| Part onePartial liver resection in rats and establishment of lentivirus transfection modelObjective(s):To explore the methods and techniques of establishing a partial hepatectomy and lentivirus transfection model in SD rats,and to establish a reliable model basis for subsequent liver regeneration experimental research.Methods:Using 2/3 partial hepatectomy,the constructed lentiviral system of CaMKⅡ γoverexpression(LV-CaMKⅡγ),knockdown(LV-CaMK II yRNAi)and the corresponding blank vector were transfected into rats after hepatectomy through the caecum vein.In vivo,and establish a blank control group.Five groups of experimental rats were observed for postoperative complications,survival,and death.They were sacrificed 1,3,5,and 7 days after hepatectomy.Liver tissues were taken for lentiviral fluorescence detection;QPCR,Western Blot were used to detect the mRNA and protein of CaMK Ⅱ γ Expression;Immunohistochemistry was used to detect the expression of CaMKⅡ γ and tissue spatial position.Results:1.The use of SD rats to successfully establish a 2/3 partial hepatectomy animal model has few postoperative complications and a high survival rate.Most of the modeled rats can survive to the time of experimental liver harvest and 4 rats died.2.Fluorescence detection showed that red fluorescence was observed in the LV-CaMKⅡγ RNAi infection and its control lentivirus group,and the fluorescence expression of the two groups gradually decreased with time;green fluorescence was observed in the LV-CaMKⅡγ infection and its control lentivirus group,two The fluorescence expression of the group gradually decreased with time.3.QPCR test and Western Blot test results,that is,at the same time point,the mRNA and protein expression of the LV-CaMKⅡγ group was the highest,and that of the LV-CaMKⅡγRNAi group was the lowest.At 1 day after surgery,there was no difference between the viral intervention group and the corresponding control group.At 72 hours,5 days,and 7 days after surgery,the expression of the LV-CaMK Ⅱγgroup was higher than that of the control group(*P<0.05),and the expression of LV-CaMKⅡγRNAi was lower than Its control group(*P<0.05).The proteins of LV-CaMKⅡγ group were most obviously expressed on the 5th day.The trend of QPCR detection was the same as Western Blot detection,but the expression of LV-CaMKⅡγ group was most obvious on the third day.4.Immunohistochemical staining of CaMKⅡγ positive showed brownish yellow,with the most cytoplasmic expression,and its expression trend was consistent with Western Blot detection.Conclusion(s):1.60-70%(2/3)hepatectomy model,which can induce liver regeneration potential to the greatest extent,is less affected by pathological factors,accurately quantifies the volume of hepatectomy,easy to operate,postoperative complications,and low mortality,is to study liver proliferation Ideal animal model.2.Transcatheter intravenous injection of lentivirus transfected hepatocytes,effectively changed the expression of CaMKIIy gene protein in rat hepatocytes,achieved the purpose of experimental research,and established a model basis for further exploring the effect of CaMKIIy on liver regeneration and its mechanism.This modeling method has high reliability,good security,strong repeatability,and low interference.Part twoEffect of CaMKIIy expression on liver regeneration after hepatectomy in rats and its mechanismObjective(s):To explore the effect and mechanism of CaMKIIy on liver regeneration ability,hepatocyte proliferation cycle,and liver synthetic function recovery after hepatectomy in rats.Methods:1.Use MRI to detect the liver volume of rats for the enrollment of experimental animals and indirectly measure the regenerated liver volume after resection.Correlation analysis of the liver volume measured by drainage and MRI methods 2.The expression of Brdu and PCNA in liver tissue was detected by immunohistochemistry at different time points in each experimental group;the expression of Ki67 was detected by immunofluorescence.3.The levels of prealbumin were measured by ELISA at different time points in each experimental group.4.Observe the ultrastructural changes of liver cells by transmission electron microscope.Results:1.The volume of rat liver measured by MRI 1 day after operation,the volume of each virus intervention group and the blank control group were not statistically different(P>0.05).At 3 days,5 days,and 7 days after operation,the volume increase of the LV-CaMK Ⅱγ group was higher than that of the control group(*P<0.01),and the volume increase of the LV-CaMKⅡγ RNAi group was lower than that of the control group(*P<0.01).There was no significant difference in volume growth between the 3day,5day,7day,con group,LV-CaMKⅡγ-NC group and LV-CaMKⅡγRNAi-NC group(P>0.05).The liver volume(y)measured by drainage method(x)and MRI method,R=0.992,R2=0.983.The linear regression equation is y=0.064+0.879x,and the two are highly correlated.2.Different detection methods,PCNA and Ki67 liver proliferation trends are consistent.One day after surgery,there was expression in each viral intervention group and blank control group but there was no statistically significant difference in positive staining rate(P>0.05).At 3 days,5 days,and 7 days after surgery,the positive staining rate of the LV-CaMKⅡγ group was higher than its control group(*P<0.01),and the positive staining rate of LV-CaMKⅡγRNAi was lower than its control group(*P<0.01).There was no significant difference between the 3day,5day,7day,con group,LV-CaMKⅡγ-NC group and LV-CaMK ⅡγRNAi-NC group(P>0.05).Three days after Brdu,the expression in each group began,and the expression trend was consistent with PCNA and Ki67.3.Serum prealbumin had no statistical difference between the virus intervention group and the blank control group 1 day after surgery(P>0.05).As the number of days increases,the concentration increases,and the increase is obvious from day 3 to day 5,and the concentration on day 5 is close to normal levels.At 3 days,5 days,and 7 days after surgery,the concentration of the LV-CaMKⅡγ group was higher than that of the control group(*P<0.01),and the concentration of the LV-CaMKⅡγRNAi group was lower than that of the control group(*P<0.01).There was no significant difference in concentration between the 3day,5day,7day,con group,LV-CaMKⅡγ-NC group and LV-CaMK ⅡγRN-Ai-NC group(P>0.05).4.One day after PH,under the electron microscope,con group:mitochondria were swollen and vacuoles were visible.LV-CaMK Ⅱγ group:the number of mitochondria increased and swelled obviously,and the number of rough endoplasmic reticulum also increased.LV-CaMK ⅡγRNAi group:the number of mitochondria and rough endoplasmic reticulum is small,and the mitochondria are not swollen.Three days after the operation,the con group had enlarged hepatocyte nuclei and obvious nucleoli.LV-CaMK Ⅱγ group:enlarged nuclei,obvious nucleoli,increased chromatin,and increased mitotic cells.In the LV-CaMK ⅡγRNAi group,the nuclei are small,the nucleoli are not obvious,and the chromatin is often low.Five days after surgery,the con group:mitochondria,rough endoplasmic reticulum and free ribosomes were visible.LV-CaMKⅡγ group:abundant organelles,scattered ribosomes,increased mitochondria and rough endoplasmic reticulum structure.LV-CaMKⅡγRNAi group:fewer organelles,fewer mitochondria and rough endoplasmic reticulum,less free ribosomes,and hyperplasia of collagen fibers.Conclusion(s):The liver is an extremely proliferative organ.SD rats after hepatectomy can recover the original liver volume and function within one week,CaMKⅡγ is an important liver regeneration regulating kinase.Increasing CaMKⅡγ expression in vivo will activate hepatocyte anabolism,promote hepatocyte division and proliferation cycle,make liver volume grow faster,and then accelerate the recovery of liver function;and knock down CaMKIIy expression It will block the metabolism and division of liver cells,slow down the growth of liver volume and recovery of function.This study is an important supplement on the basis of the early research on the effect of CaMKIIy on liver regeneration.Not only provides a reference for the experimental research of liver regeneration,but also provides a theoretical basis for the clinical application of liver regeneration.It is expected that CaMKⅡγ will be an important target for regulating liver regeneration in clinical applications. |
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