Objective To explore the most effective route of mesenchymal stem cell transplantation (MSCs) in D-galactosamine (D-gal) induced porcine model with acute liver failure (ALF) and the potential mechanism.Methods BA-MA mini-pigs with D-gal-induced ALF were transplanted with porcine mesenchymal stem cells (MSCs) through four routes:intraportal injection (InP),peripheral intravenous injection (PV),hepatic intra-arterial injection (AH) and intrahepatic injection (IH).The survival time was recorded.The blood samples before and after MSC transplantation were collected for detecting liver function.Liver histology was interpreted and scored.Hepatic apoptosis and regeneration were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay and Ki-67 assay.The protein expression of cleaved caspase-3,survivin,AKT and ERK were analyzed by Western blot.Results The average survival time in each group was (10.7 ±1.6) days (InP),(6.0 ±0.9) days (AH),(4.7 ±1.4) days (PV),(4.3 ± 0.8) days (IH) when compared with D-gal group [(3.8 ± 0.8) days].The histopathological scores revealed a significantly decrease in InP group (3.17 ± 1.04,P ＜0.05) and AH group (8.17 ± 0.76,P ＜ 0.05) when compared with that in D-gal group (11.50 ± 1.32).The apoptosis rate in InP group (25.0 ± 3.4％,P ＜ 0.05) and AH group (40.5 ± 1.0％,P ＜ 0.05) was lower than that in D-gal group (70.6 ± 8.5％).The expression of active caspase-3 was inhibited,while the expression of survivin,AKT and ERK was elevated in InP group.Conclusions The intraportal injection was superior to other pathways for MSCs transplantation.Intraportal MSC transplantation could improve liver function,inhibit cell apoptosis,promote cell proliferation and prolong the survival in porcine ALF model.

ObjectiveTo study the effects of membrane molecular weight cut off on a novel bioartificial liver(BAL) system.MethodsHealthy beagles underwent 6-hour treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating.The functional changes and cell viability were characterized.ResultsHepatocyte performance levels such as albumin secretion,urea synthesis and viability were significantly higher in the 200 kDa retention rating group when compared with the 1200 kDa retention rating group (P＜0.05).Significant levels of canine proteins were detected in the BAL medium from the 1200 kDa retention rating group.Fluorescence microscopy further verified that heavy deposition of canine IgG,IgM and complement (C3) on co-culture cells were obtained after BAL treatment in the 1200 kDa retention rating group.ConclusionsSmall membrane molecular weight cut off of BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improved the performance of the BAL.

Objective To investigate the safety and therapeutic effects ot a novel multi-layer flat-plate bioartificial liver (BAL) for patients with liver failure.Methods Thirty-eight patients with liver failure from Dec.2010 to Dec.2011 were treated with a novel BAL based on multi-layer flatplate bioreactor and the co-cultured cells of the porcine hepatocytes and mesenchymal stem cells.A total of 48 treatments was performed,4 h each time.The clinical signs and symptoms,liver function,ammonia,coagulation function and complete blood count were evaluated before,during and after the treatment.DNA in the collected PBMCs was extracted for PCR with PERV specific primers and the porcine specific primer Sus scrofa cytochrome B.The RT activity was detected as well.Levels of xenoantibodies (IgG,IgM) were determined by using ELISA kit. Titers of complement were quantified by CH50 kit.Results All treatment procedures were completed successfully without any adverse reaction. All samples presented negative PERV DNA and RT activity. The levels of antibodies were similar before and after treatment.Treatment was associated with a temporary decline in levels of complement,and then the levels were recovered quickly.The clinical symptoms such as acratia,anorexia and abdominal distension were improved.The stage of hepatic encephalopathy in 16 patients was decreased. The liver function and ammonia was reduced disproportionately. Seven patients in all were bridged to liver transplantation,2 patients died and 2 patients gave up the treatment,and the others were turned better.After the outcome judgment according to the standard developed by the Artificial Liver Group,and Chinese Association of Infectious and Parasitic Diseases,there were 9 patients with clinical healing,25 patients with improvement and 4 patients with no effect,and the cure-improvement rate was 89.5％.Conclusion The novel multi-layer flat-plate BAL could be used as a safe and effective therapy for patients with liver failure.

Objective To investigate the influence of membrane molecular weight cut off in our bioartificial liver(BAL)system.Methods Beagle dogs were used for a model of acute liver failure through D-galactosamine administration.The acute liver failure Beagles were divided into two groups by the membrane molecular weight cut off.Group A was treated with BAL containing 200 kDa retention rating membrane.Group B was treated with BAL containing 1200 kDa retention rating membrane.Each group underwent two six-hour BAL treatments that were performed on day 1 and day 21.BAL medium were examined and levels of IgG,IgM,and complement hemolytic unit of 50％(CH50)antibodies were measured in all Beagles and.Results BAL treatment was associated with a significant decline in levels of CH50.1200 kDa group experienced a significant increase in levels of IgG and IgM after two BAL treatments.Significant levels of canine proteins were detected in BAL medium from 1200 kDa group.Conclusions Xenogeneic immune response in the BAL system was influenced by membrane molecular weight cut off.

Objective To study the mutation of FLCN gene in Chinese patients with sporadic and familial primary spontaneous pneumothorax. Methods A complete genetic analysis of FLCN by use of SSCP-PCR was performed in 102 unrelated Chinese patients with isolated PSP. Results Three novel mutations (c. 924_926del, c. 1611_1631del and c. 1740C.T) and a previously reported mutation (c. 1733insC) were identified in five familial and five sporadic PSP patients. Of the 21 family members of patients with PSP including 3 previous considered as sporadic, 4 ( 19% ) had history of at least one episode of PSP and 9 (43% ) were FLCN mutant carriers without PSP. Seven of the nine (78% ) mutant carriers had pulmonary cysts detected by high-resolution computed tomography ( HRCT) . Although c. 924_926del and c. 1611 _1631 del were found in eight patients from the same geographic district, haplotype analysis demonstrated that they did not share the same affected haplotype,thus excluding common ancestry. Conclusion This study first demonstrates that FLCN mutation contributes to not only familial but also apparently sporadic' patients with isolated PSP. It suggests that mutation analysis and HRCT scan may be recommended for first-degree family members of PSP patients with FLCN mutations, irrespective of their family history status of PSP.

Objective To investigate the clinical value of preoperative nutritional support (PNS) therapy in the hepatectomy of patients with nutritional risk.Methods The prospective study was conducted.The clinical data of 133 patients with nutritional risk who were admitted to the Drum Tower Hospital Affiliated to Nanjing University Medical School from August 2012 to June 2016 were collected.All the patients undergoing PNS and traditional therapy were divided into the PNS group and the control group by random number table method,respectively.Observation indicators:(1) comparisons of laboratory indexes between groups;(2) comparisons of postoperative situations between groups;(3) comparisons of postoperative complications between groups.Measurement data with normal distribution were represented as-x±s.Comparisons between groups were evaluated with the independent-sample t test.Comparisons of count data were analyzed using the chi-square test,and repeated measures data were analyzed by the repeated measures ANOVA.Results All the 133 patients were screened for eligibility,including 68 in the PNS group and 65 in the control group.(1) Comparisons of laboratory indexes between groups:alanine transaminase (ALT),aspartate transaminase (AST),total bilirubin (TBil),cholinesterase,albumin (Alb),prealbumin,transferrin and C-reactive protein (CRP) in the PNS group were respectively (36± 13) U/L,(29± 10) U/L,(18.5±2.4) mmol/L,(5 738± 1 824) U/L,(37.4±5.1) g/L,(155±48) mg/L,(2.2±0.5)g/L,(10±4) g/L at admission and (33 ± 9) U/L,(27 ± 8) U/L,(17.9± 1.8) mmol/L,(5 796± 2 016) U/L,(38.5 ± 4.7) g/L,(181 ± 40) mg/L,(2.4± 0.5) g/L,(8± 4) g/L before operation and (285±100)U/L,(218±93)U/L,(33.5±6.3)mmol/L,(4 847±1 044)U/L,(32.6±3.8)g/L,(105±34)mg/L,(1.3±0.4) g/L,(55±28) g/L at 1 day postoperatively and (149±84) U/L,(76±42) U/L,(22.7±4.9) mmol/L,(3 866±893) U/L,(34.2±2.4) g/L,(125±30) mg/L,(1.6±0.4) g/L,(51±34) g/L at 3 days postoperatively and (64±33) U/L,(44±18) U/L,(19.4±2.8) mmol/L,(4 257± 1 032) U/L,(37.0±2.1) g/L,(148±42) mg/L,(1.9±0.4)g/L,(16±11)g/L at 7 days postoperatively;ALT,AST,TBil,cholinesterase,Alb,prealbumin,transferrin and CRP in the control group were respectively (36± 15)U/L,(31± 12)U/L,(18.3±2.9)mmol/L,(5 762±1 693)U/L,(37.3±6.1)g/L,(162±51)mg/L,(2.3±0.5)g/L,(10±4)g/L at admission and (36±11)U/L,(30±11)U/L,(18.2±2.8)mmol/L,(5 789±1 673)U/L,(37.8±7.1)g/L,(166±57) mg/L,(2.3±0.6) g/L,(9±5) g/L before operation and (305±127) U/L,(246± 104) U/L,(34.2±7.8) mmol/L,(4 842±1 173)U/L,(32.0±4.1) g/L,(83±32) mg/L,(1.2±0.4) g/L,(61 ±31) g/L at 1 day postoperatively and (163±104)U/L,(82±62)U/L,(23.1±6.0)mmol/L,(3 672±937) U/L,(33.8±3.6) g/L,(106±30)mg/L,(1.4±0.4)g/L,(61±40)g/L at 3 days postoperatively and (77±48) U/L,(52±27) U/L,(20.2±3.5) mmol/L,(3 925±987) U/L,(36.6±2.8) g/L,(125±40) mg/L,(1.7±0.4) g/L,(22± 12) g/L at 7 days postoperatively,showing no statistically significant difference in changing trends of above indicators between groups (F =1.007,2.223,0.579,0.014,0.235,3.533,2.970,2.143,P＞0.05).Results of further analysis showed that there were statistically significant differences in the levels of ALT,AST and cholinesterase at 7 days postoperatively between groups (t=1.832,2.073,1.899,P＜0.05),and in the levels of prealbumin before operation and at 1,3 and 7 days postoperatively between groups (t =1.698,3.738,3.625,3.178,P＜0.05) and in the levels of transferrin and CRP at 3 and 7 days postoperatively between groups (t=2.917,2.709,1.667,2.990,P＜0.05).(2) Comparisons of postoperative situations between groups:time to initial exsufflation,time of initial defecation,infused volume of exogenous albumin and duration of postoperative hospital stay were respectively (46± 15)hours,(64±16)hours,(23±10)g,(9.2±2.6)days in the PNS group and (55±18)hours,(78±21)hours,(39±25)g,(11.7±5.3) days,with statistically significant differences in the above indicators between groups (t =2.830,4.157,5.044,3.497,P＜0.05).(3) Comparisons of postoperative complications between groups:23 and 33 patients in the PNS and control groups had postoperative complications,showing a statistically significant difference between groups (x2=3.915,P＜0.05).Eight and 17 patients in the PNS and control groups were respectively complicated with peritoneal effusion,with a statistically significant difference between groups (x2 =4.508,P＜ 0.05).Conclusion PNS therapy in the hepatectomy of patients with nutrition risk can effectively improve pre-and post-operative nutrition statuses,reduce liver damage,accelerate recoveries of liver and gastrointestinal functions,reduce complications,shorten duration of postoperative hospital stay and accelerate patients' recovery.

OBJECTIVE: To investigate the efficacy of a novel artificial perfusate based on oxygen-carrying perfluoronaphthalene-albumin nanoparticles in normothermic machine perfusion (NMP) for preservation of porcine liver donation after cardiac death. METHODS: Artificial perfusate with perfluoronaphthalene-albumin nanoparticles was prepared at 5% albumin (w/v) and its oxygen carrying capacity was calculated. The livers of 16 Landrace pigs were isolated after 1 h of warm ischemia, and then they were divided into 4 groups and preserved continuously for 24 h with different preservation methods: cold preservation with UW solution (SCS group), NMP preservation by whole blood (blood NMP group), NMP preservation by artificial perfusate without nanoparticles (non-nanoparticles NMP group) and NMP preservation by artificial perfusate containing nanoparticles (nanoparticles NMP group). Hemodynamics, tissue metabolism, biochemical indices of perfusate and bile were monitored every 4 h after the beginning of NMP. Liver tissue samples were collected for histological examination (HE and TUNEL staining) before preservation, 12 h and 24 h after preservation. RESULTS: The oxygen carrying capacity of nanoparticles in 100 mL artificial perfusate was 6.94 μL/mmHg (1 mmHg=0.133 kPa). The hepatic artery and portal vein resistance of nanoparticles NMP group and blood NMP group remained stable during perfusion, and the vascular resistance of nanoparticles NMP group was lower than that of blood NMP group. The concentration of lactic acid in the perfusate decreased to the normal range within 8 h in both nanoparticles NMP group and blood NMP group. There were no significant differences in accumulated bile production, alanine aminotransferase and aspartate aminotransferase in perfusate between nanoparticles NMP group and blood NMP group (all P>0.05). After 24 h perfusion, the histological Suzuki score in blood NMP group and nanoparticles NMP group was lower than that in SCS group and non-nanoparticles NMP group (all P<0.05), and the quantities of TUNEL staining positive cells in blood NMP group and non-nanoparticles NMP group was higher than those in nanoparticles NMP group and SCS group 12 h and 24 h after preservation (all P<0.05). CONCLUSION Artificial perfusate based on oxygen-carrying nanoparticles can meet the oxygen supply requirements of porcine livers donation after cardiac death during NMP preservation, and it may has superiorities in improving tissue microcirculation and alleviating ischemia-reperfusion injury.
Swine ; Animals ; Liver Transplantation ; Organ Preservation ; Liver ; Perfusion ; Death ; Oxygen/metabolism*