2.00 mg/mL). Part of MCF-7 cell were extracted total protein, used Western Blot method to detect the protein expression of Bax, Bcl-2 and p53. Another part of MCF-7 cell were used to extract total RNA, the RT-PCR method was adopted, β-actin as reference genes,detected the safflower polysaccharide effect in protein expression of Bax, Bcl-2 and p53. The Real-time PCR was detected Ct value of each cell (Cycle threshold), with P-actin to homogenization processing, the data obtained by analyzing the changes in cell apoptosis related gene mRNA expression. Part IV:Chose MCF-7 nude mouse transplantation tumor model of breast cancer as the research object, set up control group and different concentrations safflower polysaccharide (0.12 mg/mL,0.25 mg/mL,0.5 mg/mL,1.00 mg/mL,2.00 mg/mL) intervention group, continuous 14 d drug intervention therapy. The general condition, body weight, tumor weight, tumors volume and tumor inhibition rate were compared between the each group.[Results]Part I:450.16 g crude drugs safflower were extracted by water extract-alcohol precipitation and obtained safflower polysaccharide 26.1 g, the polysaccharide yield was 5.79%. Polysaccharide obtained after soluble in water in the absorption of proteins, peptides and nucleic acid peak at 260 nm and 280 nm ultraviolet absorption, explain get polysaccharide purity was good, not included impurities such as proteins, peptides and nucleic acids. Sulfuric acid phenol method test results showed that the polysaccharide extraction of refined by safflower in safflower polysaccharide content was 91.2%. Part II:In the condition of normal growth, MCF-7 cell was adherence and uniformity growth, cells showed typical epithelioid adherent growth, cell arranged densely, neat and outline clear, most cells in the vigorous growth period and division, the cells of death or apoptosis is less. Compared with normal cells, the breast cancer cell by safflower polysaccharide disposed, showed cell shrinkage, irregular shape, stick a wall reduced capacity and see some cells floating, see more pyknotic nucleus, intercellular space increases, nuclear was reduced. The above phenomenon showed time and dose dependent.0.06 mg/mL～1.00mg/mLafflower polysaccharide continuous effected 72h, the growth inhibition rate of human breast cancer cell line MCF-7 significantly rose, and showed obviously dose dependent. When the safflower polysaccharide concentration of 1.00 mg/mL, the inhibition rate was highest, for 75%.0.50 mg/mL and 1.00 mg/mL safflower polysaccharide concentrations in human breast cancer cell line MCF-7 the inhibition rate of no significant difference (P> 0.05). But when the concentration of 2.00 mg/mL, safflower polysaccharide on MCF-7 cell inhibitory rate declined, and 1.00 mg/mL safflower polysaccharide groups had significant difference (P< 0.05). According to the different concentrations of safflower polysaccharide MCF-7 cell growth inhibition, with IC50 calculation software safflower polysaccharide is used in the human breast cancer cells MCF-7,72 hours after half inhibitory concentration of IC50:0.4154 mg/ml.In a certain concentration range, safflower inhibitory effect of polysaccharides and polysaccharide concentration and action time were positively than relationships. Safflower polysaccharide concentrations within 0-1.00 mg/mL, acting on the MCF-7 within 48 h, with the increase of safflower polysaccharide concentrations, the MCF-7 significant inhibition (P< 0.05). When the polysaccharide concentrations greater than 1.00 mg/mL, inhibition rate of MCF-7 no obvious increase. When the concentration of safflower polysaccharide reached 1.00 mu g/mL, acting on the MCF-7 cell 48 h, best inhibition effect. Safflower polysaccharides on human breast cancer cells in vitro can significantly induce MCF-7 cell apoptosis, and to a certain concentration range, its role and safflower polysaccharide concentration and action time higher than relationships.Safflower polysaccharide concentrations at 0-1.00 mg/mL, acting on the MCF-7 within 48 h, with the increase of concentration of safflower polysaccharide, can significantly apoptosis induction of MCF-7. When safflower polysaccharide concentrations greater than 1.00 mg/mL, the inhibition rate of MCF-7 no obvious increase. When the concentration of safflower polysaccharide reached 1.00 mg/mL, ACTS on the MCF-748 h, inducing cell apoptosis rate is highest.As safflower polysaccharide concentrations increased, S phase and G2 cell corresponding to reduce, but G1 phase cells gradually increased.At low concentration (0.12,0.25 mg/mL), safflower polysaccharide has no impact on the number of G1 phase cells.When the red flower polysaccharide concentration of 0.5 mg/mL, the number of G1 phase cells increased significantly (P< 0.05).Of breast tumor cell proliferation activity weakened due to the stagnation of the cell cycle, block most of the cells in G1 phase. Part Ⅲ:Used agarose gel electrophoresis and spectrophotometric method respectively detected the spectrophotometry, concentration and purity of RNA sample. By the spectrophotometer at 260 nm and 280 nm wavelength determination of RNA samples OD260/280 ratio are within the range of 1.8～2.0, according to OD260 calculating RNA samples concentration at the same time, adjust the concentration of RNA samples to 500 ng/mu L;Agarose gel electrophoresis results showed that 28 s rRNA and 18s rRNA the brightness of the ratio of about 2:1, this research institute to extract RNA sample quality is good, is not degraded. Rt-pcr detection Bax, BCL-2, p53 mRNA expression, according to the blank control group and safflower polysaccharide group after 48 h, Bax, BCL-2, p53 mRNA in MCF-7 cell have different degree of expression.The 1.00 mg/mL, compared with the control group, and 2.00 mg/mL safflower polysaccharide effect after 48 h, Bax mRNA expression increased significantly, and has significant difference (P< 0.05);The 0.12,0.25, and 0.5 mg/mL safflower polysaccharide group, there was no significant difference (P> 0.05);Compared with the blank group,0.25, 0.50,1.00, and 2.00 mg/mL of four groups of safflower polysaccharide group after after 48 h function, intracellular p53 mRNA expression were significantly increased, compared with the blank group significant difference (P< 0.05), only 0.12 mg/mL group no significant difference (P> 0.05). By 1.00,2.00 mg/mL safflower polysaccharide effect after 48 h, the BCL-2 mRNA expression quantity significantly reduced compared with blank group, the difference was statistically significant (P< 0.05), and 0.12,0.25, and 0.50 mg/mL safflower polysaccharide group no significant difference in the blank group (P> 0.05).This study applies Western blot method to analysis the Bax, Bcl-2 and p53 protein in safflower polysaccharide MCF-7 cell expression in breast cancer.The result shows:the in vitro, safflower polysaccharide can improve the MCF-7 cell of Bax and the expression of p53 protein, inhibit the BCL-2 protein expression.It can be seen in the concentration of 1.00 and 1.00 mg/ml, can significantly reduce the BCL-2 protein expression of Bax and p53 increased significantly, this result was consistent with the check result of mRNA expression. Part Ⅳ:The appetite of control group was significantly decreased, coat messy, different degree of angular, slow response and slow action. The living quality of experimental group are not affected, and the mental state, response speed and action capability of experimental group were superior to control group, and showed dose-dependent, the difference showed statistically significant (P<0.05)[Conclusion]Water extract-alcohol precipitation using the legal system in safflower polysaccharide, polysaccharide yield, high purity, and is simple and reliable, strong stability, high sensitivity and good reproducibility, can provide technical support for safflower polysaccharide extraction, and provide theoretical basis for the full development and utilization of safflower resource and data support. Safflower polysaccharide on MCF-7 cell has significant inhibitory effect of breast cancer, and in a certain range (<1.00 mg/ml,< within 48 h), with the increase of concentration of safflower polysaccharide, the extension of time is stronger inhibitory effect. Safflower polysaccharide homo habilis breast MCF-7 block in G1 phase, cell apoptosis in the phase of the cell, and the degree of apoptosis associated with the concentration of safflower polysaccharide. Safflower polysaccharide from breast cancer cells MCF-7 cell, can significantly increase the p53 mRNA and Bax mRNA expression level, cut the Bel-2 mRNA expression level, and the concentration dependence relationship in the regulation.Regulating apoptosis gene expression may be safflower polysaccharide is one of the main mechanisms play a role of anti-tumor. Safflower polysaccharides can effectively improve the MCF-7 cell subcutaneous nude mouse transplantation tumor of breast cancer quality of life, curb weight loss and tumor growth, and did not cause obvious toxic effects, has good body tumor suppression effect. |
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