| Objective: To establish the model of liver resection in mice,in order to group them and give them different doses of Gurigumu-13,which reflected the effects of Gurigumu-13 on liver regeneration.Methods:1.To construct a 70%(2/3)liver regeneration(PH)model in mice after hepatectomy: in this experiment,a traditional large-part ligation and resection method was used to establish a mouse liver resection model.Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate under sterile environment conditions,abdominal fur was cut off and disinfected,and about 3 cm of incision was made transversely along the median line of abdomen after the surgical plate to fully expose the mouse liver and observe whether the anatomical structure of the mouse liver was normal.the left lobe of the mouse liver was gently squeezed out by hand,gently lifted,and then cut the sickle ligament,liver and stomach ligament and part of the left coronal ligament of the liver with ophthalmic scissors.at this time,the middle lobe and left external lobe of the liver were basically free.Take 4.0 medical non-absorption suture line,ligate and cut the liver pedicle of left outer lobe,left middle lobe and right middle lobe of mice in turn(about 68% weight of the original liver of the mice was removed),sterile operation,and after suture abdominal incision by layer,wait for mouse resuscitation under electric oven lamp.mice can successfully wake up and move freely as a sign of successful surgery.2.Effect of Gurigumu-13 on liver function and liver regeneration after 70%(2/3)hepatectomy in mice :(1)effect of Gurigumu-13 on liver repair after 70%(2/3)hepatectomy in mice.KM male mice,ageing 6-8 weeks,were randomly divided into five groups:the sham group,the model group,the control group(tiop Ronin,90mg/kg),low doses group(Gurigumu-13,400mg/kg)and high doeses group(Gurigumu-13,1600mg/kg).The shame and model group did not take any medicine,and the control group was given tiopronin(tiopronin,90mg/kg).Besides,high doses group was given Gurigumu-13,whose doses was1600mg/kg,and the low doses was 400mg/kg.The dosage of administration was0.3ml/20 g,and the frequecy was once a day.After 7 days of taking medicine,we conducted the rsection of liver except the shame group the traditional method of excision following mostareas of ligation,As for the shame group,we only open their abdominal cavities,but the livers were not injured and removed.As for the shame group,we only opened abdominal cavtiy,but liver resction was or taking medicine not perform.The dosage of administration was0.3ml/20 g,and the frequecy is once a day.After stitching up the wound,these three goup then took these medicines.Nextlly,we repectively collected serum and livers from mice in the 1st,3rd,5th,9th day.Based on this,we measured the content of ALT,AST,ALB and LDH at different time via serum.Beside,we observed the cell morphology and pathological changes via HE stain on samples from different time.In addition,the expression levels of PCNA and Ki67 in regenerated tissue were detected through immunohistochemistry.During these days,we also recorded the weight of mice and their livers,calculatde the liver indec and.observed the liver regeneration.According to these indicators,we elevated the effects of Gurigumu-13 on liver regeneration.3.More than that,Western blot was used to detect STAT3 protein expression in liver tissue after hepatectomy.Results: 1.After removing the 70%(2/3)of liver,we could successfully established the PH models: Anaesthesia mice,under aseptic condition,open abdomen ligates liver leaf in turn,and suture abdomen incision according to layer,complete the operation time of one hepatectomy mouse is 15?20 min..the success rate of liver resection model surgery in mice was over 80% on the premise of ensuring the survival number of mice in each group,and the dead mice were related to the operation.Successful mice were able to return to free movement,normal diet and good wound healing in the abdomen.2.the effects of Gurigumu-13 on PH models:(1)The biochemical consequences showed that in comparison to the shame group,the content of ALT,AST,LDH from the model group were higher,but the ALB lower.Compared to the model group,the content from the high doses group and low doses group were lower at each tme,but ALB higher.(2)The results from HE results indicated that cell edema,inflammation and lipid droplet appeared in the hepatocytes of mice from four groups,except sham group;In the sham group,the hepatocytes were normal in morphology,the cell cords were arranged,neatly.The lobule structure was intact,and there was no denatured or necrotic area.The hepatocytes from the model group presented degeneration and vacuolar necrosis,and its seatosis was severe,accompanied by inflammatory cells infiltration,the cords were arranged in disorder at thesame time.Compared with,the steatosis in hepatocytes reduced from two groups treated with Gurigumu-13,which means the liver injury was improved.The consequences in immunohistochemistry showed that PCNA and Ki67 mainly expressed in the nuclei of hepatocytes.Compared with,PCNA and Ki67 from the model group expressed more accroding to time(p<0.05);In the 1st,3rd,5th days after operation,the PCNA and Ki67 from two groups treated with Gurigumu-13 obviously expressed more(p<0.05).In the 3rd day,the expression of PCNA from the high and low doses groups reached a peck.3.According to western blot,in the 1st,3rd,5th day after operation,the content of STAT3 from the model group is lower than the shme group(p<0.05).At the same point of time,STAT3 expressed more in the high doses group than the model group(p<0.05).The results showed that the expression of STAT3 protein in regenerated liver tissue was significantly increased in the high dose group after administration of Gurigumu-13(p<0.05).Conclusion: Mouse model of liver resection by 75% was successfully established.Gurigumu-13 had benificial effects on liver injury resulting from hepatectomy,and the mechanism may be related to the activation of STAT3. |
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