| BackgroundThe liver is involved in many important physiological activities such as metabolism, detoxification, immune to gobble up, etc.Liver cell is the complete these physiological structure foundation. Liver regeneration is very strong. In PHx or severe hepatitis, liver cells proliferate to recover its quality/volume. It is very important to explore the mechanism of liver regeneration.In recent years, the biochemistry theory and biomechanics theory is the study of liver regeneration mechanism mainly two theories. Biochemical signaling pathways such as HGF, EGF promote liver cells proliferation studies have very deep And biochemical factors compared with the biomechanics research is less. According to Clinical and experimental study, the changes of portal venous blood flow can affect liver regeneration. But the biomechanical factors effect on liver cell proliferation of related research is not much. Through the study of biomechanics of other organizations for found that biomechanical signals can affect cell proliferation through FAK receptor mediated reaction. FAK also through "Crosstalk" interact with growth factor receptor pathway common affect cell proliferation.Through our preliminary research results showed that the liver regeneration is affected by the portal vein blood flow change and the expression of FAK. Blood flow mechanics may be by the way of "ECM-integrin-CSK" signaling pathway regulating the proliferation of liver cells. In this premise, the biomechanics of change can affect liver cell proliferation is unclear. Also in the role of biomechanics, liver cell proliferation response whether the corresponding signal changes in molecular expression is not clear. Main purpose of this study was to explore under the biomechanical the action of liver cell proliferation and related mechanical signal path.PurposeThis experiment is divided into two parts:The first part the influence of shear stress on liver cell proliferation:We chose immortalized rats BRL-3 liver cell lines as the research objects; Built fluid stress loading cell culture devices; Loaded different size of fluid shear stress (0dyn/cm2-30 dyn/cm2)and observed the cell proliferation, and discussed the difference of cell proliferation between the condition of size of fluid shear stress. The second part shear stress through the activation of the Ras/MAPK signaling pathways key protein ERK1/2, promote the research of liver cell proliferationWe chose immortalized rats BRL-3 liver cell lines as the research objects; Built fluid stress loading cell culture devices; at the same time with ERK1/2 inhibitors inhibit Ras/MAPK signal pathway, by observing the cell proliferation and detection of Cyclin D protein expression and gene expression level, to study the shear stress in the process of liver cell proliferation’s relationship with the Ras/MAPK signal pathway.Methods1. the influence of shear stress on liver cell proliferation:1) Objects of study:immortalized rats BRL-3 liver cell lines;2) Build fluid shear stress loading cell culture device;3) The experimental groups:The control group (BO group):cut will not be recorded should (B-0 dyn/cm); Add 12 dyn/cm2 group (B12):cells in culture to exert 12 dyn/cm2 shear stress (B-12 dyn/cm2);Add 24 dyn/cm2 (B24 group):the cultured cells on 24 dyn/cm2 shear stress (B-24 dyn/cm2).Used the direct cell counting and CCK-8 (cell counting kit-8) technology to detect the cells proliferation dynamics in different groups, observed whether the fluid shear stress promoted immortalized rats BRL-3A liver cell lines and human HepG2 liver cancer cell lines and the role of HGF in this process.4) Used t test to analyze the results of the experiment.2. Shear stress through the activation of the Ras/MAPK signaling pathways key protein ERK1/2, promote the research of liver cell proliferation:1) Objects of study:immortalized rats BRL-3 liver cell lines;2) ERK112 inhibitor PD98059 treatment intervention group of cells3) Build fluid shear stress loading cell culture device;4) The experimental groups:The control group:the cells slides into fluid stress loading system but not the same time its strength, nor give inhibitor treatment;Simple pressure group:only to the size of cells each loading 24 dyn/cm2 fluid shear stress;The intervention group:on the size of a cell load 24 dyn/cm2 fluid shear stress at the same time in advance by ERK1/2 protein inhibitor PD98059 processing;Used the direct cell counting and CCK-8 (cell counting kit-8) technology to detect the cells proliferation dynamics in different groups, observed whether the fluid shear stress promoted immortalized rats BRL-3A liver cell lines and human HepG2 liver cancer cell lines and the role of HGF in this process. Western mark analysis and fluorescence quantitative PCR detection.5) Used t test to analyze the results of the experiment.Results1. The influence of shear stress on liver cell proliferation:1) The immortalized rats BRL-3-liver cells directly counting results:a) The control group (group Bo):0-168-hour interval control cell count showed a trend of add, at 0-grew fastest in 24 hours.b) 12 dyn/cm2 group (B12):0-24 hours interval B12 group the cell number increased, peak in 24 hours to proliferation, and cell proliferation rate of decline in 24-168 hours.c) 24 dyn/cm2 group (group B24):0-24 hour’s interval B24 group the cells increased, peak in 24 hours to proliferation, and cell proliferation rate of decline in 24-168 hours.d) 12 dyn/cm (B12 group) and control group (group Bo):at the same time site, B12 cells was significantly greater than B12 group cell count (P< 0.05).e) 24 dyn/cm2 (B24 group) and control group (group Bo):at the same time site, B24 cells was significantly greater than Bo group cell count (P< 0.05).f) 12 dyn/cm2 (B12) and add 24 dyn/cm2 group (group B24):at the same time site, B24 cells was significantly greater than B12 group cell count (P< 0.05).2) The immortalized rats BRL-3-a liver cell CCK 8 absorbance value test results:a) The control group (group Bo):0-168-hour interval control cell proliferation trend, at 0-grew fastest in 24 hours.b) 12 dyn/cm2 group (B12):0-24 hours interval B12 group cell absorbance value trend, reach a peak in 24 h, and 24-168-h absorbance value decline.c) 24 dyn/cm2 group (B24 group):0-24 hours interval B24 group cell absorbance value trend, reach a peak in 24 h, and 24-168-h absorbance decline.d) 12 dyn/cm2 (B12 group) and control group (group Bo):at the same time site, B12 group of cells is bigger than the absorbance value Bo group absorbance value (P< 0.05)..e) 24 dyn/cm2 (B24 group) and control group (group Bo):at the same time site, B24 group of cells is bigger than the absorbance value Bo group absorbance value (P< 0.05).f) 12 dyn/cm2 (B12) and add 24 dyn/cm2 group (group B24):at the same time site, B24 group of cells is bigger than the absorbance value B12 group absorbance value (P< 0.05).2. Shear stress through the activation of the Ras/MAPK signaling pathways key protein ERK1/2, promote the research of liver cell proliferation:1) The immortalized rats BRL-3-liver cells directly counting results:a) The control group (group A):0-168-hour interval group A cell count showed A trend of increase, at 0-proliferation fastest in 24 hours.b) Pressure group (group B):0-24 hours interval the group B cell count increased, peak in 24 hours to proliferation, and cell proliferation rate of decline in 24-168 hours.c) The intervention group (group C):0-24 hours interval group C the cell number increased, peak in 24 hours to proliferation, and cell proliferation rate of decline in the 24-168 hours.d) Pressure group (group B) group and control group (group A):sites at the same time, group B cells was significantly greater than the cell number of group A (P< 0.05)..e) Pressure group (group B) group and intervention group (group C):sites at the same time, group B cells was significantly greater than group C cell count (P< 0.05).f) Intervention group (C group) and control group (group A):sites at the same time, group C cells was slightly larger than the cell number of group A (P< 0.05).2) The immortalized rats BRL-3-a liver cell CCK 8 absorbance value test results:a) The control group (group A):0-168-hour interval absorbance group A showed A trend of increase, at 0-grew fastest in 24 hours.b) Pressure group (group B):0-24 hours interval group B the absorbance increased, reached a peak in 24 hours, and 24-168 hours.c) Intervention group (group C):0-24 hours interval absorbance group C showed a trend of increase, reach a peak in 24 hours, and 24-168 hours.d) Pressure group (group B) group and control group (group A):sites at the same time, group B is bigger than the absorbance absorbance value of group A(P<0.05).e) Pressure group (group B) group and intervention group (group C):sites at the same time, group B is bigger than the absorbance absorbance value of group C (P< 0.05).f) Intervention group (C group) and control group (group A):sites at the same time, group C cell absorbance is greater than the absorbance vakte of group A(P<0.05).3) The Westen blot detection for biochemical rats BRL-3 a liver cell ERK1/2 protein expression level:a) Pressure group (group B) group and control group (group A):within 24 hours, B group of Cyclin D1 and beta Actin protein stripe grey value is bigger than the ratio of group A (P< 0.05).b) Pressure group (group B) group and intervention group (group C):in 24 hours, B group of Cyclin D1 and beta Actin protein stripe grey value is bigger than the ratio of group C (P< 0.05).c) The intervention group (C group) and control group (group A):group C in 24 hours, Cyclin Dl and beta Actin protein bands of grey value ratio greater than group A (P< 0.05).4) PCR detection of immortalized rats BRL-3 a liver cell ERK1/2 level of gene expression:a) Pressure group (group B) and control group (group A):within 24 hours, group B is bigger than the amount of mRNA expression in group A (P< 0.05).b) Pressure group (group B) and intervention group (group C):in 24 hours, group B is bigger than the amount of mRNA expression of group C (P< 0.05).c) The intervention group (C group) and control group (group A):amount of mRNA expression in 24 hours, group C than in group A (P< 0.05).Conclusion1. Fluid shear stress could promoted the proliferation of immortalized rats BRL-3 liver cell lines and human HepG2 liver cancer cell lines, Within the scope of the physiological can afford, the higher the fluid shear stress,, the more obviously of the cells proliferation.It shows that in the process of liver cells proliferation, mechanical factors have effects.2. Inhibit Ras/MAPK signal pathway can reduce the shear stress promotes immortalized rats BRL-3 a liver cell line proliferation rate. But can’t completely inhibitory effect of shearing stress for the promotion of liver cell proliferation. Shear stress promotes liver cell proliferation and activation of the Ras/MAPK signal pathways involved; The Ras/MAPK signal pathway is not the only path of shear stress promotes liver cell proliferation. |
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