Effect And Mechanism Of Hepatic Ischemia-reperfusion Injury On The Recurrence And Metastasis Of HCC In Nude Mice

Objective To study the effect of ischemia-reperfusion injury(I/RI) on the recurrence and metastasis of hepatocellular carcinoma(HCC), and explore the possible role of epithelial-mesenchymal transition(EMT) in the process. Experiments in vivo and vitro were employed to simulate the ischemia and hypoxia state induced by I/RI during the liver cancer surgery, Octreotide(OCT) was used for further processing.So as to provide new ideas and more experimental basis for clinical prevention and treatment of HCC recurrence and metastasis.Methods1. High invasive and metastatic HCC cell line MHCC97-H was cultured in anoxic, low serum medium,and observed the changes in cell morphology after 24 h and48h.Transwell, Wound healing test, MTT assays and TUNEL methods were used to detect invasion, migration, proliferation and apoptosis abilites of MHCC97-H cells after treated with different concentrations of OCT(1?g/m L,5?g/m L,10?g/m L,20?g/m L).2. MHCC97-H cells were used to form subcutaneous tumor in nude mice, and orthotopic liver cancer models of nude mice were established with the subcutaneous tumor. The models were treated with different hepatic I/RI time(selective blocking hepatic portal blood for 0min, 15 min, 30min), and combined with low dose of OCT(50?g/kg·d) randomly. HCC recurrence and metastasis rates among no-ischemia group, ischemia groups and OCT treated group were comapred after 30 days.3. Western Blot was used to compare the expressions of E-cadherin, vimentin, twist,and Snai 1 proteins between MHCC97-H cells treated with different concentrations of OCT and cultured in anoxic, low serum medium, and between hepatocellular carcinomas in situ and recurrent tumors, respectively. Immunohistochemistry was used to detect the expressions of E-cadherin and Vimentin between orthotopic liver cancer and recurrent hepatocellular carcinoma in nude mice.Results1. Cytological experiments:Cultured MHCC97-H cells under anoxic, low serum conditions, to simulate the conditions of I/RI in vivo, 24 h and 48 h later, observations showed that the cells morphology became spindle, lost the polygonal shape of normal cells,and cell invasion, migration abilities were enhanced(p(27) 0.05). MTT assays,Transwell tests and Wound healing tests showed that OCT had inhibitory effect on MHCC97-H cells growth, invasion ability,migration ability and positively correlated with concentrations(p(27) 0.05). TUNEL tests show that different concentrations of OCT had no obvious effect in promoting apoptosis of MHCC97-H cells(p>0.05).2. Animal experiments: Hepatic I/RI of different times(15min, 30min) could promote HCC recurrence in nude mice, and the recurrent rates were consistent with the length of the I/RI time. OCT group showed that continuous injection of a low dose OCT could reduce the recurrence rate. No distant metastasis of HCC occurred in each experimental group of nude mice.3. Western Blot showed that the E-cadherin expression of MHCC97-H cells decreased after cultured under anoxic and low serum conditions for 48 h, and the expressions of vimentin, Snai 1and twist increased, but after treated with OCT, the results reversed(p(27)0.05).After I/RI, with respect to the liver orthotopic implantation tumor,E-cadherin expression of recurrence tumor decreased, and the expressions of vimentin, Snai 1, twist increased,after combined with OCT for further processing,the results reversed in certain degree(p(27) 0.05). Immunohistochemical detection of E-cadherin, vimentin results of liver orthotopic implantation tumor and recurrence tumor were consistent with the Western Blot test.Conclusions1. Liver I/RI could promote the recurrence of HCC in nude mice, and the recurrence rates were consistent with the length of I/RI.2. Liver I/RI could promote the EMT in cancer cells, showed decreaded expression of E-cadherin and increased expressions of Vimentin, Snai1, Twist, and enhanced cell invasive and metastatic abilities ultimately.3. Octreotide could reduce the HCC recurrent rate increased by I/RI in certain degree through inhibiting the occurrence of EMT in cancer cells.