ABSTRACT
End-stage liver disease refers to the advanced stage of liver disease caused by various acute and chronic liver damage, of which decompensated cirrhosis and liver failure are most common. Currently, liver transplantation is the most effective treatment option for patients with end-stage liver disease, but several limitations regarding transplantation currently restrict its application. Mesenchymal stem cells are the potential treatment option for end-stage liver disease because of the biological properties such as multidirectional differentiation, paracrine and immunomodulatory properties, as well as anti-inflammatory, anti-apoptotic and anti-fibrosis, but the therapeutic effect is affected by low homing rate, poor colonization and low conversion rate. The researchers aimed to improve them through genetic modification, chemical or hypoxic pretreatment, and three dimensional culture. This article reviewed and discussed the pretreatment methods, their application status of treatment for end-stage liver disease and the existing problems.
ABSTRACT
Objective:To explore the effect and possible mechanism of normal temperature mechanical perfusion(NMP)plus bone marrow mesenchymal stem cells(BMMSCs)on early endoplasmic reticulum stress(ERs)and cellular apoptosis after donation after cardiac death(DCD)donor liver transplantation.Methods:BMMSCs were isolated and cultured by adherence method.Sixty Sprague-Dawley(SD)rats were randomly divided into five groups of sham operation(sham), static cold storage(SCS), NMP, BMMSC and NMP plus BMMSCs(BP)( n=12 each). Liver tissue and serum sample of each group were harvested at Day 1/7 post-operation.Hematoxylin-eosin(HE)staining was employed for observing pathological changes of liver tissue; TdT-mediated dUTP nick end labeling(TUNNEL)staining for detecting cellular apoptosis; immunohistochemistry for detecting the expression of hepatocyte transcription factor C/EBP homologous protein(CHOP); Western blot for detecting the expressions of GRP-78, p-PERK, ATF4, CHOP and cleaved caspase-3. Results:Compared with SCS group, hepatic injury and inflammation significantly declined in NMP, BMMSC and BP groups and improvement of hepatic injury was the most pronounced in BP group.Cellular apoptosis lessened markedly in BP group at Day 1/7 as compared with SCS group and the difference was statistically significant.The expressions of ERs-related proteins GRP-78, p-PERK and ATF4 spiked in SCS group and the expressions of pro-apoptotic proteins CHOP and cleaved caspase-3 were significantly elevated and declined markedly in BP group.And the difference was statistically significant.Conclusions:BMMSCs plus NMP can significantly improve hepatocyte apoptosis and inflammatory response after DCD donor liver transplantation.And its mechanism may be correlated with suppressing early endoplasmic reticulum stress of hepatocytes.
ABSTRACT
Objective:To investigate the effect of heme oxygenase-1 (HO-1) modified bone marrow mesenchymal stem cells (BMMSCs) combined with normothermic machine perfusion (NMP) on the intestinal barrier function in rats with acute rejection of liver transplantation.Methods:Specific pathogen free 2 male Brown Norway (BN) rats (4-5 weeks, 40-60 g) were used to isolat BMMSCs, and HO-1 was infected by adenovirus. Of 24 male Lewis rats (7-8 weeks old, 200-220g) were used as donors, 30 male BN rats (8-9 weeks old, 220-240 g) were used as recipients. Acute rejection models of orthotopic liver transplantation were established in rats using two cuff technique. BN recipient rats were randomly divided into five groups: sham group, abdomen of the mice was open and closed within 30 min; NMP livers were simply mechanically perfused for 4 h; the BMP group were perfused with BMMSCs through the portal vein; the HBP group were perfused with HO-1/BMMSCs through the portal vein; the FK506 livers were mechanically perfused for 4 h and administered intragastrically of tacrolimus daily following surgery, 6 per group, on days 14 after surgery, the relevant indicators were taken and the rejection activity index (RAI) changes were investigated. The changes of intestinal pathological were analyzed by HE staining and transmission electron microscope, the expression levels of zonula occludens-1 (ZO-1) and occludin protein in intestinal tissue were detected by Western blotting, the concentrations of lipopolysaccharide, D-lactic acid and diamine oxidase (DAO) in serum were detected by ELISA.Results:The RAI of HBP group (2.80±0.84) and FK506 group (2.20±0.84) were significantly lower than that of NMP group (7.60±1.14) and BMP group (6.00±1.58), the differences were statistically significant (all P<0.05). The intestinal villi in NMP group were significantly sparse, wrinkled and disorderly arranged while the degree of intestinal injury in BMP group, HBP group and FK506 group were more mitigated. Electron microscope observation showed that the microvilli of intestinal epithelial cells in HBP group were rich and orderly, and the tight junction structure between cells was complete. The protein expression levels of ZO-1 and Occludin in the intestinal tissues of HBP group [(0.87±0.06) (1.28±0.26)] were higher than those of NMP group [(0.41±0.12) (0.27±0.18)] and FK506 group [(0.52±0.15) (0.63±0.22)], the differences were statistically significant (all P<0.05). The concentration of lipopolysaccharide, D-lactic acid and DAO in serum of HBP group was lower than those of NMP group and FK506 group, the differences were statistically significant (all P<0.05). Conclusion:HO-1/BMMSCs combined with NMP protects the intestinal mucosal barrier function of BN rats with acute rejection after liver transplantation.
ABSTRACT
Objective:To investigate the role of ferroptosis in bone marrow mesenchymal stem cells (BMMSCs) combine with normothermic machine perfusion (NMP) in repairing steatotic liver donor after cardiac death (DCD) in SD rats.Methods:BMMSCs were derived from SD rats to establish the DCD model of rats steatotic liver. A total of 24 rats were randomly divided into four groups: simple steatotic liver model group (Sham), static cold storage group (SCS), NMP, BMMSCs combine with NMP preservation group (BNMP), and the preservation time was 4 hours. The donor liver function was evaluated by liver structure, liver enzymes and lactic acid content of perfusion fluid, bile secretion and inflammatory cytokines; furthermore, in order to evaluate the occurrence of liver ferroptosis, the content of Fe 2+, malondialdehyde and glutathione (GSH) in liver tissue, as well as the mRNA or protein expression changes of cyclooxygenase-2 (COX-2), prostaglandin-endoperoxide synthase 2 (Ptgs2), glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) were detected. Results:After DCD steatotic donor liver was preserved for 4 hours, the liver injury, pro-inflammatory and anti-inflammatory cytokines expression in the BNMP and NMP groups were better than those in the SCS group. During the machine perfusion preservation period, alanine aminotransferase [(189.0±12.5)U/L vs. (227.7±16.2)U/L], aspartate aminotransferase [(207.3±18.6)U/L vs. (247.0±11.8)U/L] and lactic acid [(2.3±0.3)mmol/L vs. (2.9±0.2)mmol/L] in the BNMP group is lower than those in NMP group, moreover, the amount of hepatic bile secretion in the BNMP group [(1 245.7±46.8) μl vs. (1 014.3±67.9) μl] was more than that in NMP group, the difference was statistically significant (all P<0.05). The content of Fe 2+ and malondialdehyde in the liver tissue of BNMP group was significantly lower than those of SCS and NMP groups, on the contrary, the content of GSH was significantly higher than those of SCS and NMP groups. In addition, in the BNMP group, the mRNA level of Ptgs2 and protein level of COX-2 in the liver were significantly reduced, and expression of GPX4 and FTH1 were significantly higher than those of NMP and SCS groups, the differences were statistically significant (all P<0.05). Conclusion:BMMSCs combine with normothermic machine perfusion can better repair SD rats DCD steatotic donor liver and its mechanism of action may be related to its regulation on liver ferroptosis.
ABSTRACT
Ferroptosis is a new type of cell death proposed in recent years. It refers to the excessive accumulation of reactive oxygen species under iron dependence, which leads to fatal lipid peroxidation of cells and ultimately leads to cell death. Fatty liver is extremely sensitive to ischemia-reperfusion injury and poorly tolerated. It’s still not understood well, and the therapeutic options are limited for the fatty liver patients. The pathophysiological characteristics of ferroptosis and fatty liver ischemia-reperfusion injury are similar, and the discovery of ferroptosis may provide a new treatment strategy for fatty liver to prevent ischemia-reperfusion injury. This article mainly reviews the mechanism of ferroptosis and the targets related to ferroptosis in fatty liver ischemia-reperfusion injury.
ABSTRACT
Objective:To investigate the effect of heme oxygenase-1 (HO-1) modified rat bone marrow mesenchymal stem cells (BMSCs) on fibrotic rats.Methods:110 male SD rats aged 6-8 weeks were selected randomly divided into model group, BMSCs group and HO-1/BMSCs group with 11 rats in each group after intraperitoneal injection of CCl 4, PBS, BMSCs and HO-1/BMSCs were injected respectively. Another 11 rats were selected as control group. After 4 weeks of intervention, tracer experiment was used to detect the location of BMSCs. Rats in each group were executed, and liver function were detected by biochemical analyzer, liver fibrosis indexes were detected by ELISA, liver histopathology were detected by HE and Sirius red staining. Immunohistochemistry, Western blot and RT-PCR were used to detect the protein and mRNA expression of E-cadherin and Vimentin. Results:The rat fibrosis model was successfully established. Tracer experiment showed that BMSCs were implanted in rat liver after transplantation. Compared with the model group, the liver function and liver fibrosis indexes of BMSCs group and HO-1/BMSCs group were improved, and Ishak score and stage were significantly decreased, and HO-1/BMSCs group was superior to BMSCs group. The expression of E-cadherin in HO-1/BMSCs group (0.92±0.21), (0.84±0.03) were higher than those in BMSCs group [(0.54±0.16), (0.53±0.04)] and model group [(0.49±0.06), (0.11±0.06)] both at protein and mRNA level, while protein and mRNA level of Vimentin (1.21±0.23), (3.82±0.80) were lower than that in BMSCs group [(1.32±0.17), (6.39±0.75)] and model group [(1.41±0.18), (16.94±1.30)]. The difference was statistically significant ( P<0.05). Conclusion:HO-1/BMSCs can improve liver function and liver fibrosis in fibrotic rats more effectively than BMSCs alone. The mechanism was possibly through inhibiting liver epithelial mesenchymal transition.
ABSTRACT
Dendritic cells are the key cells to regulate immunity. Tolerogenic dendritic cells (Tol-DC) with immunosuppressive function play an important role in inducing and maintaining immune tolerance. In recent years, a class of liver Tol-DC with low level of presenting antigen and inhibition of T cell activation and proliferation has become a research hotspot to reduce the rejection of liver transplantation. At the same time, there are many strategies to induce phenotypic stable Tol-DC, which lays a foundation for its clinical application. This article introduces the current understanding of liver Tol-DC and its mechanism of regulating the immunity of liver transplantation, in order to provide new ideas for immunotherapy of liver transplantation.
ABSTRACT
Objective:To investigate the effects of heme oxygenase-1 (HO-1)-modified rat bone marrow mesenchymal stem cells (BMMSCs) on T lymphocyte subsets in cirrhotic rats.Methods:A rat model of liver cirrhosis was established by intraperitoneal injection of carbon tetrachloride (CCL 4) in 110 rats. These rats were randomly divided into three groups, model group, BMMSCs group and HO-1/BMMSCs group, and injected with PBS, BMMSCs and HO-1/BMMSCs through dorsal penile vein, respectively. Another 10 rats were selected as control group. All rats were executed four weeks after intervention. Pathological changes in liver tissues were observed with HE and Sirius red staining. Serum albumin (ALB) and alanine aminotransferase (ALT) were detected by biochemical analyzer. Serum hyaluronidase (HA) and collagen type Ⅳ (Ⅳ-C) were detected by ELISA. T lymphocyte subsets in peripheral blood and spleen were detected by flow cytometry. Results:The rat model of cirrhosis was successfully established. Compared with the model group and BMMSCs group, the HO-1/BMMSCs group had significantly lower Ishak score and disease stage ( P<0.05), increased serum ALB level and CD4 + T/CD8 + T cell ratio ( P<0.05), and decreased serum ALT, HA and Ⅳ-C levels and Th17/Treg ratio ( P<0.05). Conclusions:HO-1/BMMSCs could improve liver function and liver fibrosis in cirrhotic rats more effectively than BMMSCs alone. The mechanism was possibly through regulating the immunomodulatory function of T lymphocyte subsets in cirrhotic rats.
ABSTRACT
Objective:To explore the role of heme oxygenase-1 (HO-1) modified bone marrow mesenchymal stem cells (BMMSCs) in improving hepatic microcirculation after reduced-size liver transplantation (RLT) in rats.Methods:BMMSCs were isolated and cultured in vitro by adherence method. Then HO-1/adenovirus (Adv) was transfected for constructing HO-1/BMMSCs. The "dual-sleeve" method was employed for establishing an acute rejection model after 50% RLT. Immediately post-operation, 1 ml normal saline (NS) and BMMSCs or HO-1/BMMSCs single cell suspension were injected. The changes of surviving rats were observed by parameters at Day 3/7/14 post-operation. Five rats were observed at each timepoint. The serum level of mitochondrial aspartate aminotransferase (mAST) was detected; Na+ -K+ -ATPase in transplanted liver was measured by chemical colorimetry; mitochondrial ultrastructural changes were observed under a transmission electron microscope. Portal vein pressure was detected by Power Lab at Day 7 post-operation; the expressions of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and von Willebrand factor (vWF) in liver tissues were detected by Western blot. Liver histopathological changes were observed by hematoxylin & eosin stain. The expression of vWF was detected by immunohistochemistry and serum level of hyaluronic acid (HA) detected by enzyme-linked immunosorbent assay (ELISA).Results:HO-1/BMMSCs could significantly lessen the pathological injury and rejection of 50% reduced-size transplanted liver, improve mitochondrial damage and energy metabolism, promote the expression of eNOS, suppress the expression of iNOS, reduce portal pressure, up-regulate the expression of hepatic sinus vWF and HA degradation, protect hepatic sinusoidal endothelial cells (SECs) and ultimately improve hepatic microcirculation. And the differences were statistically significant as compared with NS/BMMSCs group ( P<0.05). Conclusions:HO-1/BMMSCs may play an important role in protecting rat liver by improving hepatic microcirculation during RLT.
ABSTRACT
Liver transplantation is the most effective therapeutic options for the patients at the advanced stage, but with the amount of transplant surgery increasing, margin donors are used for transplantation in the case of severe donor organ deficiency. However, the commonly used cold storage technique has poor preservation effect on margin donors, resulting in an increase in the incidence of complications after transplantation. The donated liver quality is one the most important factors for the patients long term survival, so there is an urgent need for a new type of organ preservation technology to preserve the margin donors in vitro. This paper summarized the current research on the ex-vivo preservation methods of liver and the new mechanical perfusion preservation methods.
ABSTRACT
Objective To study the effect of bone marrow mesenchymal stem cells (BMMSCs) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) after DCD donor liver transplantation in rats.Methods The third generation of BMMSCs and the BMMSCs modified by Ad/HO-1 (Ad/HO-1/BMMSCs) were cultured,identified and expanded in vitro.To establish a stable NMP system device in vitro.The DCD liver transplantation models were constructed in rats after cardiac ischemia for 30 minutes,220 SD recipient rats were randomly divided into sham operation group (S group,n=44) static cold storage (SCS group,n =44) group,and simple NMP group (P group,n =44),BMMSCs combined with NMP group (BP group,n =44) and BMMSCs modified by Ad/HO-1 combine with NMP group (HBP group,n =44),NMP group,BP group and HBP group were subjected to vitro perfusion for 4h.The group were taken at 0,1,7 and 14 days after transplantation and the relevant indicators were detected,n =6 in each group.The survival rate of the recipient rats,liver function and pathological changes of the bile duct were observed.The expression of cytokeratin 19 (CK19) protein in BEC was detected by immunohistochemistry and Western blot.Apoptotic biliary epithelial cells were detected by TUNEL staining and the expression of apoptosis-related protein caspase-3 was detected by immunohistochemistry.Results The survival time of HBP group was significantly prolonged for (5.6 ±0.8) d in SCS group vs.(18.4 ±2.0) d in NMP group,(20.5 ± 1.5) d in BP group,(82.5 ±3.2) d in HBP group,the differences were statistically significant (all P ＜ O.05).Compared with other groups,the HBP group and the BP group were significantly improved in liver function and biliary pathology,and the expression of CK19 protein in BEC was significantly increased [(0.81 ±0.02) in S group vs.(0.35 ±0.03) in SCS group,(0.47 ±0.02) in NMP group,(0.63 ± 0.02) in BP group,(0.77 ± 0.01) in HBP group on postoperative day (POD) 14],the differences were statistically significant (all P ＜ 0.05).The number of apoptosis and the expression of apoptosis-related protein caspase-3 in HBP group were significantly decreased [(10.0 ± 1.2) in S group vs.(57.3 ±5.5) in SCS group,(40.1 ±4.6) in NMP group,(32.0 ± 2.2) in BP group,(13.7 ± 3.1) in HBP group on POD 14],the difference was statistically significant (all P ＜ 0.05).Compared with the BP group,the protective effect of the HBP group was more obvious,and the difference was statistically significant (P ＜ 0.05).Conclusion By the method of the BMMSCs modified by Ad/HO-1 combined with NMP in vitro preservation of rat,DCD donor liver can significantly improve the effect of BEC on rats and the survival rate after liver transplantation.
ABSTRACT
Toll-like receptor 4 (TLR4) is a member of the toll-like receptor family and belongs to the family of pattern recognition receptors. The role of TLR4 signaling pathway in liver ischemia-reperfusion injury has been widely studied in recent years, and its control methods in inflammatory response is becoming the most important research hotspot. In this paper, the research progress of the molecules and their regulatory mechanisms involved with TLR4 signaling pathway in liver ischemia-reperfusion injury is reviewed, which act as a new foundation of clinical research to study the pathogenic mechanisms and treatment plan.
ABSTRACT
Objective@#To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation.@*Methods@#The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples.@*Results@#The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP.@*Conclusion@#Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.
ABSTRACT
Objective To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation. Methods The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples. Results The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP. Conclusion Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.
ABSTRACT
Objective To review the development of adult and pediatric liver transplantation in Tianjin First Center Hospital, and to enhance academic exchanges, improve technological innovation, and jointly promote the progress and maturity in the field of liver transplantation. Methods The development of liver transplantation in Tianjin First Center Hospital was analyzed. The clinical data of adult and pediatric liver transplantation from September 1998 to September 2018 were collected. The important events and technological innovation achievements of liver transplantation during the 20 years were summarized. Results The first clinical liver transplantation was attempted in Tianjin First Central Hospital in April 1980. The first long-term survival adult liver transplantation in China was completed in 1994 (11 years survival after the operation). The specialized team of liver transplantation was formally established in September 1998. The 20-year clinical exploration and progress reflected the characteristics of era changes and technological innovation during the rapid development of liver transplantation in China. Our center performed liver re-transplantation in January 1999, reduced-size pediatric liver transplantation in August 2000. In May 2001, we organized the formulation for the preventive and treatment plan for hepatitis B recurrence after liver transplantation. We performed combined liver and kidney transplantation in July 2002, split liver transplantation (SLT) in April 2004, the first domino liver transplantation (DLT) in August 2005. Pediatric living donor liver transplantation (LDLT) was initiated in October 2006, adult LDLT was carried out in August 2007. In September 2007, the first living donor combined liver and kidney transplantation from the same donor in Asia was performed. The first domino+living donor double grafts liver transplantation in the world was performed in January 2009. In March 2011, we performed laparoscopically assisted right hepatic lobe liver transplantation (LDLT) with middle hepatic vein. In May 2014, living donor laparoscopic left lateral lobe procurement was successfully established. In April 2016, simultaneous liver, pancreas and kidney multi-organ transplantation was completed. Domino donor-auxiliary liver transplantation was performed in February 2017. In December 2017, extracorporeal membrane oxygenation (ECMO)-supported liver transplantation in a patient with severe pulmonary hypertension was successfully completed. Liver transplantation combined with partial splenectomy was established in April 2018. Cross-domino liver transplantation (hypersensitive kidney transplantation with auxiliary liver transplantation+pediatric liver transplantation) was performed in May 2018. During the 20 years, the team has performed or assisted other centers in Beijing, Shanghai, Guangzhou and Shenzhen to carry out more than 10 000 cases of liver transplantations. A total of 7 043 cases of various types of liver transplantation were performed in the single center of the hospital (6 005 adult liver transplantations and 1 038 pediatric liver transplantations). Concerning adult liver transplantation, the cumulative 1-year, 3-year and 5-year survival rate from September 1998 to March 2003 were 83.1%, 73.0% and 69.0%, from April 2003 to March 2009 were 85.3%, 76.2% and 72.1% and from April 2009 to September 2018 were 87.5%, 79.2% and 75.1%, respectively. The cumulative 1-year, 3-year and 5-year survival rate for pediatric liver transplantation were 93.5%, 92.2% and 90.2%, respectively. The nucleoside (acid) analogue combined with low dose hepatitis B immunoglobulin (HBIG) was developed to prevent the recurrence of hepatitis B after liver transplantation, this plan has reduced the recurrence rate of hepatitis B and the 5-year re-infection rate of hepatitis B virus (HBV) after liver transplantation significantly. The risk assessment system for tumor recurrence after liver transplantation was established and individual treatment method was established based on this assessment system. Continuous exploration and improvement of liver transplantation for liver cancer, liver re-transplantation, liver transplantation with portal vein thrombosis, SLT, DLT and multi-organ combined transplantation have significantly improved the clinical efficacy of patients and the post-operative survival rate. Conclusions The liver transplantation team of Tianjin First Center Hospital has carried out a scientific and technological exploration on the key problems and technical difficulties of clinical liver transplantation. This work strongly has initiated and promoted the rapid development of liver transplantation in China. The restrictive barrier of hepatitis B recurrence after liver transplantation has been overcome. The risk prevention and control system of tumor recurrence after liver transplantation has been established. A series of innovative achievements that can be popularized have been achieved in the field of complex liver transplantation and expansion of donor liver source. The iterative progress and sustainable development of liver transplantation have been realized.
ABSTRACT
Objective To explore the effect and mechanism of heme oxygenase-1 (HO-1) gene modified bone marrow mesenchymal stem cells (BMMSCs) on rat reduced-size liver transplantation.Methods 50 male Brown Norway (BN) rats were used to prepare BMMSCs.Male Lewis and BN rats were 75,respectively.BN rats were randomly divided into model group (n =25),stem cell group (n =25) and combined group (n =25).Acute rejection models following 50％ reduced-size transplantaton were established in rats using two-cuff technique,1 ml of normal saline,BMMSCs suspension,or HO-1/BMMSCs suspension were injected immediately after surgery.Rats were executed at an instant,3rd,7th and 14th day after surgery to identify BMMSCs and HO-1/adenovirus infection efficiency.Evaluated hepatic pathology by HE staining.Liver function indexes were detected.Portal vein pressure on 7th day after surgery was detected.The levels of endothelin-1 (ET-1) and nitric oxide (NO) in serum were detected using ELISA.The expressions of ET-1 in liver were detected by immunohistochemistry staining and Western blotting.Results High purity BMMSCs were obtained and HO-1/BMMSCs were successfully infected.Compared with model group,liver tissue injury and rejection were alleviated in stem cell group and combined group,liver function was improved,and the combined group was superior to stem cell group.The portal vein pressure in model group,stem cell group,and combined group were 21.3±0.2 mmHg,11.2±0.2 mmHg,and 10.1±0.1 mmHg,respectively.The portal vein pressure in three groups showed a decreasing trend,difference was statistically significant (P＜0.05).On the 3rd,7th and 14th day after surgery,compared with model group,the expression levels of ET-1 and NO in the stem cell group and the combined group were decreased,and the combined group was significantly lower than stem cell group (P＜ 0.05).Conclusion HO-1/BMMSCs improved liver function and portal vein pressure after reduced-size liver transplantation in rats,and may play a protective role by regulating ET-1/NO expression.
ABSTRACT
Objective To establish a stable model of reduced-size liver transplantations with rejection in rats.Methods Inbred adult male Lewis rats (weighing 220-250 g,8-10 weeks old) served as donors in liver transplantation,and inbred adult male Brown Norway (BN) rats (weighing 220-250 g,8-10 weeks old) served as recipients.Orthotopic group,50％ reduced-size liver transplantation rejection group (50％ group) and 30％ reduced-size liver transplantation rejection group (30％ group)were developed.The 1-,3-,7-and 14-survival rate was observed.Besides,the levels of ALT,AST and TBIL in the peripheral blood plasma were determined.The liver tissues were obtained,and HE staining was used to examine the pathological changes and rejection severity of the liver specimens.Results No significant difference in the operation-related indicators was found among the three groups.The 1-,3-,7-and 14-day survival rate in orthotopic group was as follows:91.20％,86.50％,81.10％ and 75.70％;85.00％,80.00％,62.50％ and 45.00％;65.00％,50.00％,35.00％ and 17.50％,respectively.The 1-,3-,7-and 14-survival rate in 30％ group was significantly lower than in other groups (P＜0.05),and showed no statistical difference between 50％ group and orthotopic group (P＞0.05).The ALT,AST and TBIL levels were significantly higher in 30％ group than in control group at all time points (P＜0.05),but there was no statistical difference between 50％ group and orthotopic group (P ＞ 0.05).All groups were the models of liver transplantations with rejection in rats,and rejection activity index score was increased with time delay.Conclusion Lewis→BN rats are chosen to establish liver transplant rejection model,and by comparing the survival rate and pathological changes among the groups,finally 50％ group is selected to be the model for a follow-up study.
ABSTRACT
Objective To study the repairing effect of bone marrow mesenchymal stem cells (BMMSCs) on the donor liver after cardiac death (DCD) under normal temperature mechanical perfusion (NMP) in rats.Methods BMMSCs of Wistar rats were cultured in vitro,and 45-min warm ischemia after cardiac death model was established.The 30 Wistar rats were randomly divided into NMP,NMP + BMMSCs (N + B),cold storage (CS) groups,and the parameters were detected at 2 h and 4 h (n =10).Results N + B group was superior to NMP group and CS group in repairing the liver function and liver pathology including ultrastructure,improving the perfusate acidic environment,and increasing adenosine triphosphate level (P ＜ 0.05).The oxygen consumption of NMP group and N + B group were significantly different after 2h [2 h:(24.35 ±0.64) ml/min vs.(29.33 ±0.47) ml/min;3 h:(25.33 ±0.86) ml/min vs.(30.34 ± 0.49) ml/min;4 h:(26.88 ± 1.07) ml/min vs.(31.76 ± 0.96) ml/min;P ＜ 0.05],suggesting that the liver condition in N + B group was significantly better than that in the other two groups.Conclusion Bone marrow mesenchymal stem cells could obviously repair the DCD grafts under normal temperature mechanical perfusion.
ABSTRACT
Objective To investigate the in vitro effects of bone marrow mesenchymal stem cells (BM MSCs) on the replication of HBV with the participation of lymphocytes and to analyze the possible mechanism.Methods The HBV genomic DNA transfected HepG2.2.15 cell line was used to evaluate the HBV replication.Bone marrow and spleen samples were collected from BN rats for the isolation of BM MSCs and T lymphocyte cells, respectively.Five groups of co-culturing with different cells were designed in this study.The cellular activities of lymphocytes and HepG2.2.15 cells were detected at the time of 24 h, 48 h and 72 h after co-culturing by using MTT method.The levels of HBV DNA and HBV cccDNA were detected by real-time polymerase chain reaction ( PCR) .T cell subsets in co-culture were measured by using fluores-cence labeled antibodies and flow cytometry analysis.ELISA was used to detect the levels of cytokines in the supernatant of cultured cells.Results Compared with HepG2.2.15 cells group, BM MSCs+HepG2.2.15 cells and splenic lymphocytes+HepG2.2.15 cells co-culture groups, the levels of HBV DNA and HBV cccDNA were significantly decreased in splenic lymphocytes+BM MSCs+HepG2.2.15 cells co-culture group after 48 h of culture [ HBV DNA: ( 181.000 ±14.731 ) IU/ml vs ( 6270.000 ±300.450 ) IU/ml, (2564.000±231.058) IU/ml, (2433.300±302.379) IU/ml;HBV cccDNA: (4.330×105 ±0.464×105 ) IU/ml vs (11.100×105±0.375×105) IU/ml, (8.930×105±0.778×105) IU/ml, (9.850×105±0.810× 105) IU/ml;P<0.01].The secretion of IFN-γin the supernatant of co-cultured cells was negatively corre-lated with HBV DNA level, but the levels of IL-10 and IL-22 were positively correlated with HBV DNA.The ratio of CD4+/CD8+cells was increased in splenic lymphocytes+BM MSCs+HepG2.2.15 cells co-culture group.The percentage of CD8+cells showed a positive correlation with HBV DNA.Conclusion BM MSCs could inhibit the expression of HBV DNA to enhance the clearance of HBV strains.It might be possibly due to rebalancing of Tc1/Tc2 cells and regulating the expression of autocrine agents and cytokines.
ABSTRACT
Objective To establish method of constructing lentiviral vectors to express microRNA (miRNA) ''tough decoy''(TuD)and to detect the effects of the TuD on cellular endogenous miRNA level and cellular phenotypes. Methods Two-step cloning strategy was utilized to first generate a universal miRNA TuD frame vector,followed by con-structing the TuD expression vector specially targeting miR-203. The package of the recombinant lentivirus was per-formed in 293T cells. Then the rat bone marrow mesenchymal stem cells(BM-MSCs)were infected by the miR-203 TuD expression lentivirus. The pSIH1-H1-copGFP vector was also packaged and the BM-MSCs infected by this lentivirus were served as control. Endogenous miR-203 level in BM-MSCs was measured by quantitative RT-PCR,and cellular vi-ability and apoptosis were detected by CCK-8 test and Annexin V-PI staining respectively. Results The miR-203 TuD expression vector was successfully constructed and inserted sequence was validated. At the 3rd,6th and 9th days after in-fected by the miR-203 TuD expression lentivirus,rat BM-MSCs exhibited a repressed endogenous miR-203 level. The miR-203 TuD also promoted viability and inhibited apoptosis of BM-MSCs. All these differences between miR-203 TuD group and control group were statistically significant. Conclusion The two-step cloning method for the construction of miRNA TuD expression vector is simple and efficient. The miRNA TuD can effectively suppress the level of the target miRNA and affect cellular phenotypes.