| BackgroundThe phenomenon of liver regeneration is often seen in liver resection, portal vein thrombosis and living donor liver transplantation (LDLT). Liver has a complicated physiological function and a high regeneration ability, which is closely related with the surgical prognosis. Thus it’s very important to explore the liver-regenerating mechanism to prevent postoperative liver failure and improve the prognosis.Related studies showed that the high-pressure portal vein infusion was of great significance in promoting liver regeneration and regulating liver function. In liver resection or liver transplantation, changes of the blood flow in portal veins may be a necessary factor in initiation, sustaining, and even termination of the liver regeneration process. After Partial Hepatectomy, every unit of the remaining liver got more blood supply as it needed to hold the portal vein flow which belong to the resected lobe.In the dog partial liver resection united with the portacaval shunt experiment, liver regeneration decreased obviously. In recently years, we also found out through the clinical living donor liver transplantation and partial liver resection that, immediately increased "portal vein blood flow/liver volume" and PVP after operation stimulated and regulated the regeneration of liver; To prevent the occurrence of small liver syndrome, if we carried out the "overdo" portacaval shunt, the significantly decreased blood perfusion and stress would lead to the occurrence of liver atrophy.People observed another phenomenon, liver cells proliferated successively from portal vein gradually to lobular area around, liver cells around the central vein proliferated the latest; In the portal vein to hepatic venous pressure gradient, the liver cells around the portal vein suffered more stress than the liver cells around the central vein, we speculated further based on this phenomenon:the liver cells which suffered more stress proliferated earlier than the liver cells which suffered less stress.But the concrete mechanism is still unknown, and it’s presumably related with the activation of the relevant mechanical signal pathway caused by PVP. Under stress, ligand of ECM combined with the receptor of integrin on cell membrane, "ECM-integrin-CSK" signaling pathway was the first pathway which be activated, mechanical signal transduced into the cells and regulated the cells growth and proliferation through the downstream signaling molecules. Focal Adhesion Kinase (FAK) is the most. important component in the mechanical signal transduction pathway. After Integrin binded accordingly with the ligand of ECM,they gathered into a cluster, which caused the Tyr residues of FAK phosphorylated and FAK activated, and further catalyzed several information mollecules of downstream in cytoplasm phosphorylated, and combined with growth factor receptor pathway through the "crosstalk" and transducted teh cell signals into the cells. In mechanical signal transduction, FAK is regarded as "promoter" of this process.However, in the process of liver regeneration, there still no report about whether blood flow mechanics signal stimulation causes the expression and the change of activity of FAK to regulate hepatocytes proliferation or not.In addition, we already knew that Hepatocyte growth factor (HGF) is the most important mitogen which could promoted liver cell proliferation.After combined with c-Met, HGF activated the PTK receptor, made the receptor self-phosphorylated and made the cytoplasm messenger protein phosphorylated, launch several signaling pathways at the same time, and transmitted the proliferation signals into the cells, promoted liver cells prolifereating by regulating the expression of specific genes. Ras/MAPK signal transduction pathway which initiated by HGF/c-Met was considered to be the most important pathways about liver cells proliferation. Rats Genetic lack of urokinase or rats lack of c-Met had very poor abilities of liver regeneration; Liver cell proliferation cycle would be bogged down if we shut down c-Met by RNA interference.However, some scholars found out that liver cells proliferation caused by fluid shear stress may be related to this chemical channel too: fluid shear stress changed the microenvironment surrounding liver cells, and transducted the extracellular chemical/physical signals (HGF... c-Met) into the intracellular, after a series of cascade amplification, showed the proliferation and other effects. Webber et al found that injected HGF into the portal vein of normal rats, the proliferation of liver cell is not obvious, but injected HGF into the portal vein of rats under 30% hepatectomy, liver cells proliferated significantly.Thus,we speculated that chemical signal pathway mediated by HGF and the mechanics signal pathway mediated by fluid shear stress may have a coordinated stimulus effect on liver cells proliferation, however, there is no report about this assumption yet.This experimental research focus on the above hypothesis.PurposeWe divided this experiment into two parts, respectively discussed the effects of biological mechanics(fluid mechanics) on liver regeneration in vivo and in vitro.1. Animal experiment:We chose SD rats as the research objects; built different partial hepatectomy models; Measured portal venous pressure (PVP) value at different time points and got the liver tissue at the same time; Used immuno histochemical method to detect the expression of FAK in the liver tissue and liver proliferation nucleus antigen (PCNA); To explore the role of changes in portal vein pressure in rats liver regeneration and the expression of the mechanical signal factor FAK.2. Cell biology experiment:We chose immortalized rats BRL-3 liver cell lines and human HepG2 liver cancer cell lines as the research objects; Built fluid stress loading cell culture devices; Loaded different size of fluid shear stress and observed the cell proliferation, and discussed the difference of cell proliferation between the condition of HGF existed or not.Methods1. Research about the effect of PVP on liver regeneration and the expression of its key protein FAK:1) Chose 64 clean level health Sprague Dawley (SD) rats as research objects;2) Built different proportion (30%,50%,70%) liver resection models;3) Experimental group:The rats were divided into control group,30%,50%,70% proportion liver resection groups;4) Got data and obtained specimens:Measured PVP in postoperative 24 h,48 h,72 h,168h between groups;Got liver tissue samples at the same time;5) Used immunohistochemical method to detect the expression of proliferation nucleus antigen (PCNA) and FAK in each group;6) Statistical analysis, using the t-test and Pearson correlation coefficient test, explored the relationship among PVP, PCNA, FAK as well as the liver tissue regeneration.2. Research about fluid shear stress and HGF synergy promoted liver cell proliferation:1) Objects of study:immortalized rats BRL-3 liver cell lines and human HepG2 liver cancer cell lines;2) Build fluid shear stress loading cell culture device;3) The experimental groups:Control group:Injected cells into the device but did not give them any shear stress;Group A (BRL-3 A only pressure group):Cells under different sizes of fluid shear stress (0,12 dyn/cm2,24 dyn/cm2);group B (BRL-3A pressure+HGF group):Cells under different sizes of fluid shear stress with HGF (0,12 dyn/cm2,24 dyn/cm2);Group C (HepG2 only pressure group):Cells under different sizes of fluid shear stress (0,12 dyn/cm2,24 dyn/cm2);group D (HepG2 pressure+HGF group):Cells under different sizes of fluid shear stress with HGF (0,12 dyn/cm2,24 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-3 A 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.Results1. Research about the effect of PVP on liver regeneration and the expression of its key protein FAK:1) Compared to the control group, All the PVP of each group increased postoperatively. For 70% and 50% PH groups:The PVP value reached peak in 24 h postoperatively (P<0.01), decreased gradually in 24 h-72 h (but still higher than the normal level (P<0.05)), and was close to or slightly higher than the control group (P>0.05) in 168 h postoperatively. For 30% PH group, the PVP increased a little in 24 h (P<0.05), and then was close to or slightly higher than the control group (P>0.05) in the rest time points postoperatively.2) PCNA tested by immunohistochemical:If the PCNA testing result is positive, the staining part is brownish yellow and distributed in the nucleus, while the cytoplasm is slightly colored or non-colored. For the liver tissues in the control group of rats, the number of cells with positive PCNA and slightly color was small. For the liver tissues in groups of rats subjected to liver resection of different proportions, the number of cells with positive PCNA and dark stains increased significantly in 24 h postoperatively (P<0.01); the number of positive cells decreased gradually in 24 h-72 h, but was still higher than the control group (P<0.05, P<0.01); in 168 h postoperatively positive cells gradually decreased while for 70% PH group the number of positive cells were still higher than the control group (P<0.05) and for the rest two groups the number were close to or slightly higher than the control group (P>0.05). At the same time point, the higher the proportion of liver resection, the greater the number of PCNA positive cells, and the deeper the staining3) FAK tested by immunohistochemical:FAK is expressed mainly in the cytoplasm. In the control group, the cytoplasm was seen light yellow and a few brown expressions were observed. For the liver tissues in groups of rats after liver resection of different proportions, the number of FAK expressions increased significantly (P<0.01), the stains were darker and the number of FAK positive cells increased significantly; For the 50% and 70% PH groups the number of positive cells gradually decreased in 24 h-72 h postoperatively, but was still higher than the control group (P<0.05, P<0.01); in 168 h the number of positive cells gradually decreased and was close to the control group (P>0.05). For 30% PH group, the number of positive cells was close to or slightly higher than the control group (P>0.05) in 24 h-72 h. At the same time point, the higher the proportion of liver resection, the greater the number of FAK positive cells and the deeper the staining4) Correlation Analysisa. Correlation analysis between PVP and PCNAFor rats group of 70% PH:Positive correlations were found between the PVP in rats in 24 h,48 h,72 h and 168 h postoperatively and the liver regeneration time(P<0.05).For rats group of 50% PH:Positive correlations were found between the PVP in rats in 24 h,48 h and 72 h postoperatively and the liver regeneration time,(P<0.05); No correlation were found between the PVP in 168 h postoperatively and the liver regeneration time (P>0.05).For rats group of 30% PH:Positive correlations were found between the PVP in rats in 24 h and 48 h postoperatively and the liver regeneration time(P <0.05); No correlations were found between the PVP in 72 h or 168 h postoperatively and the liver regeneration time (P>0.05).b. Correlation Analysis between PVP and FAKFor rats group of 70% PH:Positive correlations were found between the PVP in rats in 24 h.48 h and 72 h postoperatively and the FAK expressions in the remaining liver cells(P<0.05); No correlation were found between the PVP in 168 h postoperatively and the FAK expression (P>0.05).For rats group of 50% PH:Positive correlations were found between the PVP in rats in 24 h,48 h and 72 h postoperatively and the FAK expressions in the remaining liver cells(P<0.05); No correlation were found between the PVP in 168 h postoperatively and the FAK expression (P>0.05).For rats group of 30% PH:No correlations were found between the PVP in each time point postoperatively and the FAK expression (P>0.05).c. Correlation Analysis between PCNA and FAKFor rats group of 70% PH:Positive correlations were found between the PCNA in rats in 24 h,48 h and 72 h postoperatively and the FAK expressions in the remaining liver cells, and the correlation coefficients r are 0.948,0.934 and 0.965, respectively (P<0.05); No correlation were found between the PCNA in 168 h postoperatively and the FAK expression (P>0.05).For rats group of 50% PH:Positive correlations were found between the PCNA in rats in 24 h,48 h and 72 h postoperatively and the FAK expressions in the remaining liver cells(P<0.05); No correlation were found between the PCNA in 168 h postoperatively and the FAK expression (P>0.05).For rats group of 30% PH:Positive correlation was found between the PCNA in rats in 48 h postoperatively and the FAK expression in the remaining liver cells (P<0.05). No correlations were found between the PCNA in the rest time points postoperatively and the FAK expression (P>0.05)2. Effects of fluid shear stress and fluid shear stress/HGF synergy promoted liver cell proliferation:1) The changes of proliferation dynamics of immortalized rats BRL-3A liver cell lines under different experimental conditions:Group A0 with Bo:The cell proliferation dynamics of rats BRL-3 A cell lines performed for time dependence, at every same time point, the proliferation of group B was obviously higher than that of group A (P<0.05).Group A0,A12 with A24 at the same time point:During 12 h to 72 h, the cells proliferation of group A12 and group A24 were obviously higher than that of group Ao (P<0.05), the cells proliferation of group A24 was obviously higher than that of group A12 (P<0.05); After 168 h, the cell proliferation is not obvious compared to control group(P>0.05).Group B0,B12 with B24 at the same time point:During 12 h to 72 h, the cells proliferation of group B12 and group B24 were obviously higher than that of group Bo (P<0.05), the cells proliferation of group B24 was obviously higher than that of group B12 (P<0.05); After 168 h, the cell proliferation is not obvious compared to control group(P>0.05).Group A12 with B12,group A24 with B24 at the same time point:During 12 h to 72 h, the cells proliferation of group B12 was obviously higher than that of group A12 (P<0.05), the cells proliferation of group B24 was obviously higher than that of group A24 (P<0.05); After 168h, the cell proliferation is not obvious compared to control group(P>0.05).Group A12 with A24,group B12 with B24 at the same time point:During 12 h to 72 h, the cells proliferation of group A24 was obviously higher than that of group A12 (P <0.05), the cells proliferation of group B24 was obviously higher than that of group B12 (P<0.05); After 168 h, the cell proliferation is not obvious compared to control group(P>0.05).In every same group, cells under fluid shear stress all proliferated obviously,and reached the peak at 24h, the it goes down slowly during 24 h to 72 h,but it still higher than the control group (P<0.05),After 168 h, the cell proliferation is not obvious compared to control group(P>0.05).2) The changes of proliferation dynamics of immortalized human HepG2 liver cancer cell lines under different experimental conditions are consistant with the changes of proliferation dynamics of immortalized rats BRL-3A liver cancer cell lines.Conclusion1. Animal experimental results showed that the more resection we did, the greater the PVP value increased;Correspondingly, the more obviously of the expression of PCNA and FAK, and they reached the peak value consistently at 24 h.Along with the extension of the time after the surgery, when the liver volume of rats were closed to normal sizes, the changes of PVP, PCNA and FAK of each group gradually converged.Statistical correlation analysis results suggest the sizes of the lobe resection, PVP values and the expression of PCNA and FAK presented consistency.It means that the changes of the portal vein blood flow and mechanical signal transduction pathway mediated by FAK played a crucial regulatory role in the process of liver regeneration.2. 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;Under the same stress, cells with HGF proliferated better than the cells without HGF.It shows that in the process of liver cells proliferation, mechanical and chemical factors have synergy effects. |
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