Immunological classification of hepatitis B virus-positive hepatocellular carcinoma by transcriptome analysis.

BACKGROUND: Hepatitis B virus (HBV) infection is a major factor responsible for HBV+ hepatocellular carcinoma (HCC). AIM: An immunological classification of HBV+ HCC may provide both biological insights and clinical implications for this disease. METHODS: Based on the enrichment of 23 immune signatures, we identified two immune-specific subtypes (Imm-H and Imm-L) of HBV+ HCC by unsupervised clustering. We showed that this subtyping method was reproducible and predictable by analyzing three different datasets. RESULTS: Compared to Imm-L, Imm-H displayed stronger immunity, more stromal components, lower tumor purity, lower stemness and intratumor heterogeneity, lower-level copy number alterations, higher global methylation level, and better overall and disease-free survival prognosis. Besides immune-related pathways, stromal pathways (ECM receptor interaction, focal adhesion, and regulation of actin cytoskeleton) and neuro-related pathways (neuroactive ligand-receptor interaction, and prion diseases) were more highly enriched in Imm-H than in Imm-L. We identified nine proteins differentially expressed between Imm-H and Imm-L, of which MYH11, PDCD4, Dvl3, and Syk were upregulated in Imm-H, while PCNA, Acetyl-a-Tubulin-Lys40, ER-α_pS118, Cyclin E2, and ß-Catenin were upregulated in Imm-L. CONCLUSION: Our data suggest that "hot" tumors have a better prognosis than "cold" tumors in HBV+ HCC and that "hot" tumors respond better to immunotherapy.

Epigenetic silencing of GCH1promotes hepatocellular carcinoma growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting tetrahydrobiopterin de novo biosynthesis.

Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo, while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC.

Relationships among cigarette smoking, urinary biomarkers, and urothelial carcinoma risk: a case-control study.

Cigarette smoke is a known risk factor for urothelial carcinoma (UC). However, there is limited information about the distributions and effects of volatile organic compounds (VOCs) on smoking-related UC risk. With this hospital-based case-control study, we explored the associations between urinary levels of cotinine and VOC metabolites (acrylamide, 1,3-butadiene, and benzene) and the risk of UC. Urological examinations and pathological verifications were used to confirm the diagnoses of UC. All study participants provided smoking-related information via questionnaires and face-to-face interviews; they also provided urine samples for the measurement of VOC metabolites, cotinine, and 8-hydroxydeoxyguanosine (8-OHdG), which was used as an indicator of oxidative stress. We applied multiple logistic regression analysis to estimate the risk of UC, and we found that levels of urinary cotinine and 8-OHdG were higher in the UC group than in the control group. Furthermore, urinary levels of VOC metabolites, including N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine, N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine-3, trans,trans-muconic acid (t,t-MA), and S-phenylmercapturic acid (SPMA), increased with increasing levels of urinary cotinine. After adjusting for potential risk factors, dose-response relationships were observed between UC risk and urinary levels of AAMA, t,t-MA, SPMA, and 8-OHdG. Participants with high urinary levels of cotinine, AAMA, t,t-MA, SPMA, and 8-OHdG had risks of UC that were 3.5- to 6-fold higher than those of participants with lower levels. Future, large-scale investigations of the risks of UC should be explored, and repeated measurement of VOC metabolites should be assessed.

Liraglutide protects non-alcoholic fatty liver disease via inhibiting NLRP3 inflammasome activation in a mouse model induced by high-fat diet.

Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue that has recently become the first-line treatment for type 2 diabetes mellitus (T2DM), has also been reported to decrease fatty degeneration of the liver. The purpose of this study is to explore whether liraglutide improves high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) in mice through inhibiting the NLRP3 inflammasome in the liver. After daily intraperitoneal injection of liraglutide (0.6 mg/kg body weight) for four weeks, the liver, liver/body weight, serum levels of ALT, AST, total cholesterol, triglycerides and LDL were significantly decreased in a high-fat diet-induced NAFLD mouse model. The hepatic steatosis among sections of H&E and Oil Red O staining was also markedly reduced after treatment with liraglutide. The expressions of NLRP3 inflammasome components (including NLRP3, ASC, and caspase-1) in the liver of mice after treatment with liraglutide were decreased substantially. In vitro studies found that the mitochondrial dysfunction in Kupffer cells induced by palmitic acid was attenuated, and the protein levels of NLRP3, ASC and caspase-1 were also decrease markedly. These results demonstrate that liraglutide was able to alleviate high-fat diet-induced hepatic steatosis via inhibiting NLRP3 inflammasome activation, suggesting that liraglutide is a potent drug that can reverse the pathological hallmarks of NAFLD.

Endoplasmic reticulum stress in perivascular adipose tissue promotes destabilization of atherosclerotic plaque by regulating GM-CSF paracrine.

BACKGROUND: Perivascular adipose tissue (PVAT) accelerates plaque progression and increases cardiovascular risk. We tested the hypothesis that PVAT contributed to plaque vulnerability and investigated whether endoplasmic reticulum stress (ER stress) in PVAT played an important role in vulnerable plaque. METHODS: We transplanted thoracic aortic PVAT or subcutaneous adipose tissue as a control, from donor mice to carotid arteries of recipient apolipoprotein E deficient (apoE-/-) mice after removing carotid artery collar placed for 6 weeks. Two weeks after transplantation, ER stress inhibitor 4-phenyl butyric acid (4-PBA) was locally administrated to the transplanted PVAT and then animals were euthanized after 4 weeks. Immunohistochemistry was performed to quantify plaque composition and neovascularization. Mouse angiogenesis antibody array kit was used to test the angiogenic factors produced by transplanted adipose tissue. In vitro tube formation assay, scratch wound migration assay and mouse aortic ring assay were used to assess the angiogenic capacity of supernatant of transplanted PVAT. RESULTS: Ultrastructural detection by transmission electron microscopy showed transplanted PVAT was a mixed population of white and brown adipocytes with abundant mitochondria. Transplanted PVAT increased the intraplaque macrophage infiltration, lipid core, intimal and vasa vasorum neovascularization and MMP2/9 expression in plaque while decreased smooth muscle cells and collagen in atherosclerotic plaque, which were restored by local 4-PBA-treatment. Antibody array analysis showed that 4-PBA reduced several angiogenic factors [Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), MCP-1, IL-6] secreted by PVAT. Besides, conditioned medium from 4-PBA treated-PVAT inhibited tube formation and migration capacity of endothelial cells and ex vivo mouse aortic ring angiogenesis compared to conditioned medium from transplanted PVAT. mRNA expression and protein levels of GM-CSF were markedly elevated in adipocytes under ER stress which would be suppressed by 4-PBA. In addition, ER stress enhanced NF-κB binding to the promoter of the mouse GM-CSF gene in adipocytes confirmed by Chromatin immunoprecipitation analyses. CONCLUSIONS: Our findings demonstrate that ER stress in PVAT destabilizes atherosclerotic plaque, in part through increasing GM-CSF paracrine via transcription factor NF-κB.

Prognostic value of lymph nodes count on survival of patients with distal cholangiocarcinomas.

AIM: To evaluate the prognostic value of the number of retrieved lymph nodes (LNs) and other prognostic factors for patients with distal cholangiocarcinomas, and to determine the optimal retrieved LNs cut-off number. METHODS: The Surveillance, Epidemiology and End Results database was used to screen for patients with distal cholangiocarcinoma. Patients with different numbers of retrieved LNs were divided into three groups by the X-tile program. X-tile from Yale University is a useful tool for outcome-based cut-point optimization. The Kaplan-Meier method and Cox regression analysis were utilized for survival analysis. RESULTS: A total of 449 patients with distal cholangiocarcinoma met the inclusion criteria. The Kaplan-Meier survival analysis for all patients and for N1 patients revealed no significant differences among patients with different retrieved LN counts in terms of overall and cancer-specific survival. In patients with node-negative distal cholangiocarcinoma, patients with four to nine retrieved LNs had a significantly better overall (P = 0.026) and cancer-specific survival (P = 0.039) than others. In the subsequent multivariate analysis, the number of retrieved LNs was evaluated to be independently associated with survival. Additionally, patients with four to nine retrieved LNs had a significantly lower overall mortality risk [hazard ratio (HR) = 0.39; 95% confidence interval (CI): 0.20-0.74] and cancer cause-specific mortality risk (HR = 0.32; 95%CI: 0.15-0.66) than other patients. Additionally, stratified survival analyses showed persistently better overall and cancer-specific survival when retrieving four to nine LNs in patients with any T stage of tumor, a tumor between 20 and 50 mm in diameter, or a poorly differentiated or undifferentiated tumor, and in patients who were ≤ 70-years-old. CONCLUSION: The number of retrieved LNs was an important independent prognostic factor for patients with node-negative distal cholangiocarcinoma. Additionally, patients with four to nine retrieved LNs had better overall and cancer-specific survival rates than others, but the reason and mechanism were unclear. This conclusion should be validated in future studies.

Overexpression and correlation of HIF-2α, VEGFA and EphA2 in residual hepatocellular carcinoma following high-intensity focused ultrasound treatment: Implications for tumor recurrence and progression.

Rapid growth of residual tumors can occur as a result of their recurrence and progression. The present study aimed to investigate the expression of hypoxia inducible factor-2 subunit α (HIF-2α), vascular endothelial growth factor A (VEGFA), erythropoietin-producing hepatocellular A2 (EphA2) and angiogenesis in residual hepatocellular carcinoma (HCC), following treatment with high-intensity focused ultrasound (HIFU) ablation, in order to investigate the association between protein expression and tumor recurrence and growth. Athymic BALB/c (nu/nu) mice were subcutaneously inoculated with the HCC cell line HepG2, in order to create xenograft tumors. Approximately 30 days post-inoculation, eight mice were treated with HIFU, whereas eight mice received no treatment and acted as the control group. Residual tumor tissues were obtained from the experimental groups after one month. Levels of HIF-2α, VEGFA, EphA2 and cluster of differentiation 31 (CD31) expression was measured by immunohistochemical staining. CD31-positive vascular endothelial cells were counted to calculate microvascular density (MVD), and western blot analysis was performed to determine levels of HIF-2α, VEGFA, and EphA2 protein. It was found that the expression levels of HIF-2α, VEGFA, EphA2, and MVD proteins in residual HCC tissues were significantly higher than in the control group tissues (P<0.05). Tumor MVD was strongly correlated with VEGFA (R=0.957, P<0.01) and EphA2 (R=0.993, P<0.01) protein expression levels. Furthermore, there was a significant positive correlation between HIF-2α and EphA2 expression (R=0.991, P<0.01). The correlation between VEGFA and EphA2 expression was also positive (R=0.985, P<0.01). These data suggest that overexpression of HIF-2α, VEGFA and EphA2 is related to angiogenesis in residual HCC following HIFU ablation, potentially via their association with key mediators of recurrence.

Effect of CD16a, the surface receptor of Kupffer cells, on the growth of hepatocellular carcinoma cells.

FcγRIIIa (CD16) is a low-affinity Fc receptor of IgG. As the idio-binding receptor of IgG Fc, it plays an important role in the antibody-dependent cellular cytotoxicity of natural killer cells. The aim of the present study was to investigate the distribution of Kupffer cells (KCs) and the expression of their surface receptor FcγRIIIa in hepatocellular carcinoma. Furthermore, we also aimed to observe the functional mechanism of FcγRIIIa. Immunohistochemical analysis was employed to study KCs and FcγRIIIa. In order to explore the role of FcγRIIIa in the growth of cancer cells, KCs and H22 tumor cells were co-cultured in different serum. The mRNA expression levels of tumor necrosis factor (TNF)-α and FcγRIIIa were analyzed by RT-qPCR; the TNF-α and FcγRIIIa protein expression levels were examined by enzyme­linked immunosorbent assay and western blot analysis, respectively. Our results showed that the number of Kuppfer cells in cancerous tissues (21.6±7.8) was lower than those in para-cancerous (68.8±9.1) tissues and adjacent normal hepatic tissues (62.0±1.9) (P<0.01); this decreased with the reduction in the differentiation degree of cancer (P<0.05). FcγRIIIa-positive cells were similar in morphology to KCs, and their distributive tendency was coincident (P<0.05). The increase in CD16a mRNA levels in the group treated with immune serum was 3.9-, 4.9- and 3.9-fold greater than that in the ordinary serum group at different time points, and CD16a protein expression also markedly increased (P<0.05). However, these effects were inhibited by the addition of anti-IgG Fc serum (P<0.05). The results of the present study suggested that FcγRIIIa resided in KCs, and it contributed to the inhibition of the growth of liver tumor cells.

Connexin32­mediated antitumor effects of suicide gene therapy against hepatocellular carcinoma: In vitro and in vivo anticancer activity.

Normal hepatocytes express connexin32 (Cx32), which forms gap junctions at cell­cell contact areas. The aim of the present study was to investigate whether Cx32 mediates the cell death­inducing effects of ultrasound microbubbles carrying the herpes simplex virus thymidine kinase (HSV­TK) suicide gene against hepatocellular carcinoma cells in vitro and in vivo. HepG2 cells were exposed to different concentrations of trans­retinoic acid (ATRA) in culture, to evaluate the intrinsic antitumor effect of ATRA. Detailed in­vitro and in­vivo investigations on the antitumor effects of ATRA via Cx32 mediation were performed, and the possible underlying mechanisms of action of the compound were then examined. The gene expression of HSV­TK transfected by ultrasound wave irradiation in the HepG2 cells was quantified using reverse transcription­quantitative polymerase chain reaction analysis. The effects on cell death were assessed using an MTT assay. The protein expression levels of Cx32 in ATRA­untreated or ATRA­treated tissues were quantified by immunohistochemical analysis and Western blot assays. The HSV­TK gene was successfully transfected into the HepG2 cell using ultrasound wave irradiation, and was stably expressed. Compared with the other groups, the HSV­TK gene group treated with ATRA exhibited an increased number of apoptotic cells (P<0.05) and improved tumor suppression (P<0.05). ATRA significantly increased the expression of Cx32 in the hepatoma tissues (P<0.01). The present study demonstrated that ATRA elevated the protein expression of Cx32 and enhanced the bystander effect of the HSV­TK/GCV suicide gene therapy system, which may provide a potential strategy for hepatocellular carcinoma treatment.

High-intensity focused ultrasound combined with herpes simplex virus thymidine kinase gene-loaded ultrasound-targeted microbubbles improved the survival of rabbits with VX2 liver tumor.

BACKGROUND: To explore the anti-tumor effect of high-intensity focused ultrasound (HIFU) combined with herpes simplex virus thymidine kinase (HSV-TK) gene-loaded ultrasound-targeted microbubbles on VX2 rabbit liver tumors. METHODS: Seventy-five New Zealand white rabbits were randomly divided into five groups after the models of VX2 rabbit liver tumors were established: (a) HIFU group; (b) HIFU and HSV-TK group (HIFU + HSV-TK); (c) HIFU, HSV-TK and ultrasound group (HIFU + HSV-TK + US); (d) HIFU, HSV-TK gene-loaded microbubbles and ultrasound group (HIFU + HSV-TK-MBs + US); and (e) HSV-TK gene-loaded microbubbles and ultrasound group (HSV-TK-MBs + US). After 2 weeks of VX2 liver tumor implantation, rabbits in groups (a), (b), (c) and (d) received HIFU to establish rabbit models of residual tumor by ablating 80% of the tumor volume. After HIFU ablation, rabbits in different groups received MBs wrapped around HSV-TK or HSV-TK solution via marginal ear veins and/or local ultrasonic irradiation to the tumor. Six rabbits in each group were sacrificed 48 h after the corresponding treatment, and tumors were extracted for in vitro experiments. Thymidine kinase mRNA was detected by the real-time polymerase chain reaction. The green fluorescent protein expression in liver tumor was detected by western blotting and immunohistochemistry. Tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. The growth curves of VX2 liver tumors and survival curves of rabbits were compared. RESULTS: Forty-eight hours after treatment, TK mRNA and protein were the highest in the HIFU + HSV-TK + US + MBs group and the HSV-TK + US + MBs group (p < 0.05). At 48 h after treatment, the apoptotic index of tumor cells in HIFU + HSV-TK-MBs + US group was the highest (p < 0.05). Compared to other groups, HIFU combined with MBs wrapped HSV-TK suicide gene significantly inhibited tumor growth in vivo (p < 0.05) and prolonged the survival time of animals (p < 0.05). CONCLUSIONS: HIFU combined with HSV-TK gene-loaded ultrasound-targeted MBs significantly inhibited the growth of VX2 rabbit liver tumors in vivo and prolonged the survival time of the animals, providing a novel gene delivery method and a novel strategy for liver tumor treatment.

[Effect of ultrasound microbubble carrying herpes simplex virus thymidine kinase on hepatocellular carcinoma in mice].

OBJECTIVE: To observe the effect of ultrasound microbubble carrying herpes simplex virus thymidine kinase hepatocellular carcinoma in mice. METHODS: Kunming mice were inoculated subcutaneously with H22 tumor cells. 40 male mice bearing subcutaneous hepatoma were randomized into 4 groups: PBS (group A), HSV1-TK (group B), HSV1-TK (group C), and microbubble carrying HSV1-TK (group D) were injected into the tail vein every 3 days. Mice in group C and D were exposed to ultrasound. The expression of TK protein was detected by western blot. Ganciclovir (GCV) was intraperitoneally injected at a dose of 100 mg x kg (-1) x d(-1) in group B, group C and group D. The tumor size was measured every 2 days. RESULTS: TK gene could be injected precisely into hepatocellular carcinoma with ultrasound monitor, and the expression of TK protein was found in all 4 groups. Expression in group D was higher than others (P < 0.05). The rate of tumor growth inhibition were 0 in group A, 3.90%+/-1.80% in group B, 22.70%+/-2.86% in group C, 41.25%+/-3.20% in group D (group B vs group C, P < 0.05; group D vs group C, P < 0.05; group D vs group B, P < 0.05). CONCLUSION: Ultrasound microbubble not only improve target gene therapy, but also enhance transfection efficiency.

[Effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation].

OBJECTIVE: To explore the effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation (LT). METHODS: Brown-Norway liver allografts were transplanted into Lewis recipients. The transplanted Lewis rats were injected with EK506 (2 mg/kg) and randomly divided into two groups: rats in HBsAg-DCs group (n = 15) were intraperitoneally injected with HBsAg pulsed DCs at 14 d and 28 d after LT, and rats in the HBsAg group (n = 15) were injected with HBsAg (200 mul) once a week for 12 weeks. Rats without any immunosuppressive treatment after LT served as controls (n = 5). IL-2 and IFN-gamma mRNA expression in spleen were analyzed by RT-PCR, serum IL-2, IFN-gamma and anti-HBs were detected by ELISA. RESULTS: High dose of FK506 resulted in the immunosuppressed in LT rats, as evident by low production of IL-2 and IFN-gamma, and without liver rejection compared to rats in the control group. HBsAg-DCs induced high titer of anti-HBs antibody, however, titer of anti-HBs were seldom detectable in the HBsAg group at 1, 2 and 3 mouth after vaccination. CONCLUSION: The capacity of HBsAg-DCs to induce anti-HBs in immunosuppressed rats suggested that DC vaccine may prevent HBV recurrence in liver transplanted patients.

Prevention of common bile duct injury during laparoscopic cholecystectomy.

BACKGROUND: Since the widespread adoption of laparoscopic cholecystectomy (LC) in the late 1980s, a rise in common bile duct (CBD) injury has been reported. We analyzed the factors contributing to a record of zero CBD injuries in 10 000 consecutive LCs. METHODS: The retrospective investigation included 10 000 patients who underwent LC from July 1992 to June 2007. LC was performed by 4 teams of surgeons. The chief main surgeon of each team has had over 10 years of experience in hepatobiliary surgery. Calot's triangle was carefully dissected, and the relationship of the cystic duct to the CBD and common hepatic duct was clearly identified. A clip was applied to the cystic duct at the neck of the gallbladder and the duct was incised with scissors proximal to the clip. The cystic artery was dissected by the same method. Then, the gallbladder was dissected from its liver bed. A drain was routinely left at the gallbladder bed for 1-2 days postoperatively. RESULTS: No CBD injuries occurred in 10 000 consecutive LCs, and there were 16 duct leaks (0.16%). Among these, there were 10 Luschka duct leaks (0.1%) and 6 cystic duct leaks (0.06%). Four hundred thirty cases were converted to open cholecystectomy (OC), giving a conversion rate of 4.3%. After a mean follow-up of 17.5 months (range 6-24 months), no postoperative death due to LC occurred, and good results were observed in 95% of the patients. CONCLUSIONS: In our 10 000 LCs with zero CBD injuries, the techniques used and practices at our department have been successful. Surgeon's expertise in biliary surgery, preoperative imaging, precise operative procedures, and conversion from LC to OC when needed are important measures to prevent CBD injuries.

Odontogenic potential of bone marrow mesenchymal stem cells.

PURPOSE: This study aimed to investigate the odontogenic potential of bone marrow mesenchymal stem cells (BM-MSCs) for seeding in tooth regeneration. MATERIALS AND METHODS: In this study, BM-MSCs were co-cultured with oral epithelial cells derived from rat embryos. Expression of the odontogenic genes Pax9, DMP1, and DSPP was detected by the reverse-transcription polymerase chain reaction (RT-PCR) technique. To further characterize the odontogenic potential of BM-MSCs, the gold standard in vivo transplantation system was used. RESULTS: The results revealed that Pax9, DMP1, and DSPP expression was detected by RT-PCR only after co-culture of BM-MSCs and oral epithelial cells derived from embryos age E11.5. Histological analyses of the BM-MSCs/epithelial cell mass demonstrated the presence of tooth-like structures. CONCLUSIONS: The series of experiments both in vitro and in vivo demonstrated that BM-MSCs can differentiate into functional odontoblast-like cells. This implies that BM-MSCs may become a novel source of cells for seeding in tooth regeneration research.

[The influence with block the endotoxin signal transduction for ischemia/reperfusion injury of graft liver in rats].

OBJECTIVE: To explore the feasibility of interleukin 1 receptor associated kinase-4 (IRAK-4) as gene therapy target for liver ischemia/reperfusion injury (I/RI) and effective approach in vivo for short hairpin RNA (shRNA) interference used to gene therapy in liver graft hqappened. METHODS: Sprague-Dawley rats were randomly divided into three groups: the control group, the in vivo transfection group (IVT) and the cold ischemia transfection group (CIT). Experiments of orthotopic liver transplantation were performed by two-cuff method. CIT were perfused with IRAK-4-shRNA plasmid (pSIIRAK-4) during cold ischemia phase, IVT received the equivalent volumes (2 mL) of pSIIRAK-4 after portal vein inosculated, and the control group leaved without any treatment. At 0 min, 60 min and 180 min after reperfusion, the expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The serum TNF-alpha level was detected by ELISA. Liver histopathological changes and cell apoptosis were observed by electron microscope and TUNEL. RESULTS: After reperfusion, the expression of IRAK-4 were largely depressed in CIT than that of IVT and the control group (P < 0.01), and furthermore, the serum TNF-alpha level, proportion of hepatocyte apoptosis and severity of hepatocyte injury were also lower than the latter. CONCLUSION: These results indicate that depression IRAK-4 expression with IRAK-4-shRNA through portal vein perfusion during cold ischemia phase could effectively blunt graft hepatic I/RI.

[Retrospective analysis of 3,958 patients with facial injuries].

OBJECTIVE: To determine the causes and incidence of facial injuries by an epidemiologic retrospective study. METHODS: A total of 3 958 patients with facial injuries treated at Department of Oral and Maxillofacial Surgery, West China School of Stomatology, Sichuan University from 1955 to 2001 were investigated. Data regarding age, gender, cause of injury, pattern of fracture and associated systemic injuries were reviewed. RESULTS: The male to female ratio of the patients with facial injury was 4.27:1 and 33.4% of patients were aged between 21 and 30 years. The most common cause of injury was traffic accident (30.6%), followed by falls (21.4%) and collision (15.8%). A total of 794 patients (20.1%) showed only soft tissue injuries. 1 100 patients (27.8%) had multiple fractures in facial bones and 2,064 patients (52.1%) had single fracture. The mandibular fracture was most frequently seen, followed by the maxilla and the zygoma. The most common site of mandible fracture was the body (31.2%), followed by the symphysis (22.7%), the condylar (20.5%) and the angle (13.7%). Accompanied injuries to brain and skull happened in 916 patients (23.1%). CONCLUSIONS: Bone fractures were more common in hospitalized patients with facial injuries. The numbers and sites of fracture were related to the causes of injuries and anatomic structure of the bone. The brain and skull injuries, the most often and seriously accompanied injuries, would not be neglected.

[A study on repairing mandibular defect by means of tissue-engineering and human bone morphogenetic protein-2 gene transfection in osteoporotic rats].

OBJECTIVE: To investigate the feasibility of repairing bone defect with methods of tissue-engineering and human bone morphogenetic protein-2 (hBMP-2) gene transfection in osteoporotic rats. METHODS: Twenty-four 6-month-old female Sprague-Dawley rats underwent ovariectomy, while 8 rats received sham-operations. Three months later, bone mesenchymal stem cells (BMSC) harvested from osteoporotic rats were divided into two groups randomly. Experimental group were transfected by recombinant plasmid carrying hBMP-2 gene, and control group left untreated. All BMSC were seeded into coralhydroxyapatite scaffolds. Then the cell/scaffold constructs were implanted into the defect site created in the ramus of mandible of osteoporotic rats respectively. RESULTS: Positive results were confirmed by immunohistochemistry and in situ hybridization in experimental group. New bone formation was found at the margin of the defect treated with the BMSC modified by hBMP-2 gene transfer at 4 weeks after implantation and appeared mature 8 weeks after the treatment. However, the amount of newly formed bone was much less and there was some adipose tissue at defect margins 8 weeks after implantation in control group. CONCLUSIONS: The results of this experiment indicate that BMSC-mediated rhBMP-2 gene therapy in conjunction with bone tissue engineering may allow for successful treatment of large bone defects in osteoporosis rats.

[Expression of human interleukin-1 receptor antagonist in the transfected chondrocytes of temporomandibular joint].

OBJECTIVE: To investigate the expression of the human interleukin-1 receptor antagonist (hIL-1ra) in the transfected chondrocytes of temporomandibular joint (TMJ). METHODS: Chondrocytes of TMJ in vitro were transfected by hIL-1ra gene via cationic liposome as a medium. The stable transfected cells were selected by G418. The proliferations of the transduced cell were examined with the growth curve, cell population doubling time. The protein expressing in different periods was detected by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: The proliferation suppression of gene transfected cells fell significantly with compared to normal cells. The expression of hIL-1ra was detected in the cell plasma and the cell culture supernatant. The highest expression of IL-1ra protein was at the time of 48 hours after gene transfection. The transiently transfected cells were secreted IL-1ra protein continuously 28 days and the stably transduced cells were secreted IL-1ra protein till 72 days. CONCLUSION: This study showed that hIL-1ra protein expressed positively in the cell plasma and the culture supernatant after gene transfection within a certain periods.