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Objective:To analyze the anti effect of chimeric antigen receptor (CAR)-T cells targeting hepatitis B surface antigen (HBsAg) on hepatocellular carcinoma cells.Methods:HBsAg-CAR gene was transduced into T cells (obtained from the blood of healthy donors) through a lentiviral vector. CD19-CAR-T cells were included as mock group, and untransduced T cells were included as control group. Cells of the three groups were co-cultured with hepatocellular carcinoma cells expressing HBsAg or not to detect the anti effect and releasing level of anti-tumor cytokines (tumor necrosis factor-α, interferon-γ, interleukin-2). Subcutaneous xenograft PLC/PRF/5 tumor model using NPG mice were established and HBsAg-CAR-T cells (experimental group, n=5) or untransfected T cells (control group, n=5) were injected through tail vein. Tumor volume was measured 15 days after injection. Results:HBsAg-CAR-T cells proliferation was good under in vitro culture, and the expression rate of CAR was stable. After co-cultured with hepatocellular carcinoma cells expressing HBsAg, the level of anti-tumor cytokines released by HBsAg-CAR-T cells was significantly higher than that of the other two groups of T cells, and the difference was statistically significant (all P<0.05); the anti rate of HBsAg-CAR-T cell group on HBsAg-positive hepatocellular carcinoma cells was significantly higher than that of the other two groups, and the difference was statistically significant (all P<0.05). The tumor volume of NPG mice in the experimental group was (250.8±62.8) mm 3, which was lower than that of the control group (757.5±102.6) mm 3, and the difference was statistically significant ( P<0.05). Conclusion:HBsAg-CAR-T cells can specifically recognize and kill HBsAg-positive hepatocellular carcinoma cells and release high level of anti-tumor cytokines.
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Objective To study the clinical application of indocyanine green fluorescence fusion imaging (FIGFI) in anatomical hepatectomy.Methods The clinical data of 41 patients who underwent anatomical hepatectomy with guidance of FIGFI from March to June 2017 in our department were analyzed retrospectively.The data included:(l) Intraoperative data:surgical procedure,extent of hepatectomy,ICG fluorescent staining procedure and result,operation time,intraoperative blood loss and intraoperative blood transfusion.(2) Postoperative data:postoperative complications and pathology.Results Of the 35 patients who underwent laparoscopic anatomical hepatectomy,34 patients were successfully carried out under FIGFI guidance.One patient was converted to laparotomy.For the 6 patients who underwent laparotomy,liver resections were successfully carried out.Of all the 41 patients,37 had successful staining but 4 failed.Staining failure mostly occurred in patients who underwent anti-staining in liver segments with multiple vascular branching supply because not all the target liver pedicles were blocked before injecting ICG.Conclusions FIGFI guided anatomical hepatectomy is a very promising technique.The combination of preoperative imagings,intraoperative laparoscopic ultrasound guidance and FIGFI helped to achieve the goal of anatomical liver resection.
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Objective To detect the protective effect of extracorporeal membrane oxygenation (ECMO) on Maastricht type Ⅱ donation after cardiac death (DCD) liver transplantation in pigs.Methods Twenty mini-pigs were randomized into ECMO group (n =10) and control group (n =10).Then 10 pigs in each group were randomized into donors and recipients.Maastricht type Ⅱ DCD model was induced in all of the 10 donors.Donors of ECMO group received 2-h ECMO after cardiac death,then underwent liver graft procurement.The donors of control group underwent liver procurement directly after cardiac death.Recipients of two groups underwent orthotopic liver transplantation without venovenous bypass.During this procedure,vital signs were monitored continuously,lactate and liver biochemistry were tested,and 5-day survival rate was observed.Results Maastricht type Ⅱ DCD model was successfully built in all of the donors with consequent dark liver.For donors of ECMO group,liver turned sanguinous and soft quickly after treatment of ECMO.There were no significant differences in operation time,anhepatic time and anhepatic hemodynamic changes between these two groups (P ＞ 0.05).As compared with control group,ECMO group had better hemodynamic parameters 30 min after reperfusion,lower lactate,ALT and AST levels 30 min after reperfusion and before closing the abdomen,and higher 5-day survival rate (P ＜ 0.05).Conclusion ECMO may improve the quality of Maastricht type Ⅱ DCD liver graft,and increase the survival rate of DCD liver transplantation.
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Objective To study the repair mechanism of extracorporeal membrane oxygenation on liver after cardiac death.Methods Twelve pigs were equally randomized to ECMO group and control group.Cardiac arrest was induced by administration of 1 g KCL intravenously,followed by 30 min cardiopulmonary resuscitation according to the standard guideline Cannulas were placed through inferior vena cava and abdominal aorta,then connected to ECMO extracorporeal circulation pipes in ECMO group for 4 h.The livers were stored in cold UW for 4 h in control group.ATP,superoxide dismutase (SOD),glutathionein (GSH),malondialdehyde (MDA),heat shock protein 70 (HSP70) and intercellular cell adhesion molecule-1 (ICAM-1) were detected in liver tissue.Pathological change was observed by optical microscope and electron microscopy.Results Tissue ATP decreased to less than 40％ of baseline after 30 min of warm ischemia,then restored to 70％ after 2 h of ECMO and returned to baseline after 4 h,while ATP of control group continued a further decline As compared with control group,SOD,GSH and HSP70 increased significantly in ECMO group (P＜0.05),while MDA and ICAM-1 decreased significantly (P＜0.05).Pathological changes of liver tissue observed by optical microscope and electron microscopy in ECMO group were significantly were significantly alleviated as compared with those in control group.Conclusion ECMO can supply oxygen and nutrients to liver after warm ischemia and increase energy reserve.By upregulating GSH,SOD and HSP-70 and other protective proteins,ECMO alleviates oxidative stress and liver damage ECMO also improves microcirculation and reduces neutrophil infiltration by protecting sinusoidal endothelial cells.
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Objective To summarize the experience of establishing a split liver transplantation pig model using extracorporeal normothermic machine perfusion (NMP).Methods Twenty miniature pigs were purchased with ten as donors and another ten as receptors.The graft was spliced along Taira line and the right half was reserved for transplantation.Hemodynamics and bile production volume were monitored,and blood biochemical and blood gas analysis indicators were detected during machine perfusion.Pathological change was observed by HE stain.Hemodynamics during liver transplantation,5-day survival rate and the cause of death were recorded.Results Hemodynamic,biochemical and blood gas analysis indicators remained stable during NMP.All receptor pigs were successfully extubated and awake after surgery.Two receptors died on the second day after the operation.The 5-day survival rate was 80％.Conclusion The split liver transplantation pig model using extracorporeal normothermic machine perfusion is feasible and appropriate,and it lays the foundation for further investigation.
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Objective To investigate the repair and protective effect of extracorporeal membrane oxygenation (ECMO) on liver and bile duct after cardiac death in pig.Methods Eight pigs were purchased and cardiac arrest was induced by the administration of 1 g KCL intravenously,followed by 30 min cardiopulmonary resuscitation according to standard guideline.Cannulas were placed through inferior vena cava and abdominal aorta,and then connected to ECMO extracorporeal circulation pipes.ECMO was performed for 4 h.Circulation flow rate of hepatic artery and bile production were monitored and recorded.Lactate dehydrogenase (LDH),γ-glutamyl transferase (γ-GT) and direct bilirubin (DBIL) in bile were detected.Transaminase,tumor necrosis factor-α (TNF-α),interleukin-1β (IL-13),hyaluronic acid (HA),endothelin-1 (ET-1) and nitric oxide (NO) in serum were detected.Pathological change was observed by HE staining under optical microscope and cell apoptosis was detected by TUNEL.Results There was no bile production after cardiac death,which increased to 80％ of the baseline after 4h of ECMO.In addition,γ-GT,LDH and DBIL content in bile was (23.3 ± 11.8) IU/L,(15.9 ± 3.3) IU/L and (72.3 ± 21.4) mmol/ L,and IL-1,TNF-α and HA content in serum was (117.6 ± 39.0) ng/L,(120.4 ± 16.5) ng/L and (63.7 ± 4.4) ng/L,respectively,and no statistically significant differences were observed when compared with the baseline (all P ＞ 0.05).ET-1 content was (4.9 ± 1.3) ng/L and NO content was (135.3 ± 16.7)mmol/L in serum,which was statistically increased (both P ＜ 0.05).Pathological changes of liver and bile duct were significantly alleviated.Conclusion ECMO could exert protective effect on liver and bile duct after cardiac death.
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Objective To investigate the optimal time of extracorporeal membrane oxygenation (ECMO) on repairing Maastricht Ⅱ pig liver with warm ischemia injury for 30 min.Method Thirty-six miniature pigs were randomized to ECMO 2-h group,ECMO 4-h group and ECMO 6-h group,12 pigs per group,6 donors and 6 recipients.Cardiac arrest was induced by administration of 1 g KCl intravenously,followed by cardiopulmonary resuscitation (CPR) for 30 min according to the standard guideline.Cannulas was placed through inferior vena cava and abdominal aorta,then connected to ECMO extracorporeal circulation pipes.Transaminase,circulation flow rate of portal vein and hepatic artery and arterial blood gas were monitored and recorded.The hepatic tissues were cut into sections for pathological observation by HE stain under a light microscope.Apoptosis was detected by TUNEL and apoptotic index was calculated.The liver was stored in cold UW for 2 h after the ECMO circulation,then orthotopic liver transplantation without veno-venous bypass was performed.The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured in the peripheral blood for 5 days after the operation.Result With the increase of the running time of ECMO,transaminase and lactate levels were decreased continuously.Circulation flow rate of portal vein and hepatic artery in ECMO 6-h group was significantly lower than that in the other two groups (P＜0.05).Pathological change in ECMO 4-h group was milder than the rest two groups.Apoptosis index in ECMO 2-h,4-h and 6-h groups was (40.20 ± 7.22)％,(18.60 ± 4.04)％ and (29.25 ± 5.98) ％,respectively.The 5-day suvival rate in ECMO 2-h,4-h and 6-h groups was 83％,100％ and 83％,respectively.The transaminase level in ECMO 4-h group at 5th day after the operation was lower than in ECMO 2-h group and 6-h group (P＜0.05).Conclusion The optimal time of ECMO on repairing Maastricht Ⅱ liver was 4 h.The effect of restoration is not ideal when circulation time is not enough.Liver function and liver cell viability decline beyond 4 h.
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<p><b>OBJECTIVE</b>To investigate the kinetic rate constants ozone and hydroxyl radicals towards two groups of antimicrobials -sulfadiazine (SD) and sulfamethoxazole (SMX).</p><p><b>METHODS</b>The solute consumption method was used to detect the rate constants of ozone alone with sulfadiazine and sulfamethoxazole, and tertiary butanol was selected as a scavenging agent and pH was adjusted to 2.5 by adding orthophosphate buffers (OB); and the competition kinetics studying methodwith nitrobenzene as a reference was applied to measure the rate constants of hydroxyl radicals towards sulfadiazine and sulfamethoxazole, and pH was adjusted to 7.0 by adding OB.</p><p><b>RESULTS</b>The rate constants of SD and SMX with ozone alone were 261 mol(-1) · dm(3) · s(-1) and 303 mol(-1) · dm3 · s(-1) by calculating in low reaction system. The rate constants of hydroxyl radicals with SD and SMX were 2.2×1010 mol(-1) · dm(3) · s(-1) and 2.7×1010 mol(-1) · dm(3) · s(-1), respectively. Moreover, the rate constants of hydroxyl radicals with SMX were found to have increased from 3.6×109 mol(-1) · dm(3) · s(-1) to 2.8×1010 mol(-1) · dm(3) · s(-1) with pH value rising from 5.0 to 7.8.</p><p><b>CONCLUSION</b>SMX and SD are both refractory to ozone oxidation alone, and are liable to be degraded by hydroxyl radicals, and the rate constants of SMX with the hydroxyl radical slightly increases with pH rise.</p>