Clinical Performance And Related Fundamental Study On Ex-vivo Liver Resection And Autotransplantation

Objective:1. This study is designed to explore the indication, feasibility, safety, and effectiveness of ex-vivo liver resection and autotransplantation(ELRA) in patient with end-staged alveolar echinococcosis(AE); to evaluate the clinical application of three dimensional imaging(3D) and personalized virtual surgery, and to explore the liver regeneration pattern after ELRA as well as its social and economic value;2. To assess liver regeneration after partial liver resection with different volume and analyze its survival rate in Wistar rats resection model;3. To explore the corelation between bone marrow mesenchymal stem cell (BMSCs) and chronic fibrosis,regeneration and repair by using chemical liver injury model in Wistar rats;4. To explore the possible fibrosis inducing, liver regenetation and repair mechanisms and interactions between TLR3signaling pathway and BMSCs.Methods:Part one:1. The clinical data of10end-staged AE patients underwent autotransplantation from August2010to August2013were retrospectively analyzed. The ELRA indication for end-staged AE patients was proposed;2. To compare the accuracy and feasibility of personalized virtual surgical plan and real operation by using preoperative CT and3D reconstruction imagings, and meanwhile estimated real graft weight;3. Perioperative parameters including operative time, morbidity, mortality and hospitalization costs were analyzed to assess feasibility and safety;4. The liver regeneration was evaluated through GV/SLV,CT scan and PET scan at1,3, and6months postoperatively. Part two:70%,80%and90%liver resection were successfully performed and survival rate was followed-up. The serum ALT and AST level were detected postoperatively. The the liver weight was compared on the14th day postoperatively. HE staining was used to evaluate the liver tissue morphological changes. Part three:Femur and tibia bone marrow were extracted, and the BMSCs were cultured, purified and amplified. The morphology of BMSCs was observed under optical microscope and the surface marker CD31, CD45, CD44, CD105, CD11b, and CD29were detected. Experimental rats was fed by0.01%DEN for16weeks to set up liver chemical injury model.1×106BMSCs were infusioned in the tail vein on the4th,8th,12th and16th week, simple model group and the blank control group were also built. Liver was examined by MRI on the20th week. All rats were euthanized after21weeks to exam the liver tissue morphological changes by HE staining. In situ hybridization technique was used to detect the expression of SrY in liver tissues, VG staining for collagen fiber, immunohistochemistry for a-SMA and TLR3. The protein expression of a-SMA, TLR3, NF-kBp65, TRIF and IRF3were also confirmed by Western-blotting. TLR3and IRF3mRNA were detected by FQ-RT-PCR.Results:Part one:1. The survival rate was100%. The operation was safe according to the inclusion criteria;2. Invasion and volume were consistent in operation with CT scan and3D imagings. The GV/SLV among10parents varied from41%to78%and the average was60.6%. The volume of virtual liver resection was695.11±142.14cm3and the actual volume687.78±130.81cm3(p>0.05);3.There was one case which was implemented by the umbilical vein infusion at low temperature, the other9cases with portal vein perfusion. In addition there were no bypass implemented in2cases, autologous inferior vena cava bypass in3cases, artificial blood vessel and temporal portal vein bypass in5cases. Operation time among10surgeries last from11.5to20.5hours and the average time was15.2hours. Anhepatic phase was200-435min, with median of285.5min. Red blood cell suspension was infusioned from0to15units. Hospitalization stay after surgery was18-58days. The total incidence of complications was60%(6/10), bile leakage was most common. Hospitalization cost was101.6-434.8thousand yuan RMB and174.4thousand yuan for median;4. The average growth of liver was14.75%in the first month,3.75%per month between the2nd-3th,3.08%in4th-6th, GV/SLV was87.5%in averge in the first6months. SUVave Rate of18F-FDG was0.9-1.91tibetan AE patient died of unknown fever cause after six months, the rest of the9cases recovered to normal life with2-37months follow-up,the longest survival time has been27months. Part two:1. The experimental rats began to die after patial liver resection from6hours, with peak in8-12hours. The survival rates were86.7%,80%and7%while liver resection volume was70%,80%,90%respectively after two weeks;2. ALT and AST level showed statistical differences between70%group with the others in8h,12h after surgery, no differences between80%and90%groups. There were statistical difference between70%group with80%in24h,72h postsurgically. ALT and AST returned to normal in 7days in each group;3. The residual liver volume was bigger in the80%resection group than in the70%group with statistical differences after14days. There were no differences in HE stain among the three groups. Part three:1. Positive rate of CD105was62.2%on the surface of BMSCs in P3; CD29was16.7%; CD31was12.0%; CD45was8%; CD11b was1.5%; CD44was1.2%;2. In situ hybridization technique showed only transplantation group of BMSCs expressed SrY;3.MRI showed the quantity of liver nodules in BMSCs transplantation group were more than simple model group(p<0.05).4. HE staining indicated8rats had mild fibrosis in simple model group.9rats had significantly fibrosis and1produced liver cancer in BMSC transplantation group. There was no fibrosis in blank control group.5. Immunohistochemistry showed a-SMA had higher expression in BMSCs transplantation group than simple model with statistical differences between two groups(p<0.05), none expressed in black control group,TLR3expressed in the cytoplasm of liver celll.6. Fibrosis percentage was higher in BMSCs transplantation group than simple model with significantly differences.7.a-SMA had higher expression in BMSCs transplantation group according to Western-blotting technique(p<0.05). TLR3, NF-kBp65, TLR3and IRF3mRNA were higher expressed in BMSCs transplantation group than orther two group (p<0.05),Simple model was more than black control group (p<0.05). There were no differences in TRIF among the three groups (p>0.05). Conclusions:1.ELRA can be considered feasible and practicable for those with difficulty in resection of end-staged AE with inferior vena cava invasion, the indications was the inclusion criteria of study. CT scan and3D technique could be used as an reliable prediction method to evaluate the relationship of lesion with tube, level of invasion and volume of graft, which improves operation safety. Operation can be safe and effective during anhepatic phase by integrated application of autologous or artificial blood vessel bypass, portocaval shunt temporarily, inferior vena cava reconstruction and ex-vivo liver resection vascular reconstruction technology. The liver graft regeneration occurred mainly in the half year after operation, especially in first month and then slow down in the2nd-6th months. it closes to the patient standard liver volume in six months after operation. ELRA has the huge economic and social benefits to cure AE and it is worthy of popularization and application at home and abroad.2.It was safe in rat with80%liver resection, which indicates over70%liver resection may be acceptable in human being. It has positive correlation between resection with hyperplasia.3.BMSCs promotes fibrosis in liver injury model caused by DEN even leads to cancer.4. Related protein of TLR3signaling pathways may be involved in chronic liver injury, liver regeneration and repair, BMSCs in the microenvironment of TLR3pathway activated might tend to hepatic stellate cells, myofibroblasts, TLR3and BMSCs have influence and interaction with each other., and ultimately lead to liver fibrosis after chronic liver injury.