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PURPOSE: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02. METHODS: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry. RESULTS: With the MACS method, (5-10) x 10(4)/mL hMAPCs could be separated from 1 x 10(6)/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5. CONCLUSION: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.
Subject(s)
Adult , Humans , alpha-Fetoproteins , Bone Marrow , Cell Differentiation , Cell Line , Coculture Techniques , Flow Cytometry , Hepatocytes , Immunohistochemistry , Keratins , Microspheres , Stem CellsABSTRACT
Objective:To investigate the effectiveness of immunoFISH technology in detecting tumor cells in cerebrospinal fluid (CSF) for adjuvant diagnosis of meningeal metastasis from lung cancer. Methods:Circulating tumor cells (CTCs) were detected by im-munoFISH technology in CSF samples from 16 patients with meningeal metastasis from lung cancer and 8 patients with non-tumorous brain diseases. Meningeal metastasis from lung cancer was diagnosed on the basis of neurological symptoms and confirmed by en-hanced magnetic resonance imaging and CSF cytological examination. Results:The number of CTCs was significantly greater in pa-tients with meningeal metastasis from lung cancer than in those with non-tumorous brain diseases (P<0.01). The critical point of the maximum correct diagnosis index (Youden index) was regarded as the judging criterion for the positive tumor cells in CSF based on the receiver operating characteristic curve. When one tumor cell existed in 7.5 mL of CSF, the area under the curve was 0.875, and the 95%confidence interval ranged from 0.705 to 1.000. The diagnostic sensitivity, specificity, effectiveness, positive predictive values, and neg-ative predictive values were 75.0%, 100.0%, 83.3%, 100.0%, and 66.7%, respectively. Conclusion:ImmunoFISH technology provides great significance in detecting CTCs in CSF to diagnose meningeal metastasis from lung cancer.
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Objective To investigate the effective strategies to prevent and treat biliary complications after orthotopic liver transplantation.Methods The clinical data of 316 patients who received orthotopic liver transplantation at the Fuzhou General Hospital of Nanjing Military Command from November 2001 to March 2012 were retrospectively analyzed.Cold perfusion with HTK + UW solution was applied when obtaining the liver graft,and then the liver graft was preserved in the UW solution.The bile duct was perfused with UW solution thereafter.Orthotopic liver transplantation or piggyback liver transplantation were adopted in the cadaver liver transplantation.Left liver transplantation and right liver transplantation were adopted in the living donor liver transplantation.Choledochojejunal Roux-en-Y anastomosis or duct-to-duct choledochostomy were used for biliary reconstruction.Ordinary T tubes were used for drainage before 2006,and then 6 F pediatric suction catheter or epidural catheter were applied for drainage thereafter.The Ttube was pulled out 3-6 months after the operation.Enteral nutrition was applied to patients at the early phase after operation.The immunosuppressive agents used including tacrolimus + mycophenolatemofetil + adrenal cortical hormone,and for some patients,tacrolimus + mycophenolatemofetil + sirolimus + hormone were used.Patients were followed up for 2 years to learn the incidence of biliary complications and guide the medication.The difference in the incidence of bile leakage between patients who wcrc admitted before 2006 and those admitted after 2006 were compared using the chi-square test.Results The warm ischemia time was 2-6 minutes,and the cold ischemia time was 3-10 hours.For patients who received cadaver liver transplantation,orthotopic liver transplantation was carried out for 291 times and piggyback liver transplantation for 24 times; biliojejunal Roux-en-Y anastomosis was carried out for 5 times and bile duct end-to-end anastomosis for 310 times.For patients who received living donor liver transplantation,1 received left liver transplantation and 1 received right liver transplantation,and they received bile duct end-to-end anastomosis.A total of 311 patients received immunosuppressive treatment with tacrolimus + mycophenolatemofetil + adrenal cortical hormone,and 5 patients reveived tacrolimus + mycophenolatemofetil + sirolimus + hormone.Of the 316 patients who received orthotopic liver transplantation,38 had biliary complications after the operation,including bile leakage in 18 patients,intra-and extra-hepatic bile duct stricture in 6 patients,anastomotic stricture in 6 patients,biliarycomplications included cholangitis in the portal area and cholestasis in 4 patients,choledocholithiasis and cholangitis in 2 patients and biliary infection in 2 patients.The incidence of bile leakage before 2006 was 14.00％ (7/50),which was significantly higher than 4.12％ (11/267) of bile leakage after 2006 (x2-7.676,P ＜ 0.05).Of the 38 patients with biliary complications,the condition of 35 patients was improved,and 3 patients died.Of the 18 patients with bile leakage,15 was cured by conservative treatment,3 received surgical treatment (the condition of 1 patient was improved by drainage,anti-infection treatment and nutritional support,but died of peritoneal hemorrhage at postoperative 1 month; 2 patients received peritoneal drainage,1 was cured and 1 died of peritoneal infection).For the 6 patients with intra-and extra-hepatic bile duct stricture,1 was cured by liver retransplantation and 5 were cured by conservative treatment,endoscopic retrograde cholangio-pancreatography (ERCP) or balloon dilation.For the 6 patients with anastomotic stricture,the condition of 3 patients was improved by conservative treatment,balloon dilation or stent implantation,1 gave up treatment due to hepatic cancer recurrence and died thereafter,1 received anastomosis + T tube drainage,1 was cured by recurrent tumor resection and choledochojejunostomy.Four patients with cholangitis in the portal area and cholestasis were cured by conservative treatment.For the 2 patients with choledocholithiasis and cholangitis,1 was cured by stent implantation with ERCP,and 1 received conservative treatment,and the level of total bilirubin was decreased.Two patients with biliary infection were cured by anti-infection treatment.Conclusions Most of the biliary complications could be treated by non-surgical treatments.For patients with severe biliary complications or those could not be treated by non-surgical treatment,re-exploration of the bile duct is effective.Liver re-transplantation is the only choice for patients with dysfunction of liver graft caused by severe ischemic biliary injury.
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Objective To investigate the methods for reconstructing portal vein in liver transplantation patients with grade Ⅳ portal vein thrombosis.Methods Clinical data of 6 patients with grade Ⅳ portal vein thrombosis who underwent liver transplantations were analyzed retrospectively.Different portal vein reconstructing approaches were applied: 4 patients underwent portal vein anastomosis with internal organ varicosis vein (group A),and 2 patients underwent portal vein arterialization (group B). Portal venous flow was monitored by intraoperative ultrasound and postoperative liver function was tested periodically during follow-up.Results In group A,one patient died of celiac infection 2 months post-transplantation.The remaining three patients were followed up for 14-17 months,and their portal veins remained smooth without thrombosis and with mitigated esophageal varicosity.In group B,one patient,with recurrent upper gastrointestinal bleeding,died of celiac infection 47 days after liver transplantation.The patient was followed up for 33 months with satisfactory liver and kidney functions although stomach esophagus varicosity was aggravated.Portal vein blood flow in groups A and B was 1258 ± 345 and 2275 ± 247 ml/min respectively after anastomosis by intraoperative color Dopplar ultrasound monitoring. Aspertate aminotransferase (AST) in group B was significantly lower on the fourth day after liver transplantation,and alanine aminotransferase (ALT) in group B was significantly lower on the 3rd,4th,5th and 6th day after liver transplantation than in group A (all P＜0.05).Serum total bilirubin (TBIL) had no statistically significant difference during the 10 days post-operation (P＞0.05).Conclusion Patients with grade Ⅳ portal vein thrombosis may achieve a satisfactory clinical effect by reconstructing portal vein through anastomosis of donor portal vein with internal organ? varicosis vein.PVA may be associated with early recovery of graft function and may be an effective remedial measure for patients with grade Ⅳ portal vein thrombosis who undergo liver transplantation.
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Objective: To investigate the protective effect of astilbin on warm ischemia/reperfusion-induced liver injury and to study the related mechanism. Methods: C57BL/6 mice were randomly divided into four groups (n=8): sham-operated group (Sham), model control group (I/R), low dose astilbin treatment group (10 mg/kg) and high dose astilbin (40 mg/kg) treatment group. Mice in the two treatment groups were intraperitoneally injected with astilbin 24 hours and one hour before ischemia. Then 70% hepatic ischemia/reperfusion model (the left and middle hepatic lobe) was established. Mice in the I/R model control group and the sham operation group were administered with the same volume of normal saline. The blood sample and liver tissue samples were collected 90 min after ischemia and 6 h after reperfusion. Serum ALT activity was detected as an indicator of liver function damage and the content of MPO in liver tissues were detected by ELISA. The pathological changes of the liver were observed. The expression of IL-10 in liver tissues was detected by Western blotting and the expression of IL- 10 mRNA was detected by semi-quantitative RT-PCR. Results: Astilbin treatment can effectively lower the serum ALT level and MPO level in the liver tissues in some I/R mice. It could also improve the pathological manifestations of the liver. Compared with the I/R model control group, IL-10 protein levels gradually increased in the two treatment groups, which was consistent with the result of RT-PCR (low dose group P<0.05; high dose group, P<0.01). Conclusion: Astilbin can effectively reduce the inflammatory response after liver warm ischemia-reperfusion induced injury, effectively improve the mouse liver function and pathological damage, which might be related to the upregulation of IL-10 expression in the liver tissues.
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<p><b>OBJECTIVES</b>To investigate the effects of astilbin on the expressions of TNF alpha and IL-10 during liver warm ischemia-reperfusion injury.</p><p><b>METHODS</b>C57BL/ 6 mice were randomly divided into 4 groups (n = 8): sham-operated group (Sham), model control group(I/R), low dosage of astilbin treatment group (10 mg/kg) and high dosage of astilbin (40 mg/kg) treatment group. The treatment group mice were intraperitoneally injected with 10 or 40 mg/kg astilbin 24 hours and one hour before Ischemia, the hepatic ischemia-reperfusion model were thus established. After jn90 of min ischemia and 6 h reperfusion of the partial hepatic lobe, the expressions of TNF alpha and IL-10 in liver tissues collected from the experimental groups were detected by Western blot and semiquantitative RT-PCR.</p><p><b>RESULTS</b>The expression of TNF alpha protein in liver tissues gradually decreased in treatment groups (low and high dosages of astilbin treatment groups) as compared to the I/R model control group. Similar results were observed in the mRNA expressions of these genes as determined by semiquantitative RT-PCR (P less than 0.05 for low dosage group; P less than 0.01 for high dosage group). Compared with the I/R model control group, the expression of IL-10 was increased in both treatment groups (low dosage group P less than 0.05; large dosage group P less than 0.01).</p><p><b>CONCLUSION</b>Treatment with astilbin decreases TNF alpha expression but induces IL-10 expression in liver during warm ischemia-reperfusion injury.</p>
Subject(s)
Animals , Male , Mice , Flavonols , Pharmacology , Interleukin-10 , Metabolism , Liver , Metabolism , Mice, Inbred C57BL , Reperfusion Injury , Metabolism , Tumor Necrosis Factor-alpha , Metabolism , Warm IschemiaABSTRACT
Objective To study the effects of Sinomenine on the dendritic cells after hepatic ischemia-reperfusion. Methods Forty-eight BN rats were equally divided into control group, low dose (40 μg/g) and high dose (80 μg/g) of Sinomenine groups after the liver transplantation models were established by two cuff tech-nique. Three days after the orthotopic liver transplantation, the livers were resected, then the dendritic cells were separated and purified. The phenotypes [OX62, major histocompatibility complex Ⅱ (MHC-Ⅱ) and CD86] of dendritic cells were examined by FACS, the expression of IL-12, IL-1, and TNF-a mRNA by RT-PCR, and the expression of Toll-like receptor 4 (TLR4) by Western blot. Results The dendritic cells treated with Sinomenine showed immature phenotypes. The expressions of MHC-Ⅱ and CD86 were significantly deceased. The expressions of IL-12, IL-1, TNF-a mRNA and TLR4 were low. Conclusions Sinomenine can significantly inhibit the maturity and immunologic function of dendritic cells after hepatic ischemia-reperfusion.
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<p><b>OBJECTIVE</b>To investigate the possibility of marrow derived multipotent adult progenitor cells (MAPCs) differentiating into hepatocytes by co-culturing with human hepatocyte line L02, and to evaluate the potential use of MAPCs in tissue-engineering either experimentally or clinically.</p><p><b>METHODS</b>(1) Co-culturing without cell-to-cell contact: MAPCs and L02 hepatocytes were spread on coverslips separately (both with a cell density of 1x10(5)/ml), and then they were put in a culture dish (10 cm). The expressions of Alb, AFP, CK18, and CK19 in MAPCs were detected by immunocytochemistry at different time points. A separate culture of L02 hepatocytes served as a positive control and a separate culture of MAPCs served as a negative control. (2) Co-culturing with cell-to-cell contact: MAPCs labeled with CFSE were mixed with L02 hepatocytes (both with a cell density of 1x10(4)/ml), and then the mixed cells were seeded on specific dishes for detection by laser scanning confocal microscope (LSCM). Five days later, the cells were double-stained with SABC-Cy3. The expressions of Alb, AFP, CK18 in MAPCs were observed under LSCM. Similarly, separately cultured L02 hepatocytes served as a positive control and separately cultured MAPCs served as a negative control.</p><p><b>RESULTS</b>(1) Results of co-culturing without cell-to-cell contact: On the first day, the MAPCs expressed a high level of AFP. Then AFP expression tapered daily and there was hardly any expression of AFP on day 7. The expression of Alb was very weak on day 1, but increased significantly by day 3, reached its peak on day 5, and still maintained a high level on day 7. The initial expression of CK18 appeared on day 5 and reached a higher level on day 7. The expression of CK19 was always negative. The positive control cells had a high expression of Alb and CK18, while there was a weak expression of AFP and a negative expression of CK19. The negative control cells had no expressions for the four markers. (2) Results of co-culturing with cell-to-cell contact: On day 5, there were three colors of fluorescence under LSCM: yellow cells were MAPCs differentiating into hepatocytes; green cells were undifferentiated MAPCs; red cells were L02 hepatocytes. The result showed that Alb and CK18 were expressed in many cells and AFP appeared in only a few cells.</p><p><b>CONCLUSION</b>Human MAPCs can be induced to differentiate into mature hepatocyte-like cells by co-culturing with L02 hepatocytes, either with or without cell-to-cell contact, but the former way may be more effective.</p>
Subject(s)
Humans , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cells, Cultured , Coculture Techniques , Hepatocytes , Cell Biology , Multipotent Stem Cells , Cell BiologyABSTRACT
Objective: To establish a method for isolation, purification, and culture of human multipotent adult progenitor cells (MAPCs) in vitro, so as to lay a foundation for the application of hMAPCs in tissue-engineering research and clinical medicine. Methods: The mononuclear cells were obtained from bone marrow of healthy volunteers by gradient centrifugation and were subjected to adherence culture. The adherent cells were then subjected to magnetic activated cell sorting (MACS) (depletion selection with CD45 and GlyA microbeads). Then the purity of selected cells was identified by flow cytometer. The growth of the purified cells was observed and the expression of CD29, CD44, CD34, and HLA-DR was analyzed by flow cytometers. Results: (1) Averagely (5-10) × 104/ml hMAPCs could be separated from 1 × 106/ml bone marrow mononuclear cells by MACS. The cell viability was similar before ([96.7±1.7]%) and after ([96.0±2.4]%) isolation. (2) The isolated MAPCs grew well in the self-designed culture medium and could be passaged for 20 generation. (3) The purity of the CD45- and GlyA- cells separated from bone marrow adherent cells was more than 98% as determined by flow cytometer. (4) In hMAPCs, the positive rate was 99.2% for CD29, 98.3% for CD44, 1.2% for CD34, and 5.3% for HLA-DR. Conclusion: (1) The bone marrow-derived hMAPCs can be purified by MACS through depletion selection of CD45 and GlyA microbeads. (2) The hMAPCs can be expanded in vitro in self-designed medium, maintaining a non-differentiation state for a long time.
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Objective:To establish a method for isolation,purification,and culture of human multipotent adult progenitor cells(MAPCs) in vitro,so as to lay a foundation for the application of hMAPCs in tissue-engineering research and clinical medicine.Methods: The mononuclear cells were obtained from bone marrow of healthy volunteers by gradient centrifugation and were subjected to adherence culture.The adherent cells were then subjected to magnetic activated cell sorting(MACS)(depletion selection with CD45 and GlyA microbeads).Then the purity of selected cells was identified by flow cytometer.The growth of the purified cells was observed and the expression of CD29,CD44,CD34,and HLA-DR was analyzed by flow cytometers.Results: (1)Averagely(5-10)?10~(4)/ml hMAPCs could be separated from 1?10~(6)/ml bone marrow mononuclear cells by MACS.The cell viability was similar before(96.7?1.7%) and after(96.0?2.4%) isolation.(2)The isolated MAPCs grew well in the self-designed culture medium and could be passaged for 20 generation.(3)The purity of the CD45~-and GlyA~-cells separated from bone marrow adherent cells was more than 98% as determined by flow cytometer.(4) In hMAPCs,the positive rate was 99.2% for CD29,98.3% for CD44,1.2% for CD34,and 5.3% for HLA-DR.Conclusion:(1)The bone marrow-derived hMAPCs can be purified by MACS through depletion selection of CD45 and GlyA microbeads.(2)The hMAPCs can be expanded in vitro in self-designed medium,maintaining a non-differentiation state for a long time.