OBJECTIVE: To observe effects of polyanhydride-three-dimensional vector-glucan material on the fetal liver stem cell adhesion and proliferation.METHODS: The two-step collagenase perfusion digestion and bliquid percoll discontinuous density gradient centrifugation was used to isolate fetal liver stem cells. Fetal liver stem cells at the third passage were incubated on the polyanhydride-three-dimensional vector-glucan material. Inverted microscope was utilized to observe cell adhesion and growth status. Cell adherent rate, proliferation activity were calculated, and cell number was counted. Cell-vector was obtained for tissue section. Using hematoxylin-eosin staining, cell growth in the vector was observed under the optical microscope. At 7 days,immunofluorescence staining and flow cytometry were used to determine marker expression.RESULTS: Polyanhydride-three-dimensional vector-glucan promoted grow and adhesion of liver stem cells. There was the active function of the liver stem cells within carrier materials. In the three-dimensional surface and the internal culture, liver stem cell proliferation was sustained. After 10 days, the polyanhydride common culture-three-dimensional vector-glucan on stem cells was non-toxic, and human fetal liver stem cells could be attached to the polyanhydride-three-dimensional vector-glucan stent. The cell proliferation was better and dynamic sustained expression of markers. 7-days training received 19.7 percent increase in the number of cells.CONCLUSION: Polyanhydride-three-dimensional vector-glucan promotes the proliferation of liver stem cells, and liver stem cells can be used as the vector in liver tissue engineering.

Objective To observe the influence of peripheral serum came from patients with hepatectomy at different time points on hepatocyte proliferation in vitro. Methods According to the different types of cultured serum, cultured HL-7702 cells were divided into fetal bovine serum (FBS) group, preoperative serum group, 0.5 h, 3 h, 24 h and 72 h post operative serum groups. All groups of cells were cultured for 72 hours in the Cell-IQ unmarked living cell image analysis system, and the amplification curves of each group were mapped by continuous counting of cells. The cell amplification multiple was compared between all groups after culturing for 72 hours. BrdU immunofluorescence staining was performed and BrdU positive rate was calculated for comparing the cell proliferation of all groups. Results Amplification curves showed that HL-7702 cell proliferation rates of all human serum groups except for 72 h post operative group were higher than those of FBS group. Human serum 0.5 h and 3 h postoperative groups were more obvious. The amplification multiples of human serum groups, except for 72 h post operative group were all significantly higher than those of FBS group (P<0.01), and 0.5 h and 3 h post operative groups were both significantly higher than those of preoperative group (P < 0.05). BrdU positive rates of all human serum groups were significantly higher than those of FBS group (P < 0.01), which were significantly higher in 0.5 h and 3 h post operative groups than those of preoperative group (P < 0.05), but there were no statistical differences between 24 h and 72 h post operative groups and the preoperative group. Conclusion Human serum can promote the proliferation of hepatocytes compared with that of FBS. The influence of serum acquired post hepatectomy is closely associated with the post operative time.

Objective To detection the methylation of p16INK4A in primary hepatocellular carcinoma, a nested bisulfite sequencing and methylation-specific polymerase chain reaction (BS-MSP) protocol was designed and used.Methods Bisulfite-modified DNA were amplified to evaluate the quality of templates with a pair of bisulfite sequencing primers in the first round of PCR, then subjected to methylation assay with corresponding methylation or unmethylation specific PCR primers.Representative PCR products were sequenced to confirm its correctness.Results 3 of 40 cases (7.5%) were failed to assay due to poor quality of templates, and 29 of 37 cases (78%) were detected p16INK4A methylation.Sequencing results confirmed that templates were correctly amplified.Conclusion BS-MSP technique might be valuable for methylation study on carcinogenesis and clinical assay.

Acute-On-Chronic Liver Failure ; complications ; virology ; Adult ; DNA Mutational Analysis ; Female ; Genetic Variation ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; complications ; virology ; Humans ; Male ; Middle Aged

Objective To investigate the levels of serum Golgi protein(GP73) (sGP73) in patients with HBV-related liver disease and investigate the role of sGP73 as an indicator for diagnosis of hepatocelluar carcinoma (HCC).Methods The concentration of sGP73 in patients with chronic hepatitis B (CHB,n =31),liver cirrhosis (LC,n =60),HCC (n =71),self-limited HBV infectors (n=21 ) and healthy controls (n =42) were tested by enzyme-linked immunosorbent assay (ELISA) assay and statistically analyzed in combination with relevant clinical indicators.Results The median of sGP73 in HBV-related liver disease group was significantly higher than that in the groups of self-limited HBV infectors and healthy controls respectively (P＜0.01).Among the groups with HBV-related liver disease,the median of sGP73 in LC group (231.13 ng/ml) was significantly higher than that in HCC without treatment group ( 117.63 ng/ml) (P ＜ 0.01 ) and CHB group (93.09 ng/ml) (P＜0.01).No significant difference was shown between HCC (without treatment) group and CHB group (P＞ 0.05),neither between self limited HBV infectors and healthy controls (respectively,36.79 ng/ml and 45.40 ng/ml) (P ＞ 0.05). The median of sGP73 in post-operation group (175.12 ng/ml,n=52) was significantly higher than in pre-operation HCC group (107.28 ng/ml,n=52) (P＜0.01).Along with the decreasing of liver function,sGP73 level was elevated in groups with HCC or LC.The receiver operating curve (ROC) constructed with the ratio of AFP and GP73 (AFP/GP73) showed a sensitivity of 78.87 ％ and specificity of 86.21 ％ with an area under the receiver operating curve (AUROC) of 0.878 (95％ CI:0.817-0.938) for diagnosis of HCC; comparably,a sensitivity of 67.61％ and specificity of 85.12％ were shown with a AUROC of 0.826 (95％ CI:0.755-0.897) when performed with AFP.Conclusion The level of sGP73 in HBV-related liver disease group is higher than that in the groups of self-limited HBV infector and healthy control,and it is positively correlated with the degree of hepatic impairment.For the diagnosis of HCC,joint detection of AFP and GP73 could achieve a better combination of sensitivity and specificity than the independent AFP test.

OBJECTIVE: To investigate biological characteristics of human umbilical cord-derived mesenchymal stem cells, and to explore the possibility of hepatocyte-like cells differentiation.METHODS: The umbilical cord was provided by healthy term birth woman in Tianjin Third Central Hospital. Mesenchymal stem cells were isolated from human umbilical cord by enzyme digestion method. Cells were passaged at 80%-90% confluent. The ninth passage of cells at a density of 5×10~(10)/L were seeded in 12-well culture plate and incubated with DMEM containing hepatocyte growth factor, fibroblast growth factor-4 and oncostatin for 28 days. Cell growth activity was detected by MTT method; cell cycle was detected by flow cytometry; surface immunological marker in MSC was detected by immunocytochemical stain and flow cytometry; specific surface phenotype of hepatocyte was detected by immunocytochemical staining. Function characteristic of hepatocyte was determined by staining for glycogen.RESULTS: MSCs were isolated from human umbilical cord and presented with fibroblastic morphology. 80% of cells were at G_0/G_1 phase with good growth activity and stably passaged over 20 times. These cells were positive for CD29, CD105, and Vimentin, but negative for CD34 and CD31. MSCs were induced to hepatocyte-1 ike cells that were positive for alpha fetoprotein, CK18, CK19 at 1 week and albumin at 3 weeks. At 4 weeks, induced cells were positive for glycogen staining.CONCLUSION: MSCs isolated from human umbilical cord can be cultured in a long periods time in vitro and are able to differentiate into functional hepatocyte-like cells.

ObjectiveTo investigate the therapeutic effect of human umbilical cord mesenchymal stem cell-paracrine substance on fulminant hepatic failure (FHF) rat, and to study the effect on liver function and hepatocyte proliferation. MethodsMesenchymal stem cells(MSCs)were separated from human umbilical cord, and surface makers of cells were detected by flow cytometry. Human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) was prepared. FHF rat model was induced by intraperitoneal injection of D-galactosamine and they were randomly diveded into three groups: MSC-CM group, NS group, PHGF group. 24 h later, 1 ml MSC-CM, 1 ml 0. 9% NaCl solution and lml PHGF solution was injected into the tail vein of MSC-CM, NS, and PHGF rats, respectively. In each group (n=8 per group), blood samples were collected at 12, 24, 36, and 60 h after treatment from inner canthus for analysis of blood ALT and TBIL levels. We used five rats per group for tissue collection after sacrifice at 36 h after treatment and 10 animals per group for survival analysis. PCNA immunohistochemical staining was used in the sections of liver tissue to detect hepatocyte proliferation. Results24 h after treatment, the levels of ALT and TBIL in the MSC-CM and PHGF groups were lower than those in the NS group(P＜0. 05), but there was no significant difference between the MSC-CM and PHGF groups. There were more PCNA-positive hepatocytes in the MSC-CM and PHGF groups than in the NS group(P＜0.01), but there was no significant difference between MSC-CM and PHGF group. Survival analysis found that the survival rate of rats in the MSC-CM and PHGF groups was higher than that of rats in the NS group (P=0. 049), but there was no significant difference between the MSC-CM and PHGF group. ConclusionsThe paracrine substance of human umbilical cord mesenchymal stem cells can stimulate hepatocyte proliferation and improve liver function of FHF rats, potentially creating a new avenue for the treatment of FHF.

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.

Objective To study the distribution of the specific fragment R049 of uropathogenic E. coli(UPEC) 132 in UPEC and fecal E. coli strains. Methods The specific fragment R049 was amplified by PCR from 20 UPEC strains and 40 fecal E. coli strains, and 5 genes encoding virulence factors (papC, fimH, hly, aer, cnf1) were detected from fragment R049 positive strains. Pulse field gel electronphoresis (PFGE) was applied for isolating the Xba Ⅰ restriction fragments of the genomes of fragment R049 positive strains, and then Southern blot was applied for analyzing the distribution features of the positive hybridization bands by digoxin-labeled R049 ORF probe. Results The specific fragment R049 was amplified from 8 of 20 UPEC strains (40%) and 3 of 40 fecal E. coli strains (7.5%), and statistics analysis showed significant difference (P<0.01). The specific fragment 11049 was closely related with 5 virulence factors of UPEC in the fragment R049 positive strains. Southern blot showed the sizes of positive bands were 150 kb, 15 kb and 240 kb in 3 fecal E. coli strains, 350 kb in 6 of 8 UPEC strains, and 280 kb and 25 kb in the rest two UPEC strains. Conclusion The specific fragment R049 of UPEC132 possessed the feature of clustering distribu-tion in domestic isolated UPEC strains.

ObjectiveTo investigate hepatocyte growth factor (HGF) levels in the tissue and serum of patients with chronic hepatitis,cirrhosis or hepatocellular carcinoma (HCC),and analyze the clinical significances of HGF for HCC.MethodSurgical specimens from 97 patients were collected during Dec.2003 to Aug.2008 in the Third Central Hospital.The patients were prospectively enrolled and categorized into four groups:normal subjects ( n =11 ),chronic hepatitis B or C ( n =6＝,cirrhosis ( n =20)and HCC ( n =60 ) including well-differentiated ( n =21 ),moderately differentiated ( n =23 ),poorly differentiated (n =16) specimens.N0 (n =24),N1 (n =21 ),N2 (n =54) and N3 (n =43) were tissues respectively removed from liver at 0,1,2 or 3 cm beyond the margin of tumor.HGF mRNA expression in liver tissues was determined by real-time quantitative reverse transcription- (RT)-PCR.Serum HGF levels in the other cases of normal subjects ( n =20),chronic hepatitis B or C ( n =20),cirrhosis ( n =20) and HCC (n =57) were measured by ELISA.The Kaplan-Meier method with log-rank test was employed for survival analysis.Univariate and multivariate analyses were performed to identify prognostic factors in each group.ResultsThe HGF mRNA in normal subjects,chronic hepatitis,cirrhosis,N3,N2,N1,N0 and HCC were0.99(0.78-1.66),2.15(1.06-3.40),1.78(1.18-2.73),4.59(2.67 -8.63),3.86 ( 2.25 - 6.45 ),3.12 ( 1.59 - 5.74 ),2.92 ( 0.88 - 5.99 ) and 0.48 ( 0.19 - 1.06 ) respectively.The serum concentration of HGF in the normal subjects,chronic hepatitis,cirrhosis and HCC patients were (0.31 ± 0.05 ),(0.65 ± 0.07 ),( 1.27 ± 0.30 ) and ( 2.06 ± 0.66) μg/L respectively.The highest level of HGF mRNA was found in N3,while the HGF mRNA expression in HCC was [ (2.14 ± 0.52 ) μg/L] lower than that not only in the non-tumor tissues,but also in the normal control ( U =196.50,P =0.03 ).The serum concentration of HGF was significantly higher in patients with chronic hepatitis,cirrhosis or HCC than in normal subjects.The serum HGF level of HCC was bounced after hepatectomy (t =2.70,P ＜0.01 ).On the logistic regression analysis,the tumor numbers and Child-pugh were related with the levels of the tissue HGF mRNA and serum HGF of HCC,OR were0.15 (95％CI:0.03-0.72,P＜0.05) and0.13 (95％CI:0.27 -0.89,P ＜0.05 ),respectively.Univariate analysis using the Cox proportional hazards model in the complication groups revealed that the levels of the tissue HGF mRNA and serum HGF were significant risk factors of death for HCC,OR were 0.02 (95％ CI:0.00 - 0.52,P ＜ 0.05 ) and 10.01 (95％ CI:1.16 -86.23,P ＜ 0.05 ),respectively.On the Log-rank analysis,no statistically difference in the cumulative survival was found between the two groups categorized by median (0.49) of tissue HGF mRNA 2 - AACT (X2 =0.13,P =0.72).While the HCC patients were dichotomized by their the median(0.69 μg/L) of serum HGF concentration,the death risk for the patients with higher levels of HGF was increased 2.84 fold than those with lower levels (95％ CI:1.03 - 7.92,P ＜ 0.05 ).ConclusionHGF mRNA expression is decreased in tumor tissues,while its level in tumor adjacent live and serum is significantly elevated and is in association with shortened postoperative survival of HCC patients.