Study On The Effect Of Ropivacaine On Prognosis Related Factors Of Breast Cancer And Its Mechanism

The process of tumorigenesis and its development are very complicated,the part of tissue cells of body are affected by carcinogenic factors,at the gene level,the ability to lose with the normal control of growth,then the occurrence of abnormal proliferation lead to cancer.At present,Surgery is still the preferred and most commonly used method for the treatment of malignant tumors.After resection of primary tumor,local tumor recurrence and distant metastasis treatment effect is poor.Metastasis and recurrence are the main causes of death in patients with malignant tumors.Intraoperative dissemination of cancer cells and blood transfusion can not be completely avoided,anesthesia and surgery affect metabolism and nerve secretion factor,causing infection and immune stress,and then affect the prognosis after the operation of malignant tumor.Breast cancer is one of the most common malignant tumors in women,the prevalence rate is highest among women,mortality is second only to lung cancer.With the changes in people's lifestyles and the persistence of survival and ecological environment in recent years,the incidence and mortality of female breast cancer are increasing year by year in China,serious harm to women's health,has become the"number one killer".New breast cancer cases increase to 3-4%per year,and the trend of getting younger is becoming more and more.In recent years,as perioperative medicine receives more and more attention,the relationship and influence of narcotic drugs and methods with malignant tumor during and after operation has gradually become a new hotspot and concern.Now clinical research data and animal experiments have been shown,the influence of narcotic drugs and anesthesia on the microenvironment of malignant tumors development is of great significance,latest research tips,anesthetics can also act directly on malignant tumor lesions,the study involved a small number of narcotic drugs such as halothane,thiopental,propofol and so on,however,there are few studies on the effects of other general anesthetics and local anesthetics on the prognosis of malignant tumors.Because it is a new research field,each one is still in dispute.Since ancient times,science is always moving forward in the process of breaking and setting up,malignant tumor is still the medical profession and even life science problems,it is hoped that the study of the effect of anesthesia on malignant tumors can contribute to the treatment of malignant tumors.Ropivacaine?ropivacaine,RP?,called?S?-N-?2,6-dimethylphenyl?-1-propyl-2-piperidine carboxyamide,is a pure levorotatory?S?-enantiomeric local anesthetic,it is the most commonly selected local anesthetic in spinal anesthesia and peripheral nerve block in clinical practice.Existing studies show that,local anesthetic has a positive effect on the prognosis of patients with malignant tumors,while the domestic research on ropivacaine in anti-tumor research is still very small in China,local anesthetics still have a lot of unexplored benefits,waiting for us to make a deeper study of it.ObjectiveThis study first observed the effect of ropivacaine on proliferation,apoptosis,invasion and migration of human breast cancer MCF-7 cells,and then established a human breast cancer MCF-7 cell transplant tumor,to observe the growth,morphological changes and molecular effects of ropivacaine in the development of breast cancer,finally,the effects of combined use of ropivacaine on prognostic factors in breast cancer patients under clinical anesthesia were observed,to provide a new scientific basis for selecting more suitable anesthesia methods for patients with breast cancer during clinical operation anesthesia.This research was divided into three parts:Part ?:Effects of ropivacaine on proliferation,apoptosis,invasion and migration of breast cancer cellsMethods1.MTT method was used to detect the incubation of breast cancer MCF-7 cells after 12h with different concentrations of ropivacaine?100?g/ml,200?g/ml and400?g/ml?,effect on proliferation of breast cancer cells,the control group received the same amount of normal saline.2.Flow cytometry was used to detect the effects of different concentrations of ropivacaine?ibid.?on human breast cancer MCF-7 cells,effect on breast cancer cell cycle,quantitative and rapid quantitative analysis of individual cells or other biological particles by monoclonal antibodies at the cellular and molecular levels.3.The effect of different concentrations of ropivacaine?ibid.?on human breast cancer cell line MCF-7 was observed by microtubule tracing method.4.Hoechst 33258 single staining and Hoechst 33258/PI double staining method to detect the effects of different concentrations of ropivacaine?ibid.?On apoptosis and death of human breast cancer MCF-7 cells.5.Transwell chamber method was used to determine the effect of different concentrations of ropivacaine?supra?on the invasive ability of human breast cancer MCF-7 cells.6.Western blotting was used to detect the effect of different concentrations of ropivacaine?ibid.?On the expression of MMP-2 protein and MMP-9 protein in human breast cancer MCF-7 cells,and then study the impact of its ability to attack.7.Cell scratch assay was used to detect the effects with migration of human breast cancer MCF-7 cells after treatment with different concentrations of ropivacaine?ibid.?8.The effects of different concentrations of ropivacaine?ibid.?on the changes of microfilament and mitochondrial number in human breast cancer cell line MCF-7were observed by microfilament and mitochondria tracing method.Results1.The result of MTT test showed,different concentrations of ropivacaine?100?g/ml,200?g/ml and 400?g/ml?significantly inhibited the proliferation of human breast cancer MCF-7 cells?P<0.05?.2.Flow cytometry test results showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,breast cancer cells are decreased at the time of division or are prepared for the period of division,while the cells in the quiescent period increased significantly,and it's associated with the concentration of ropivacaine?P<0.05?.3.The microtubule tracer method showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,the mitotic phase of cells decreased,it's associated with the concentration of ropivacaine,the higher the concentration,the more obvious the decrease of mitotic phase?P<0.05?.4.Hoechst 33258 single staining and Hoechst 33258/PI double staining test results showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,the apoptosis rate of cells was increased,the cell mortality rate also increased,and increased with the increase of ropivacaine concentration.?P<0.05?5.Transwell chamber test showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,the number of cells in each field of the breast cancer cell was significantly reduced through the bottom and on the back of of the microporous membrane filter,the degree of reduction was related to the concentration of ropivacaine?P<0.05?.6.Western blotting test results showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,the expression of MMP-2 protein and MMP-9 protein decreased with the increase of the concentration of ropivacaine?P<0.05?.7.Cell scratch test showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,the relative healing distance decreased gradually with the increase of ropivacaine concentration?P<0.05?.8.Microfilaments and mitochondria were observed by tracer method showed,after the different concentrations of ropivacaine?ibid.?were acting on human breast cancer MCF-7 cells,with the increase of the concentration of ropivacaine,the morphological changes of microfilaments and mitochondria develop in the direction that is not conducive to the movement of tumor cells.Part ?:Effect of ropivacaine on breast cancer xenograftsMethods1.To establishment of xenograft model of human breast cancer MCF-7 in nude mice.All 30 nude mice were tumorigenic,nude mice with the smallest diameter of 2mm<b<10 mm were included in the group,finally,24 nude mice were selected to enter the experiment,were randomly divided into two groups:control group?C'group?,ropivacaine treatment group?R'group?,C'group was injected with normal saline around the tumor,1ml/times,the R'group was injected with 2mg/ml ropivacaine 1ml/times around the tumor,1 time/d,15 consecutive days.The tumor size was measured every 3 Days,until 1 d after the end of medication.To recorded the average volume of tumor was measured every time in each experimental group.On the 15th day,the nude mice in each group were killed by cervical dislocation,complete dissection of MCF-7 breast cancer tumor tissue,to measure the size of the tumor,immediately fixed embedding or stored at-80°C for subsequent experiments.2.To measure the volume of transplanted tumor,draw the growth curve,observe the effect of ropivacaine on the growth of transplanted tumor.3.Hematoxylin eosin?H.E?staining method,to observe the effect of ropivacaine on the morphology of breast cancer xenografts.4.Immunohistochemical staining method,objective to investigate the effects of ropivacaine on the expression of epithelial mesenchymal transition protein and hypoxia inducible factor-1 alpha?HIF-1 alpha?in breast cancer xenografts.5.Immunofluorescent method,to investigate the effect of ropivacaine on the expression of epithelial mesenchymal transition protein in breast cancer xenografts.6.The effect of ropivacaine on the expression of TGF-?,VEGF and MMP-9 in breast cancer xenografts was detected by Western blot.Results1.To measure the volume of transplanted tumor,draw a growth curve display,the growth of breast cancer xenografts in the R'group was significantly slower than that in the C'group?P<0.05?.2.Hematoxylin eosin?H.E?staining method,observation of R'group found that the pathological changes to the degree of malignancy to reduce the direction of development.3.Immunohistochemical staining was showed:in the R'group,the expression of E-cadherin protein were markedly increased,the expression of vimentin was markedly decreased,the expression of HIF-1?was markedly decreased.4.Immunofluorescence observation was showed,in R'group,E-cadherin protein was expression bright,the vimentin was expression dark,the inhibitory effect of ropivacaine on epithelial mesenchymal transition marker protein was verified again.5.Western blot detection was showed,compared with the C'group,the infiltration of tumor around the graft of ropivacaine significantly inhibited the expression of TGF-,beta,VEGF and MMP-9 in breast cancer xenografts?P<0.05?.Part?:Effect of ropivacaine local infiltration anesthesia combined with pectoral nerve block on the prognosis of patients with breast cancer1.68 patients undergoing radical surgery for breast cancer,randomly divided into two groups,the general anesthesia group?Ggroup?and the experimental group?E group?,each consisted of 34 patients.The Ggroup was treated by total intravenous anesthesia with laryngeal mask airway and PCIA,the Egroup was treated by ropivacaine local infiltration anesthesia combined with pectoral nerve block combined with laryngeal mask airway total intravenous anesthesia and continuous incision infusion.Before the operation?T₀??at the beginning of surgery?T₁??When the operation was 1.5h?T₂??at the end of surgery?T₃?and 3 days after surgery?T₄?,4 time points,6ml venous blood was extracted from each patient,store in-80centigrade refrigerator.2.Serum levels of PRL,GH,IL-8 and cortisol?Cor?were measured by high performance liquid chromatography?HPLC?at the time of T₀,T₁,T₂,T₃ and T₄ with two groups of patients,which informed of changes in the body's stress response.3.Two groups of patients were examined by flow cytometry at the time of T₀,T₁,T₂,T₃ and T₄,and the subtypes of T lymphocytes?CD₃⁺,CD₄⁺and CD₈⁺?and NK cells were detected.To compare the percentage of CD₃⁺T lymphocytes and CD₄⁺/CD₈⁺ratio,in order to reveal the changes of the immune function of the patients.4.Two groups of patients were examined by ELISA chemical immunoassay to changes of VEGF-C and TGF-?were recorded at time points T₀,T₁,T₂,T₃ and T₄.5.We recorded the data of the two groups:tumor size,grade,type,if the patient received chemotherapy,radiation therapy,or endocrine therapy.In addition,we calculated a Nottingham Prognostic Index.The follow-up time were 36 months in both the two groups,and we recorded the recurrence,metastasis and treated during the follow-up time.Results1.There were no significant differences in tumor sizes,grade,type and prognostic factors between the two groups?p>0.05?.Both groups had a mean Nottingham Prognostic Index score in the intermediate range.More-over,the incidence of chemotherapy,radiation therapy,and endocrine therapy after the initial surgical treatment did not differ significantly between the two groups?p>0.05?.2.The two groups of PRL T₀ is the lowest,T₁ is the highest?T₂-T₄ gradually decreases,the Egroup was significantly lower than the Ggroup?p<0.05?;GH gradually increases at T₁-T₂,T₃-T₄ gradually decreases,and the Egroup was significantly lower than the Ggroup?p<0.05?;IL-8 gradually decreases at T₁-T₄,And the Egroup was significantly lower than the Ggroup?p<0.05?;Cor gradually increased at T₁-T₂,T₃-T₄ gradually decreases,and the Egroup was significantly lower than the Ggroup?p<0.05?.3.There was no significant difference in the percentage of CD₃⁺T lymphocytes in venous blood between the Ggroup and the Egroup at T₀.The percentage of CD₃⁺T lymphocyte in the Ggroup was significantly lower than that of the Egroup at T₁,and the change range of the former is larger than that of the latter?p<0.05?.The percentage of CD₃⁺T lymphocyte in T₂ and T₃ time points Ggroup was still lower than that in Egroup?p<0.05?.At the time of T₄,the percentages of CD₃⁺T lymphocytes in the Ggroup and Egroup increased in varying degrees,but still lower than the preoperative level.In addition,The percentage of CD₃⁺T lymphocytes in the Egroup was still higher than that in the Ggroup?p<0.05?.There was no significant difference in CD₄⁺/CD₈⁺ratio between the two groups before operation,at T₁,the CD₄⁺/CD₈⁺ratios of the two groups all decreased significantly?p<0.05?,and the Ggroup decreased even more than the Egroup?p<0.05?.The CD₄⁺/CD₈⁺ratio decreased significantly at T₂ and T₃,however,the ratio of the Egroup was still higher than that of the Ggroup?p<0.05?.At T₄ time point CD₄⁺/CD₈⁺ratio decreased significantly,however,the ratio of the Egroup was still higher than that of the Ggroup?p<0.05?.At the same time,the CD₄⁺/CD₈⁺ratios in each group increased in different degrees at T₄time points,but it is still below the preoperative level.Besides,the ratio of CD₄⁺/CD₈⁺in the Egroup was still higher than that of the Ggroup?p<0.05?.There were no significant changes in NK cells at T₀-T₄,when compared with two groups,the Egroup was significantly higher than the Ggroup at T₃-T₄?p>0.05?.4.There were no significant differences in VEGF-C and TGF-?between the two groups?p>0.05?.5.Recurrence or metastasis was documented in 2 of 34 patients?6%?in the Egroup and in 8 of 34patients?24%?in the Ggroup during the follow-up period?p<0.05?.Analysis using kaplan-meier showed that recurrence time was slow in the Egroup?p<0.05?.Conclusions1.Ropivacaine has a significant inhibitory effect on proliferation,invasion and migration of human breast cancer MCF-7 cells,it can significantly promote the apoptosis of human breast cancer MCF-7 cells.2.Ropivacaine can inhibit the growth of human breast cancer cell line MCF-7 in transplantable tumor of nude mice,it can also inhibit the occurrence of epithelial mesenchymal transition,reduce the expression of HIF-1,TGF-,VEGF and MMP-9.3.Ropivacaine local infiltration anesthesia combined with pectoral nerve block,and continuous incision infusion can reduce stress response and improve immune suppression.The anesthetic method reduced the risk of recurrence or metastasis fourfold during a 36 months follow-up period.