Objective:To compare the clinical efficacy of percutaneous transhepatic choledochoscope lithotomy (PTCSL) with laparoscopic choledocholithotomy (LD) in treatment of choledocholithiasis.Methods:Data of 132 patients with choledocholithiasis treated at the First Affiliated Hospital of Guangzhou Medical University from July 2012 to December 2018 were retrospectively analyzed. There were 75 males and 57 females, with an average age of 62.7 years. For 76 patients underwent PTCSL (the PTCSL group) and 56 underwent LD (the LD group). The data of the patients the success rate of lithotomy, stone residual rate, operation time, postoperative complications and stone recurrence, chronic cholangitis, and acute cholangitis 1 month after operation were compared between the two groups.Results:The ratio of upper abdominal operation history and biliary tract infection in the PTCSL group was higher than that in the LD group, and the difference was statistically significant (both P<0.05). In the PTCSL group, the calculi were successfully removed in 64 patients in one treatment session, while residual calculi were removed through subsequent sinus choledochoscopy in 9 patients. In the remaining 3 patients, the residual calculi were removed with LD or laparotomy operations. Postoperative complications occurred in 14 patients (19.2%, 14/73). In the LD group, the calculi were successfully removed in one session in 46 patients while in 8 patients the residual calculi were removed by choledochoscopy (1 patient still had residual calculi after choledochoscopy). The remaining 2 patients underwent open surgery due to anatomical difficulties. Postoperative complications occurred in 11 patients (20.4%, 11/54). There were no significant differences between the two groups in the one-off stone removal rate, postoperative stone residual rate, final stone removal rate and postoperative complication rate (all P>0.05). The operation time of the PTCSL group was (156±60) min, which was significantly shorter than the LD group (203±59) min ( P<0.05). There was no significant difference between the two groups in the incidence of postoperative chronic cholangitis and recurrence rate of calculi (both P>0.05). The incidence of acute cholangitis in the PTSCL group was significantly higher than that in the LD group ( P<0.05). Conclusion:PTCSL was as safe and effective as LD, with fewer complications and faster recovery. It is especially suitable for patients with previous upper abdominal surgery, recurrence of calculi and repeated biliary tract infection.

Objective To investigate the curative effect on patients with choledocholithiasis by percutaneous transhepatic rigid choledochoscope lithotomy (PTCSL) vs endoscopic retrograde cholangiopancreatography (ERCP) plus EST.Methods From Jan 2010 to Dec 2015,92 cases of choledocholithiasis were treated by one-stage PTCSL (n =23) vs ERCP (n =69).The curative effects and postoperative complications in two groups were observed and analyzed.Results In PTCSL group,the complete stone clearance at one-time achieved in all 23 cases (100％).While in ERCP group stone clearance was achieved in 72.46％ cases at first attempt and the final clearance rate was 82.60％,leaving 12 cases with residual stones and among those 12 cases 5 cases were converted to surgical operation.The average intra-operative hemorrhage in two groups was (20.6 ± 4.6) ml vs (3.0 ± 0.3) ml,and the average hospital stay after operation was 6.8 d and 7 d respectively.The post-operative complications (30.43％) and stone recurrence (13.04％) were similar in the two groups.Conclusions PTCSL is safe,effective,and more suitable to patients with large stones and those with a history of biliary surgeries.

Aim According to various target sites of HLA-E mRNA,to design and synthesize 3 pieces of HLA-E siRNA chain,to compare quantitatively their efficiency of silencing gene in BEL-7402 with HLA-E(+)in order to select the dominant siRNA.Methods The hepatocarcinomal BEL-7402 cells,induced by 5×10~5 IU·L~(-1) IFN-γ,expressed HLA-E(+) and was pured by flow cytometry selecting as target cells for research.3 pieces of specific siRNA(A,B,C group)were designed and chemically synthesized,then the concentration of which(0.1 mmol·L~(-1))was respectively transfected through Lipofectamin 2000 into target cells.After 48 h,the gene silent effect on HLA-E gene in A,B and C groups was quantitatively observed by cytoimmunofluorence,flow cytometery,Western blot and real-time PCR,as well as on NK cytotoxicity to target cells tested by NK killing rate.Results Compared with those of control or non-specific siRNA group,HLA-E antigen,protein product,HLA-E mRNA and HLA-E molecule on cell surface were statistically down-regulated in A,B,and C group(P<0.01),whose were silenced more (above 90%) in B or C group than in A group (P<0.01).The NK killing rate in A,B and C groups was dominantly improved(P<0.01),which in B or C group was higher than in A group (P<0.01).Conclusion The targeted siRNA can specifically and high-efficiently silence HLA-E expression in hepatocarcinomal cells,and may keep them from immunoescape through non-classic HLAⅠ pathway to imply new strategy for hepatocarcinomal gene-immunotherapy.

BACKGROUND: In vitro differentiation of embryonic stem cells into hepatocytes has been successfully reported to a certain degree; however, whether embryonic stem cells are able to effectively enter hepatic plate of host after intrahepatic transplantation, whether embryonic stem cells can further differentiate into hepatocytes and express hepatocyte function, and risk factors for neoplastic formation are still unclear at present. OBJECTIVE: To study the intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation models, and to investigate the liver tissue replacement, growth and differentiation in vivo, and neoplastic formation.DESIGN: Randomized controlled animal study.SETTING: Department of Pediatric Surgery, the Second Hospital affiliated to Sun Yat-sen University. MATERIALS: Twenty-four BALB/c mice, 6-8 weeks old, weighing 20-35 g, irrespective of gender, were provided by Guangzhou Experimental Animal Center. Embryonic stem cells-derived hepatic stem cells were differentiated from embryonic stem cells. E14 was provided by Stem cell Center of our hospital. METHODS: This study was performed at the Stem Cell Center, the Second Hospital affiliated to Sun Yat-sen University from July 2006 to June 2007. Twenty-four mice were randomly divided into a liver repopulation model + stem cell transplantation group (group A) and a liver resection + stem cell transplantation group (group B), with 12 mice in each group. Mice in the group A were intraperitoneally injected with 50 mg/kg retrorsine once every two weeks for totally twice. Four weeks after the second injection, about 70% liver was resected. And then, the embryonic stem cells-derived hepatic stem cells, labeled by 1×105 carboxy fluoresce in diacetate succinimidyl ester (CFDA-SE), were transplanted into mouse liver through portal vein. On the other hand, 70% liver of mice in the group B was resected and embryonic stem cells-derived hepatic stem cells were transplanted into mouse liver. MAIN OUTCOME MEASURES: The distribution, incorporation, and proliferation of transplanted cells were observed under fluorescent microscopy. Two weeks later, hepatic function was stained with albumin fluorescence immunoassay (double fluorescence staining) and assayed by level of serum albumin. Embryonic stem cells-derived hepatic stem cells were poured into liver of remedial liver regeneration mice, and undifferentiated embryonic stem cells were transplanted into subcutaneous tissue in axillary region as the controls to observe neoplastic formation in embryonic stem cells-derived hepatic stem cells. RESULTS: ① Growth of hepatic stem cells in recipient mice: One week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, some scattered region was green under fluorescent microscopy. The area of green region increased apparently in 2 weeks, and cord-like structure could be observed. ② Liver function: Immunofluorescent staining of albumin (double fluorescence staining) demonstrated that labeled cells expressed positive albumin (yellow fluorescence) in liver tissue of recipient mice, but there was not significant difference in serum albumin level between group A and group B (P > 0.05). ③ Reliability of hepatic stem cell transplantation: Teratoma did not form over 6 months; however, transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks. CONCLUSION: The transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation model mice can effectively and further grow and differentiate, or even partially express hepatocyte function; in particular, the transplantation is safe.

BACKGROUND: Recently, little attention has been paid to how to induce and identify the functions of differentiated cells in the methods for embryonic stem (ES) cells differentiation into hepatocytes. Whether the differentiated cells express functional characteristics of hepatocytes should be one of the markers to identify the hepatic differentiation of ES cells.OBJECTIVE: To direct mouse embryonic stem cells in vitro differentiation into functional hepatocytes by introduction of murine cholestatic serum in hepatocyte growth factor (HGF)-induced system.DESIGN: A controlled observation and in vitro cytological trial.SETTING: Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Sun Yat-sen University.MATERIALS: The experiment was carried out in the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from October 2004 to February 2007. The mouse E14 ES cell line was kindly provided by the Stem Cell and Tissue Engineering Center of Sun Yat-sen University. Twenty male SD rats, aged 2 weeks, were purchased from the Experimental Animal Center of Sun Yat-sen University. All animal experimental procedures were abided by the rules of animal ethnics.METHODS: The SD rats were undergone common bile duct ligation to induce cholestasis. Ten days after the operation, the whole blood of rats was collected to prepare cholestatic serum. The ES cells were cultured using hanging-drop method for 5-7 days to develop embryonic bodies (EBs). The dissociated EBs cells were then induced hepatic differentiation with spontaneous system, HGF (20 μg/L) system and cholestatic serum (5%) plus HGF (20 μg/L) system, respectively.MAIN OUTCOME MEASURES: The cellular morphologic changes were observed using transverse microscopy dynamically. (2) The cell staining for albumin, α-fetoprotein, CK18/19, glycogen, indocyanine green (ICG) and fluorescein diacetate (FDA) was done after 4 weeks differentiation. (3) The hepatocyte-specific metabolic functions of synthesizing albumin, triacylglycerol and urea nitrogen were assayed at 3 days interval.RESULTS: (1) The differentiation of ES cells cultured in spontaneous system was uncontrolled and the cells could grow into a wide range of three-germ cells. The HGF could promote ES cells differentiation into endoderm and mesoderm (myocardium). But the differentiated cells only expressed low levels of hepatic specific functions in these two induced systems. (2) Under cholestatic serum plus HGF system, the ES cells could differentiate into polygonal cells with very uniform morphology which were positive in glycogen, ICG and FDA staining and showed higher capabilities of synthesizing albumin, triacylglycerol and urea nitrogen than the differentiated cells in the other systems (P＜0.05-0.01).CONCLUSION: The cholestatic serum, a mimic pathological microenvironment in vitro, could effectively promote ES cells-derived hepatocytes induced by HGF to express high level of liver-specific metabolism functions.

Objective To investigate the functional role of mur in e hepatocyte growth factor (HGF) in inducing in vitro the mouse embryonic st em cells (ESC) to differentiate unidirectionally into hepatocyte. Method s Both spontaneous and HGF induced differentiations of the primordial c ell elusters formed by suspended culture in vitro of ESC were daily observed with microscope for 5-7 days. After 4 weeks of culture, the cells were stained with HE and glycogen staining, their morphology were observed, The synthesis of urea nitrogen and triglycerides in the culture medium, the expression of myocard ial MHC, albumin, AFP and CK18, and the indocyanine green (ICG) staining were a lso detected. Results It was hard to control the spontaneous di fferentiation of ESC. HGF could promote the differentiation of ESC into endoderm , and more likely into myocardium. HGF could also induce the expression o f albumin, AFP and CK18, and positive staining of ICG and FDA. Conclusio ns HGF may induce the differentiation of ESC into hepatocyte, but the f unctional role is limited. It implies that a comprehensive effort of multiple fa ctors might be needed in inducing the hepatocyte differentiation.

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro . METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.

AIM: To explore a new method of hepatocyte growth factor (HGF) inducing bone marrow mesenchymal stem cells (MSC) to differentiate into cardiomyocytes. METHODS: Bone marrow MSC was cultured with DMEM media (10% fetal calf serum) 4-6 passages, and induced by HGF (10 ?g/L) for 30 d. Automatical beating of the differentiated cells was observed daily with transverse microscopy, or under condition of 0.1% isoproterenol or cal (cium-deprived) incubation. Specific cardiac myosin in the cells was indentified by immunochemistry. RESULTS: At 14-20 d of differentiation, bone marrow mesenchymal stem cells formed clones, in 10%-50% of which spontaneous beating cell-mass had come to continuously exist. Isoproterenol increased the beating rate and calcium-deprived media inhibited the beating. The cells were identified to be cardiomyocytes by expression of cardiac myosin heavy chain. CONCLUSION: HGF may induce bone marrow mesenchymal stem cells into cardiomyocytes with high efficiency, but the differentiating pathway of stem cells remains to be further studied. [

0.05).No teratoma was formed in the experimental group,while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION:The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure,proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.

To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispersed method. After exposure of the AECs to cigarette smoke extract(CSE), the ECD expression in the cells was detected by using immunocytochemistry and in situ hybridization. The results showed that ECD was distributed on the plasma membrane at the cell junctions of AECs. After exposure to 20% CSE, the membranous ECD expression was decreased, the cytoplasmic ECD expression was increased (P＜0.01) as the exposure time went on.But the content of ECD mRNA in the AECs did not chang. It suggests that the change of ECD expression is regulated at the posttranslational level and plays a role in the injury and repair of AEC caused by smoking.