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Objective To explore the synergetic effect of HBX protein and M2 macrophages in inflammatory microenvironment on invasion and metastasis of hepatocellular carcinoma cells.Methods Hep3B cells were infected with recombinant lentivirus carrying HBx gene,following co-culture with THP-1 original M2 macrophages.The cells were divided into six groups:two infected groups (Hep3B +and Hep3B + + M2),four non-infected groups (Hep3B-,Hep3B-+ LV5,Hep3B-+ M2,Hep3B-+LV5 + M2).Western blot (WB) was used to assess the expression changes of E-cadherin and N-cadherin,markers of epithelial-mesenchymal transition (EMT).The cellular location of EMT markers was observed by immunofluorescence confocal microscopy.Transwell assay was used to evaluate the invasion ability of Hep3B cells.Results HBX protein overexpressed in Hep3B cells by lentivirus infection.After 72 h co-culture with M2 macrophages,WB results showed that E-cadherin descreased significantly in Hep3B+ (0.42 ±0.11) when compared with Hep3B-(1.00 ±0.18) (t =4.762,P ＜0.05),while N-cadherin was significantly higher in Hep3B + (2.85 ± 0.44) than in Hep3B-(1.00 ± 0.17) (t =4.762,P ＜ 0.05).M2macrophages decreased E-cadherin expression in Hep3 B + + M2 (0.1 ± 0.13) compared with Hep3 B + (t =3.255,P ＜0.05),while N-cadherin expression increased in Hep3B+ + M2 (4.18 ± 0.52) (t=10.009,P ＜ 0.05).Non-Infected groups didn't change the markers of E-cadherin and N-cadherin.It was suggested that invasion ability of Hep3B increased by HBx overexpression.Conclusions HBX protein and M2 macrophages synergetically mediated the invasion and metastasis of hepatocellular carcinoma cells by EMT.
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Objective To observe recombinant plasmids were constructed with the macrophage colony-stimulating factor ( GM-SCF) , interleukin -21 (IL-21) and retinoic acid early transcription factor-1 (Rae-1), and observe the inhibitory effects in subcutaneous liver cancer model in mice with the recombinant plasmids.Methods The recombinant plasmids of GM-SCF, IL-21 and Rae-1 were constructed with RT-PCR method, mouse model was constructed, the model mice were randomly divided into six groups including control, IRES/GFP, IRES/IL21, IRES/GM-SCF, IRES/GM-SCF-IL21 and IRES/combination with 10 mice included in each group, each groups (15 mice) were treated with the corresponding gene therapy.The survival rate were observed after 60 days.The blood levels of interferon -γ(IFN-γ) and interleukin -2 (IL-2) were detected in each group.Results The pGM-CSF-GFP-IRES-Rae-1-IL-21 has been successfully constructed.All mice had demised 14 and 16 days after treatment in the control and IRES/GFP groups, respectively.There were 2, 1, 11 mice remaining after 60 days of treatment in the IRES/GM-SCF, IRES/IL21 and IRES/GM-SCF-IL21 groups respectively.The survival rate of mice at 60 days of treatment was 73.33%, 13.33%, and 6.67% for groups IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21, respectively.The survival rate of the mice was significantly higher in IRES/GM-SCF-IL21 than the other groups.The levels of IL-2 and INF-γof mice 1-6 days after treatment gradually increased in the IRES/combination groups, including IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21.They were highest in the IRES/combination group and lowest (P<0.01) in the IRES/GM-SCF and IRES/IL21 groups, with the IRES/GM-SCF-IL21 group showing intermediate levels.By 6-10 days after treatment, IL-2 and INF-γlevels had stably increased in the IRES/combination groups, but had gradually decreased in the IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21 groups.At the end of treatment, IL-2 and INF-γlevels were significantly (P<0.01) higher in the IRES/GM-SCF-IL21 than were found in either the IRES/GM-SCF group or IRES/IL21 group, which were also significantly (P<0.01) higher than either the IRES/GFP or control groups.The levels of IL-2 and INF-γwere highest in the IRES/combination group ( P<0.01) and not significantly different among the IRES/GM-SCF, IRES/IL21, IRES/GFP, and control groups.Conclusion The inhibitory effects in subcutaneous liver cancer model in mice were obvious significantly, and its mechanism maybe be related to the activation of the body's immune.
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Objective To evaluate the prognostic impact of a wide spectrum of pathologic parameters in a consecutive series of homogenously treated and well-characterized patients with stage Ⅰ and Ⅱ colorectal cancer, and to investigate the prognostic value of lymph node occult disease (micrometastasis) in disease-free survival rate detected by immunohistochemistry with epithelial membrane antigen and carcinoembryonic antigen. Methods The study included 126 patients operated on by a single surgeon for stage Ⅰ and Ⅱ colorectal tumors. The postoperative follow-up was performed for 64 to 106 months. At least 10 lymph nodes were harvested and examined in all the specimens. The prognostic value of 10 pathologic parameters, including lymph node occult disease (micrometastasis) detected by immunohistochemistry was investigated. Results Multivariate analysis identified lymphatic vessel invasion (absent or present;P=0.009) in lymph node positive and negative by immunohistochemistry. The five-year disease-free survival rates were 78.7%, 65.5% and 43.8% for the lymph node negative, isolated tumor cells and micrometastasis groups, respectively. There was significant difference between the lymph node negative and micrometastasis groups (P=0.005). However, the difference between the lymph node negative and isolated tumor cells groups was not statistically significant (P=0.144). Conclusions We propose that for patients found micrometastasis in lymph node with high-risk stage Ⅰ and Ⅱ colorectal cancer, adjuvant therapies are justified and effective.
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Objective To evaluate the impact of pathologic parameters and lymphatic mierometastasis on 5-year disease-frtee survival in patients with stages Ⅰ and Ⅱ colorectal cancer.Methods Surgical operation was performed in 126 patients with stage Ⅰ and Ⅱ colorectal cancer.Sixteen (range,10-28)lymph nodes were harvested in each specimen and immunohistochemical staning was performed. Theimpact of pathologic parameters and lymphatic micrometastases in survival was estimated by KaplanMeier.Results The mean follow up time was 64.11 (range,64-106) months. Multivariate analysisrevealed that lymphatic vessel invasion and depth of tumor invasion were correlated with positive CEA in lymph node,and unrelated with clinical pathologic factors.There was no significant difference between pathologic parameters and five year disease-free survival rates. The five-year diseasse-free survival rates was 75.4 percent in CEA negative patients,68.2 percent in patients with isolated tumor cells,and 46.2 percent in patients positive for micrometastasis.There was no significant difference in 5 year disease-free survival between CEA negative patients and patients with isolated tumor cells (P=0.245).However,the5-year disease-free survival was lower in patients positive for micrometastases compared to CEA negativepatients (P=0.003).Conclusions The presence of micrometastases in patients with stages Ⅰ and Ⅱ colorectal cancer may result in poor prognosis and high recurrence,and adjuvant chemotherapy will bejustified and effective.