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 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.

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 study the expression and effect of macrophage inflammatory protein-2(MIP-2) in liver injury induced by ischemia/reperfusion(I/R). Methods Thirty-two rats were randomly divided into 4 groups(8 rats in each group):false operation (control) group and 3, 9, 24 hours reperfusion group.The expression of MIP-2 mRNA in hepatic tissue, MIP-2 protein in plasma, the neutrophil infiltration in liver tissue and serum ALT were measured. Results The expression of MIP-2 mRNA in the ischemic tissue was significantly higher than that in nonischemic tissue (P

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 feasibility of Wilms’tumor gene 1 (WT1)-specific CD8+T cells from periph?eral blood for the treatment of breast cancer by detecting the killing activity of WT1 specific CD8+T cells on breast cancer cells. Methods Flow cytometry was used to detect WT1-specific CD8+T cells in the peripheral blood of 20 samples from HLA-A2 seropositive healthy donors, which were isolated by WT1/MHC streptamer magnetic beads and cultured. The func?tion of WT1-specific CD8+ T cells were analysis by cytotoxicity assay. Results Twelve of 20 healthy donors had naive WT1-specific CD8+T-cell frequencies of>0.5%, and 4 of 20 even>1.0%of all CD8+T cells. After positive selection by magnetic cell separation, a purity of up to 80%can be achieved. WT1 specific CD8+T cells can specifically kill breast can?cer cell line with WT1 polypeptide. Conclusion WT1 specific CD8+T cells can be detected in peripheral blood of healthy volunteers. WT1 specific CD8+T cells have killing effect on breast cancer cells, suggesting the feasibility of adoptive immu?notherapy for breast cancer.

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.

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 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.

Objective:This study aimed to detect the special methylation profile in peripheral blood for hepatocellular carcinoma (HCC). Methods:The methylation status of 12 tumor suppressor genes (TSGs) in the plasma of 55 HCCs and 54 chronic liver diseases (CLDs) was tested by methylation-specific PCR (MSP). Results:In HCC, the methylation frequencies were 78.18%in APC, 63.64%in cyclin D2, 58.18% in TFPI2, 49.09% in DKK3, 49.09% in GSTP1, 47.27% in p16, 40.00% in Sigma 14-3-3, 18.18% in SFRP2, 16.36% in ppENK, 9.09% in DKK2, 7.27% in NPTX2, and 5.45% in LHX1. In CLD, the methylation frequencies were 27.78% in APC, 22.22%in cyclin D2, 7.41%in TFPI2, 3.70%in DKK3, 16.67%in GSTP1, 37.04%in p16, 37.04%in Sigma 14-3-3, 11.11%in SFRP2, 20.37%in ppENK, 7.41%in DKK2, 7.41%in NPTX2, and 9.26%in LHX1. The methylation frequencies of APC, cyclin D2, TFPI2, DKK3, and GSTP1 were higher in HCC than in CLD (P<0.01). The methylation index (MI) of the five-gene methylation profile was statistically higher in HCC (median, 0.6;IQR, 0.4-0.8) than CLD (median, 0.2;IQR, 0-0.2) (P<0.01). In HCC, MI was statistically related to the patient's age. Older patients with HCC had a higher MI. No significant correlation was observed between MI and other clinicopathological data. Moreover, MI was not related to the disease free survival and the overall survival in HCC. Conclusion:This five-gene methylation profile may be a promising biomarker for the assistant diagnosis of HCC.