Objective To investigate the efficacy and safety of umbilical cord blood ( UCB) - derived or autologous cytokine- induced killer ( CIK) cells combined with interferon therapy in patients with chronic hepatitis B ( CHB) . Methods Thirty CHB patients hospitalized in the Department of Infectious Diseases from October 2010 to June 2013 were included in the study. These patients were randomly and equally divided into first and second treatment groups and control group. The first or second treatment group underwent transplantation of UCB- derived or autologous CIK cells combined with interferon therapy; the control group received interferon therapy alone. At 4 and 12 weeks after treatment, alanine aminotransferase ( ALT) , serum hepatitis B virus ( HBV) marker, HBV DNA, and CD4+/CD8+ratio in peripheral blood were measured. Comparison of means was made by paired t test or independent- samples t test. Results At 12 weeks after transplantation of UCB- derived or autologous CIK cells, the first or second treatment group had ALT, HBeAg, and HBV DNA levels of ( 22. 6 ± 14. 4) or ( 32. 9 ± 15. 3) U /L, ( 12. 5 ± 5. 8) or ( 18. 4 ± 8. 8) PEIU /ml, and ( 3. 2 ± 0. 7) or ( 3. 7 ± 0. 6) log10 copies /ml, respectively, significantly lower than those for control group ( t = 2. 80 ~ 5. 45, P < 0. 05) . At 4 weeks after transplantation, the two treatment groups had significantly increased CD4+levels and CD4+/CD8+ratios compared with the control group ( t = 2. 21 ~ 2. 43, P < 0. 05) . No severe adverse reactions and transplantation- related severe complications were found in the two treatment groups. Conclusion For CHB patients, transplantation of UCB- derived or autologous CIK cells significantly inhibits virus replication rapidly, improves liver function, relieves clinical symptoms, improves cellular immunity, and has high safety.

Objective To observe the in vitro proliferation capacity and killing efficacy of cytokine- induced killer ( CIK) cells from umbilical cord blood ( UCB) and versus chronic hepatitis B ( CHB) patients of different ages. Methods Five samples of UCB from healthy fetuses born in our hospital from January to December, 2012, as well as 20 samples of peripheral blood from CHB patients, were divided into group A ( 5 samples of UCB) , group B ( 12 samples of peripheral blood from CHB patients aged 20- 35 years) , and group C ( 8 samples of peripheral blood from CHB patients aged over 35 years) . Mononuclear cells were separated by Ficoll- Hypaque centrifugation. Peripheral blood mononuclear cells were induced into CIK cells in the presence of cytokines. The immunophenotypes of CIK cells were determined by flow cytometry. The killing efficacy of CIK cells against K562 cells was measured by MTT assay. Results On day 15, CIK cells from group B multiplied 589. 15 ± 237. 6 times, versus 600. 93 ± 249. 1 times for group A ( P > 0. 05) ; CIK cells from group C multiplied 370. 45 ±165. 2 times, which were significantly lower than those for groups A and B ( P < 0. 05) . The killing efficacy of CIK cells from groups A, B, and C against K562 cells was ( 77. 3 ± 5. 1) %, ( 54. 5 ± 3. 5) %, and ( 44. 1 ± 3. 4) %, respectively; there were significant differences in killing efficacy between group A and groups B and C ( P < 0. 01) . Conclusion UCB- derived CIK cells have high in vitro proliferation capacity and killing efficacy. The in vitro proliferation capacity of CIK cells declines in CHB patients aged over 35 years, so autologous transplantation of CIK cells is not suitable for this group of patients.

Objective To investigate the relationship between Toll- like receptor 2 ( TLR2) in peripheral blood mononuclear cells ( PBMCs) and T helper 17 ( Th17) cells among hepatitis B patients and to provide a theoretical and experimental basis for the study on the mechanism of inflammatory response to hepatitis B virus ( HBV) infection. Methods Thirty- four previously untreated patients with hepatitis B ( 24 cases of chronic hepatitis B and 10 cases of acute hepatitis B) who visited or were hospitalized in the Center of Infectious Diseases, Tangdu Hospital, from July 2012 to July 2013, as well as ten healthy controls, were enrolled in this study. PBMCs were isolated and stimulated by HBV ( genotype C) envelope peptides ( specific) or propylene glycol monomethyl ether acetate ( PMA) plus ionomycin ( nonspecific) . Flow cytometry was performed to measure the expression of TLR2 and the percentage of Th17 cells. PBMCs were further stimulated by TLR2 agonist Pam3Csk4, and the changes in percentage of Th17 cells were evaluated. Comparison between groups was made by Kruskal-Wallis H test. Results When stimulated by PMA plus ionomycin, patients with chronic hepatitis B had a significantly higher percentage of Th17 cells than patients with acute hepatitis B and healthy controls [ ( 4. 08 ± 1. 78) % vs ( 1. 85 ± 1. 28) %, P = 0. 0009; ( 4. 08 ±1. 78) % vs ( 2. 09 ± 0. 53) %, P = 0. 0004], while the percentages of TLR2+and IL- 17A+TLR2+T cells in peripheral blood CD3+CD4+T cells showed no significant differences between patients with acute hepatitis B, patients with chronic hepatitis B, and healthy controls ( P > 0. 05 for all) . When stimulated by HBV envelope peptides, patients with chronic hepatitis B had significantly higher percentages of IL- 17A+T cells and TLR2+T cells than patients with acute hepatitis B [ ( 5. 45 ± 1. 61) % vs ( 3. 20 ± 1. 13) %, P = 0. 0006; ( 5. 19± 3. 18) % vs ( 1. 88 ± 1. 30) %, P = 0. 0006]. After the addition of Pam3Csk4, patients with chronic hepatitis B had a significantly increased percentage of Th17 cells, while patients with acute hepatitis B had a nonsignificantly increased percentage of Th17 cells. Conclusion TLR2 could directly influence Th17 cell responses, thereby playing a proinflammatory role in the immune response to HBV infection.

Objective To study the changes in peripheral blood T cell subsets among chronic hepatitis B ( CHB) patients and asymptomatic hepatitis B virus ( HBV) carriers ( ASC) , who have different viral loads, and to investigate the correlation of T cell subsets with transforming growth factor beta ( TGFβ) , alanine aminotransferase, and total bilirubin. Methods A total of 175 HBV infection patients admitted to our hospital from July to December, 2012 were recruited and divided into ASC group ( n = 112) and CHB group ( n = 63) ; 84 healthy controls who underwent physical examination in the same period were selected as control group. The percentages of T cell subsets in serum were determined by flow cytometry, and liver function parameters were measured. For normally distributed continuous data, comparison between groups was made by analysis of variance; for non- normally distributed continuous data, comparison between groups was made by Kruskal- Wallis H rank sum test. Results Among the 175 HBV infection patients, the percentages of CD3+, CD4+, and CD8+T cells and CD4+/CD8+ratio were72. 14%- 74. 07%, 38. 43%- 39. 47%, 30. 74%- 31. 42%, and 1. 31- 1. 34, respectively. Compared with the control group, the ASC group and CHB group had significantly increased TGFβ levels ( P < 0. 05 for both) , significantly reduced percentages of CD3+and CD4+T cells ( P < 0. 05 or P < 0. 01) , significantly reduced CD4+/CD8+ratios ( P < 0. 01) , and significantly increased percentages of CD8+T cells ( P<0.05 or P <0.01) .="" in="" the="" asc="" and="" chb="" both="" hbeag-="" positive="" negative="" patients="" had="" reduced="" percentages="" of="" t="" increased="" ratios.="" no="" correlation="" was="" found="" between="" hbv="" dna="" loads="" cell="" subsets="" two="" groups="" p="">0. 05) . Conclusion TGFβ may be involved in the pathogenesis of CHB, and its immunosuppressive effect may be exerted by inhibiting T cells and antigen- presenting cell maturation.

Objective To investigate the effects of splenectomy on peripheral immune cells including Th17 cells in patients with portal hypertension and to analyze the influence of splenectomy on their immune function. Methods Twenty- five portal hypertension patients treated with splenectomy in No. 180 Hospital of PLA from June 2012 to June 2014 were selected as observation group, and 25 healthy controls who underwent physical examination in the same period as control group. Flow cytometry was used to determine the percentages of CD3, CD4, and CD8 T lymphocytes, CD4 /CD8 ratio, and percentage of Th17 cells in the peripheral blood of portal hypertension patients ( 1 day before operation and at 7 days, 1 month, and 3 months after operation) and healthy controls. ELISA was used to evaluate the changes in serum interleukin- 6 ( IL- 6) , interleukin- 17 ( IL- 17) , and interleukin- 23 ( IL- 23) . Comparison between groups was made by one-way ANOVA and LSD- t test. Results Before splenectomy, the portal hypertension patients had significantly lower percentages of CD3, CD4, and CD8 T lymphocytes and CD4 /CD8 ratio ( P < 0. 01 for all) and significantly higher percentage of Th17 cells and levels of their associated inflammatory cytokines ( IL- 6, IL- 17, and IL- 23) ( P < 0. 01 for all) , as compared with the control group. At 7 days, 1month, and 3 months after operation, the percentages of CD3, CD4, and CD8 T cells and CD4 /CD8 ratio in portal hypertension patients first decreased and then increased, and these values were significantly lower at 7 days after operation than before operation ( P < 0. 01 for all) ; at3 months after operation, the patients showed no significant differences in the percentages of CD3, CD4, and CD8 T cells and CD4 /CD8 ratio compared with the control group ( P > 0. 05 for all) . At 7 days, 1 month, and 3 months after operation, the percentage of Th17 cells and levels of their associated inflammatory cytokines ( IL- 6, IL- 17, and IL- 23) in portal hypertension patients decreased gradually ( P <0. 05 for all) . Conclusion Th17 cells and their associated inflammatory cytokines ( IL- 6, IL- 17, and IL- 23) are reduced in portal hypertension patients treated with splenectomy, so their immune function can be improved.

Objective To investigate the inhibitory effect of inhibition of antigen presentation attenuators ( iAPA) - based dendritic cells ( DC) and cytotoxic T lymphocytes ( CTL) ( iAPA- DC/CTL) on HepG2 xenograft in nude mice. Methods Human hepatocarcinoma HepG2 cells were subcutaneously implanted into nude mice on nude mice to establish a HepG2 xenograft model transplanted tumor model of human HCC. Twelve nude mice were randomly and equally divided into two groups: normal saline control group ( C group) and iAPA- DC /CTL group ( DC group) . After four times of treatment with iAPA- DC /CTL ( once a week) , all mice were sacrificed. Tumor growth was evaluated by measuring the long and short diameters and delineating the tumor growth curve. The tumors were weighed, and the tumor inhibition rate was calculated. In addition, histopathological examination was performed. Comparison of means between the two groups was made by independent- samples t test. Results The HepG2 xenograft model was successfully established in 92. 31% of all mice. The tumor volume was 697. 69 ± 143. 99 mm3 in C group and 485. 64 ± 188. 75 mm3 in DC group; the tumor growth was significantly slower in DC group than in C group ( t = 2. 28, P < 0. 05) . The tumor weight was 0. 32 ± 0. 07 g in C group and 0. 22 ± 0. 08 g in DC group; DC group had a significantly lower tumor weight than C group ( t = 2. 31, P < 0. 05) , and the tumor inhibition rate was 30. 39%. After immunohistochemical staining, T lymphocyte count was 0 cell /high- power field ( HPF) in C group and 39. 74 ± 5. 11 cells /HPF in DC group; the number of T lymphocytes in DC group was significantly higher than that in C group ( t = 19. 05, P < 0. 05) . Conclusion iAPA- DC /CTL can effectively inhibit the growth of subcutaneous HepG2 xenograft in nude mice.

Objective To investigate the risk factors for non- alcoholic fatty liver disease ( NAFLD) and to provide a basis for the prevention of NAFLD. Methods A total of 190 patients with NAFLD who visited the First Affiliated Hospital of Soochow University from January2011 to January 2013 were included in the study. The investigated factors included sex, age, height, weight, dietary habit, smoking and alcohol consumption, educational level, occupation, intensity and duration of physical exercise, bedtime, previous history, and family history. Univariate and multivariate analyses were performed using SPSS 18. 0 to determine the risk factors for NAFLD. Results The univariate analysis showed that sex, age, dietary habit, occupation, body mass index ( BMI) , and educational level were associated with NAFLD ( P <0. 05) . The logistic regression analysis showed that the risk factors for NAFLD were sex ( OR = 5. 692, P = 0. 029) , age ( OR = 0. 423, P =0. 041) , occupation ( OR = 0. 698, P = 0. 008) , BMI ( OR = 3. 939, P = 0. 003) , educational level ( OR = 5. 463, P = 0. 030) , and dietary habit ( OR = 9. 235, P = 0. 039) . Conclusion NAFLD may be related to many factors, and corresponding preventive measures may reduce the development of NAFLD.

Objective To investigate the collection and preservation of blood specimens from patients with nonalcoholic fatty liver disease ( NAFLD) and the establishment and information management of biobank. Methods Whole blood samples were collected from 1226 patients who were diagnosed with NAFLD based on B- mode ultrasound and blood tests from October 2009 to October 2013. Biochemical parameters were measured. Plasma and whole- blood genomic DNA was extracted from the samples, and the purity and concentration of DNA were determined. Specimens were preserved in a refrigerator (- 80℃) . An information management system for NAFLD biobank was established. Results Specimens of 1226 NAFLD patients, including those of 83 twins and 100 families, were collected. The success rate was100% for extraction of plasma and whole- blood genomic DNA. One hundred DNA samples were randomly selected for testing, and the results showed that the collected specimens met the requirements of following experiments. Conclusion The NAFLD Biobank has been successfully established in this study. It has the standard information management system and enables the quality control and information management of specimens, laying a solid foundation for further research on NAFLD.

Objective To analyze and compare the clinical effects of comprehensive treatment and liver transplantation for hepatocellular carcinomas ( HCC) exceeding Milan criteria. Methods A retrospective analysis was performed on the clinical data of 157 patients with HCC ( exceeding Milan criteria) who received comprehensive treatment ( 99 patients, including 48 cases meeting Shanghai criteria) and liver transplantation ( 58 patients, including 26 cases meeting Shanghai criteria) in our hospital from January 2006 to January 2010. The median survival times and 1-, 2-, and 3- year survival rates were calculated by the Kaplan- Meier method, and the log- rank test was used for survival difference analysis. Results For all patients, the median survival times of the comprehensive treatment group and liver transplantation group were 18. 00 ± 1. 15 and 23. 40 ± 4. 44 months, respectively, and their 3- year survival rates were ( 18. 2 ± 3. 9) % and ( 39. 7 ±6.4) %, respectively; the survival was significantly improved by the treatment of liver transplantation ( P =0.009) . For patients meeting Shanghai criteria, the median survival times of the comprehensive treatment group and liver transplantation group were 20. 00 ± 1. 17 and 36. 00 ± 0. 00 months, respectively, and their 3- year survival rates were ( 25. 0 ± 6. 3) % and ( 57. 7 ± 9. 7) %, respectively; there was significant survival difference between the two groups ( P = 0. 008) . For patients exceeding Shanghai criteria, the median survival times of the comprehensive treatment group and liver transplantation group were 16. 00 ± 1. 78 and 16. 00 ± 1. 69 months, respectively, and their 3- year survival rates were ( 11. 8 ± 4. 5) % and ( 25. 0 ± 7. 7) %, respectively; there was no significant survival difference between the two groups ( P = 0. 221) . Conclusion Compared with comprehensive treatment, liver transplantation leads to a significant higher long- term survival rate in HCC patients exceeding Milan criteria but meeting Shanghai criteria. However, the two therapies cause no significant survival difference in HCC patients exceeding Shanghai criteria.

Objective To assess the efficacy of transcatheter arterial chemoembolization ( TACE) in patients with hepatocellular carcinoma ( HCC) before hepatectomy. Methods PubMed, Embase, the Cochrane Library, CNKI, VIP, and Wanfang Data were searched to identify randomized controlled trials ( RCTs) evaluating the efficacy of preoperative TACE plus hepatectomy ( study group) versus hepatectomy alone ( control group) in HCC patients published up to March 12, 2013. The quality of included studies was assessed, and relevant data were extracted. Statistical analysis was performed by RevMan 5. 2. Results A total of 4 RCTs involving 342 participants were included. Meta- analysis of data extracted from the included RCTs showed that there were no significant differences between the study group and control group in 1-, 3-, and 5- year disease- free survival ( DFS) , with relative risks ( RRs) ( 95% confidence intervals ( CIs) ) of 1. 07 ( 0. 92-1. 25) ( P = 0. 38) , 1. 05 ( 0. 79, 1. 41) ( P = 0. 72) , and 0. 95 ( 0. 64- 1. 42) ( P = 0. 81) , respectively, in 1-, 3-, and 5- year overall survival ( OS) , with RRs ( 95% CIs) of 1. 01 ( 0. 92- 1. 10) ( P = 0. 85) , 1. 14 ( 0. 97- 1. 34) ( P = 0. 11) , and 0. 95 ( 0. 75-1. 21) ( P = 0. 68) , respectively, and in rate of postoperative complications and mortality, with RRs ( 95% CIs) of 0. 89 ( 0. 45- 1. 75) ( P= 0. 73) and 0. 77 ( 0. 25- 2. 37) ( P = 0. 65) , respectively. Conclusion TACE before hepatectomy cannot increase DFS and OS and reduce complications and mortality in HCC patients. However, the numbers of studies and cases included in the analysis are small, and more high- quality, large- sample RCTs are needed to confirm the conclusion.

Objective To analyze the correlation of gamma- glutamyl transpeptidase ( GGT) level with alpha- fetoprotein ( AFP) level and to re- evaluate the diagnostic value of GGT for hepatocellular carcinoma ( HCC) . Methods Four hundred and seventy- two patients with HCC or liver cirrhosis, who were hospitalized in China- Japan Friendship Hospital from January 2003 to June 2009, were included in the study. The correlation between GGT and AFP was analyzed by Spearman nonparametric test. The cut- off values for the two parameters were determined based on their receiver operating characteristics ( ROC) curves, areas under the ROC curve ( AUCs) , sensitivity, and specificity, and the diagnostic values were presented using their sensitivity, specificity, and correct index. Statistical analysis was performed using SPSS 17. 0. Normally distributed continuous data were analyzed by independent- samples t test, while non- normally distributed continuous data were analyzed by Mann- Whitney U test. Categorical data were analyzed by Pearson chi- square test, continuity- corrected chi-square test, or Fisher's exact test. Results Among 472 patients, 224 were diagnosed with HCC, and 248 with liver cirrhosis. Compared with cirrhotic patients, HCC patients had a significantly higher GGT level ( 113 ( 58- 254) U /L vs 38 ( 22- 72) U /L, Z =- 11. 037, P <0. 001) and a significantly higher AFP level ( 429. 5 ( 15. 7- 1210. 0) ng /ml vs 5. 7 ( 3. 4- 18. 2) ng /ml, Z =- 10. 157, P < 0. 001) . A significant correlation was found between GGT and AFP ( r = 0. 449, P < 0. 001) . The AUC was 0. 784 for GGT and 0. 788 for AFP. The cut- off value was 60 U /L for GGT and 20 ng /ml for AFP. The sensitivity was 74. 1% for GGT, 71. 8% for AFP, and 90. 7% for a combination of the two parameters, the specificity was 70. 2%, 77. 6%, and 58. 7%, respectively, and the correct index was 0. 443, 0. 494, and0. 494, respectively. Conclusion GGT may be regarded as one biomarker for HCC, and its level is significantly correlated with AFP level.The diagnostic value of AFP may not be improved when used in combination with GGT.

Objective To investigate the relationship of the classification of Doppler ultrasound blood flow signals with matrix metalloproteinase- 9 ( MMP- 9) expression and microvessel density ( MVD) in primary hepatocellular carcinoma ( PHC) . Methods Sixty patients with PHC, who underwent surgical resection in the Second Affiliated Hospital of Dalian Medical University from April 2008 to April 2013, were included in the study. These patients underwent ultrasound examination before operation. The classification of Doppler ultrasound blood flow signals in the focus was recorded. The expression of MMP- 9 and CD31 in carcinoma tissues was detected by immunohistochemistry, and MVD was calculated. Continuous data were analyzed by t test, while categorical data by chi- square test; the relationship was evaluated by Spearman correlation analysis. Results The classification of Doppler ultrasound blood flow signals was as follows: grade 0 ( 15 cases) ;grade 1 ( 20 cases) ; grade 2 ( 15 cases) ; grade 3 ( 10 cases) . The positive rate of MMP- 9 expression was 72. 3%. The MVD was 43. 2 ±5. 4. The classification of Doppler ultrasound blood flow signals in the focus was positively correlated with MMP- 9 expression and MVD ( r =0.56, P <0.05) . Conclusion The classification of Doppler ultrasound blood flow signals can reveal vascular changes in the focus, and it can be used in the clinical evaluation of angiogenesis in tumor tissues and the clinical diagnosis and treatment of PHC.