Effects of doxorubicin-loaded tumor-derived extracellular vesicles on cell proliferation and apoptosis of human hepatocellular carcinoma / 中华肝胆外科杂志

Objective:To investigate the effects of doxorubicin (Dox)-loaded tumor-derived extracellular vehicles (EVs) on cell proliferation and apoptosis of human hepatocellular carcinoma.Methods:The extracellular vesicles loaded with Adriamycin (EVs-Dox) were prepared by the method of directly co-incubation. The morphology of EVs-Dox was detected by transmission electron microphotometer. The diameter of EVs-Dox was determined by dynamic light scattering (DLS). Western blotting was utilized to detect the expression of CD63, HSP 70 and TSG 101 in the EVs-Dox. The encapsulation efficiency of EVs-Dox was calculated by tandem mass spectrometry (LC-MS/MS). The drug release experiment in vitro was utilized to simulate the drug release of drug-loaded vesicles in vivo. PKH67-labeled EVs-Dox was showed cellular uptake. After treatment with EVs-Dox, MTS assay and flow cytometry assay were conducted to investigate the effects of EVs-Dox on cell proliferation and apoptosis of PLC/PRF/5.Results:The EVs-Dox showed an elliptical double-layer membrane structure of different sizes under transmission electron microscope. The diameter of EVs-Dox was (115.9±5.2) nm.Western blotting data showed high expression of CD 63, HSP 70 and TSG 101 in the EVs-Dox. The encapsulation efficiency of EVs-Dox was 0.77%. The in vitro release experiment showed that the drug-loaded vesicles could release the drug slowly. PKH67-labeled EVs-Dox showed that carcinoma cells can uptake EVs-Dox within 16h. MTS assay showed that the cell viability rate of (54.9±3.2) % was significantly lower than that of in the Dox group [(77.7±5.4)%, P<0.05]. EVs-Dox inhibited hepatocellular carcinoma proliferation. Flow cytometry assay showed that the apoptosis rate of EVs-Dox (47.9±7.0) % was higher than that in the Dox group [(38.0±1.5)%, P<0.05]. Conclusion:EVs-Dox inhibits cell proliferation and accelerates apoptosis of hepatocellular carcinoma cells.

Impact of portal hypertension and hypersplenism on prognosis of hepatocellular carci-noma after curative treatment / 中国肿瘤临床

Objective: To investigate the impact of portal hypertention with hypersplenism of different severity and splenectomy on prognosis of hepatocellular carcinoma (HCC). Methods: We retrospectively analyzed the clinical data of 403 patients with HCC who met the Milan criteria and received radical treatment in Tianjin Third Central Hospital from January 2008 to January 2018. Cox propor-tional risk regression analysis was performed for parameters such as platelet levels (PLT), albumin-bilirubin (ALBI) grade, aspartate ami-notransferase-to-platelet ratio index (APRI), and post-sinusoidal resistance (PSR). HCC patients with severe hypersplenism were as-signed into two groups according to treatment method: radical treatment for HCC alone and radical treatment for HCC plus splenecto-my. Clinical data were compared, and the two groups were evaluated using the Kaplan-Meier survival analysis method. Results: Univar-iate and multivariate analyses showed that PLT was an independent risk factor for overall survival (OS) and disease-free survival (DFS) in patients with HCC. OS curves differed significantly with different PLT among patients with HCC (P=0.013). Furthermore, parameters of portal hypertension in cirrhosis, such as PSR, APRI, and ALBI grade, were risk factors for HCC prognosis. The degree of portal hyper-tension and hypersplenism, liver function, and tumor-node-metastasis stage did not differ between the two groups (P>0.05). Survival analysis showed significantly longer OS in the radical treatment plus splenectomy group (P=0.025). Following were the 1-, 3-, and 5-year survival rates: radical treatment alone group 100% , 98.2% , and 68.5% and radical treatment plus splenectomy group. 97.1% , 79.4%, and 56.8%, respectively. DFS did not differ between the two groups (P=0.326). Conclusions: Clinical parameters, such as PLT, PSR, APRI, and ALBI grade, are important prognostic factors in HCC patients with portal hypertension and hypersplenism. Radical treat-ment for HCC plus splenectomy can improve OS in HCC patients within the Milan criteria with severe hypersplenism.

The application of peripheral serum interleukin-6 levels in predicting the recurrence of early hepatocellular carcinoma after receiving microwave ablation / 介入放射学杂志

Objective To investigate the clinical application of serum interleukin-6 (IL-6)and interleukin-22 (IL-22) levels in predicting the recurrence of hepatitis B virus (HBV)-related early hepatocellular carcinoma (HCC) after receiving microwave ablation (MWA).Methods Preoperative peripheral blood samples were collected in 49 patients with early-stage HBV-related HCC,and serum concentrations of IL-6 and IL-22 were measured by using ELISA.Thirty healthy volunteers were recruited and used as the control group.The xtile software was used to define the best cut-off value,and the IL-6 and IL-22 levels were divided into highlevel group and low-level group.The tumor-free survivals of high-level and low-level groups were analyzed with Kaplan-Meier analysis,log rank test was adopted to determine the difference,and Cox regression model was employed to screen the risk factors affecting HBV-related HCC recurrence.Results The serum IL-6 and IL-22 levels of HCC group were 13.20 pg/ml (11.87-15.79 pg/ml) and 42.18 pg/ml (34.39-57.44 pg/ml) respectively,which were significantly higher than 10.47 pg/ml (9.50-13.82 pg/ml) and 25.45 pg/ml (22.31-30.12 pg/ml) of the control group (P=0.001 and P＜0.001 respectively).Kaplan-Meier analysis revealed that preoperative lower IL-6,higher total bilirubin and lower albumin levels indicated a shorter disease-free survival (DFS),and IL-22 levels had no statistically significant effect on the recurrence of HCC.Cox regression multivariate analysis showed that lower serum IL-6 level (≤ 13.2 pg/ml;hazard ratio=3.721;95％ CI=1.674-8.272;P=0.001) and lower serum albumin level (≤41.0 g/L;hazard mtio=2.085;95％CI=1.101-3.950;P=0.024) were independent risk factors affecting HBV-related HCC recurrence Conclusion Preoperative serum IL-6 level and serum albumin level can be used as the predictors of HCC recurrence in patients with HBV-related early HCC who are receiving MWA treatment.(J Intervent Radiol,2017,26:232-236)

Expressions and roles of three inflammatory cytokines in chronic hepatitis B patients with nonalcoholic fatty liver disease / 中华传染病杂志

Objective To investigate the roles of cytokeratin 18 (CK18) M30 and M65,thymosin beta 4 (Tβ4) and tumor necrosis factor (TNF)-α in hepatic steatosis and development of inflammatory and fibrosis in chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease (NAFLD).Methods A total of 46 CHB patients with NAFLD and 42 CHB patients were collected.Serum CK-18 M30,M65,Tβ4 and TNF-α levels were measured by enzyme linked immunosorbent assay (ELISA) in two groups.The associations between inflammatory factors levels and biochemical or pathological indicators were analyzed.The statistical analysis was conducted by t test and chi square test of two independent samples.The correlation analysis was performed by Pearson and Logistic regression analysis.Results The mean serum CK 18 M30 level in CHB with NAFLD group was (614.48±471.43) U/L,which was significantly higher than that in CHB group (374.50±231.04) U/L (t=2.988,P＜0.01).The mean levels of CK18 M65,Tβ4 and TNF-α in CHB with NAFLD group were (369.41±262.21) U/L,(0.80±0.32) mg/L and (54.87±20.36) ng/L,respectively,and those in CHB group were (296.50±231.44) U/L,(0.68±0.30) mg/L and (51.88± 20.60) ng/L,respectively.There were no difference between CHB with NAFLD group and CHB group (t=1.378,1.810 and 0.685,respectively,all P＞0.05).In CHB with NAFLD patients,the CK-18 M30 level was positively correlated with alanine aminotransferase,triglyceride,fasting blood glucose,histology inflammation score,fibrosis score and steatosis (r=0.507,0.456,0.384,0.551,0.458 and 0.457,respectively,all P＜0.01).Tβ4 level was negatively correlated with inflammation and fibrosis score (r=0.371 and-0.308,respectively,P＜0.05).TNF-α level was positively correlated with inflammation score and steatosis (r=0.570 and 0.441,respectively,P＜0.01).CK-18 M30,Tβ4 and TNF-α were independent predictors of CHB combined with NAFLD,progressive inflammatory fibrosis and severe steatosis.Conclusions Serum CK-18 M30,Tβ4 and TNF-α levels are associated with hepatic steatosis,development of inflammation and fibrosis in CHB with NAFLD patients.

Effects of serum came from hepatectomized patient on proliferation of cultured hepatocytes / 天津医药

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.

Experimental study of WT1 specific CD8+T cells in the treatment of breast cancer / 天津医药

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.

Methylation profile of tumor suppressor genes in the cell-free DNA of plasma in hepatocellular carcinoma / 中国肿瘤临床

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.

The levels and clinical significances of hepatocyte growth factor in the hepatic tissue and serum of hepatocellular carcinoma / 中华检验医学杂志

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.

Quantitation of hepatitis B virus total DNA, covalently closed circular DNA and HBsAg in patients with different stages of hepatitis B virus infection / 中华传染病杂志

Objective To quantitatively analyze total hepatitis B virus (HBV) DNA (HBV tDNA),covalently closed circular DNA (cccDNA) and HBsAg in patients with chronic hepatitis B (CHB),HBV-related liver cirrhosis (LC) and hepatocellular carcinoma (HCC),and to analyze the characteristics.Methods HBV tDNA and HBV cccDNA in the serum and liver biopsy samples were measured in 21 CHB,23 LC and 25 HCC patients by real-time polymerase chain reaction (PCR) assay. HBsAg titer was measured by chemiluminescence. Normally distributed variables among multiple groups were analyzed by ANOVA and t-test.Correlation between two variables was tested using Pearson correlation analysis.Skewed distribution was tested using Rank sum test.Results In CHB,LC and HCC patients,the serum HBV tDNA levels were (5.38±2.08),(4.96± 1.65) and (4.18 ± 0.91) lg copy/mL,respectively; the intrahepatic HBV tDNA levels in three groups were (7.18±1.91),(6.51±1.87) and (5.87± 1.47) lg copy/ug,respectively; the intrahepatic HBV cccDNA levels were (3.53±2.03),(2.63±2.13) and (0.58± 1.40) lg copy/μg,respectively; the serum HBsAg levels were (3.30±0.65),(3.12±0.52) and (2.60± 1.03) lg IU/mL,respectively.In CHB patients,the serum HBV tDNA,intrahepatic HBV tDNA,HBV cccDNA and HBsAg levels were all significantly higher than those of HCC patients (t=2.446,P=0.013; t=2.562,P=0.014;t=5.799,P＜0.01 ; t=2.709,P=0.003,respectively).However,only intrahepatic HBV cccDNA and HBsAg levels were statistically different between LC and HCC patients (t=-3.894,P＜0.01;t=-2.237,P=0.023,respectively).HBV cccDNA was all negative in the serum of 69 patients.The serum HBsAg level was positively correlated with serum HBV tDNA (r=0.290,P=0.016),intrahepatic HBV tDNA (r=0.372,P =0.002) and intrahepatic HBV cccDNA (r=0.378,P=0.001).Conclusions The levels of HBV tDNA,HBV cccDNA and HBsAg decrease gradually with the disease progression.The serum HBsAg level is positively correlated with serum HBV tDNA,intrahepatic HBV tDNA and intrahepatic HBV cccDNA.

Studies on the relationship between the serum Golgi protein 73 and the HBV-related liver disease / 中华肝胆外科杂志

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.

Establishment of the limited cell line of skin fibroblast and their differentiation to hepatic cells / 国际生物医学工程杂志

Objective To establish the long-term culture system for fetal skin fibroblast by performing long time in vitro cultivation of the cells,and study the potential of its differentiation to hepatocytes.Methods Fibroblast was isolated from human fetus skin tissue.Surface phenotypes of cells were detected by ICC and FCM,and biological characteristics were analyzed by the karyotype analysis and soft agar colony formation observation.ALB、CK18、CK19 were detected by ICC,glycogen stain by PAS,AFP and ALB mRNA by RT-PCR after P3～30cells were induced differentiation by cytokines of HGF,FGF4 and OSM.Results CD29,CD49f,HLA- Ⅰ and CD 105 were highly expressed while CD90 hardly in skin fibroblast.The rate of induced differentiation of fibroblast into hepatocyte-like cells was approximately 5％.The cells could be cultured in vitro for almost 50 passages with normal karyotype and no oncogenic and immortalized characteristics.Conclsion The skin fibroblast possesses the characteristic of mesenchymal stem cell and can be induced into hepatocyte-like cell in vitro.

Effect of human umbilical cord mesenchymal stem cell-paracrine substance on liver function and hepatocytes proliferation in FHF rat / 中华肝胆外科杂志

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.

Effect of human umbilical cord mesenchymal stem cell-secretion on proliferation and apoptosis in hepatocytes / 中华肝胆外科杂志

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.

Investigation of polyanhydride-glucan as stem cell carriers / 生物医学工程学杂志

<p><b>UNLABELLED</b>The purpose of this study with regard to the effects of polyanhydride--three-dimensional vector-glucan material on the fetal liver stem cell adhesion and proliferation was to find a new carrier. The methods of two-step collagenase perfusion digestion and liquid Percoll discontinuous density gradient centrifugation were used for the separation of fetal liver stem cells. The fetal liver stem cells were selected and cultivated in the polyanhydride-three-dimensional vector-glucan material. Inverted microscope was used to observe cell adhesion and growth status. Also performed were: Calculation of the rate of cell adhesion; MTT assay of the cells in each group absorbance value (OD value); collecting and counting the cells on the carrier scaffold. Then the cell carriers histological sections (HE staining) were observed. On the 7th day of cell culture, the cells were subjected to immunofluorescence staining and flow cytometry.</p><p><b>RESULTS</b>polyanhydride-three-dimensional vector-glucan promoted liver stem cells growth and adhesion. There were active functions of the liver stem cells within carrier materials. In the three-dimensional surface and the internal culture of liver stem cell, proliferation was sustained. After 40 days, the polyanhydride co-culture-three-dimensional vector-glucan showed no sign of toxicity to stem cells. Human fetal liver stem cells attached to the polyanhydride--three-dimensional vector-glucan stent. The cell proliferation went on well and exhibited sustained expression of markers; 7 days training led up to an increase of 19.7 percent in the number of cells. Conclusively, polyanhydride-three-dimensional vector-glucan can be used for promoting the proliferation of liver stem cells, and liver stem cells can be used as vectors in liver tissue engineering.</p>

Growth activity of fetal liver stem cells in polyanhydride-three-dimensional vector-glucan / 中国组织工程研究

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.

Long-term in vitro culture and hepatocytic differentiation of human umbilical cord-derived mesenchymal stem cells / 中国组织工程研究

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.

Detection of methylation in hepatocellular carcinoma using SYBR Green fluorescent quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.</p><p><b>METHODS</b>Following bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.</p><p><b>RESULTS</b>The amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.</p><p><b>CONCLUSION</b>SYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.</p>

Clustering distribution of the specific fragment R049 of uropathogeulc Escherichia coil / 中华微生物学和免疫学杂志

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.

Prepare and clinical application of HCV genotyping oligochip / 中华检验医学杂志

Objective To study the preparation of hepatitis C viruses (HCV) genotyping oligochip and its application in the detection of 76 hepatitis C patients.Methods Oligonucleotide probes and primers were designed in the 5’noncoding region and core region of HCV. The HCV typing chip was prepared by spotting the modified probes onto nylon membrane. Products of the second PCR were labeled with Dig-dUTP. Furthermore, 6 PCR products were sequenced.Results Using the chip,15 subtypes in 11 types of HCV were analyzed.Results of hybridization indicates that 76 hepatitis C patients were all positive and 20 health people were negative.Among 76 patients, 64 cases were 1b type, 11 cases were 2a type and 1 case was 3a type. Mix infection was not found. The results obtained by sequencing 6 samples and chip arraying were the same.Conclusion The HCV genotyping chip could be used in detecting serum HCV RNA and analyzing its genotypes.

Detecting the methylation of p16INK4A in primary hepatocellular carcinoma using a nested bisulfite sequencing-methylation specific polymerase chain reaction / 中华检验医学杂志

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.