Expression of plasma vascular endothelial growth factor in patients with hepatocellular carcinoma and effect of transcatheter arterial chemoembolization therapy on plasma vascular endothelial growth factor level

AIM: To investigate the expression level of plasma vascularendothelial growth factor (P-VEGF) in patients withhepatocellular carcinoma (HCC) and its relationship withthe clinicopathologic characteristics, and to examine thechanges of P-VEGF in the course of transcatheter arterialchemoembolization (TACE).METHODS: Peripheral blood samples were taken from 45HCC patients before and 1, 3, 7 d, and 1 mo after TACE.Plasma VEGF level was measured with the quantitativesandwich enzyme-linked immunosorbent assay (ELISA).Twenty patients with benign liver lesions and 17 healthycontrol subjects were also included in this study.RESULTS: Plasma VEGF levels in HCC patients weresignificantly elevated as compared to those in patients withbenign liver lesions (P = 0.006) and in the normal controls(P = 0.003). Significant differences were observed whenP-VEGF was categorized by tumor size (P = 0.006), portalvein thrombosis (P= 0.011), distant metastasis (P= 0.017),arterial-portal vein shunting (P = 0.026), and InternationalUnion Against Cancer (UICC) TNM stage (P = 0.044). Therewas no correlation between plasma level of VEGF and thelevel of alpha fetoprotein (^-FP) (r = 0.068, P = 0.658) andweakly correlated with the number of platelets (r = 0.312,P = 0.038). P-VEGF levels increased significantly andreached the peak value on the first day after TACE, and thendecreased gradually. The change rate of P-VEGF concentration(one month post-TACE/pre-TACExl00%) was correlatedwith the retention rate of lipiodol oil (rs = 0.494, P= 0.001)and the tumor volume change (r s = 0.340, P = 0.034).The patients who achieved a partial or complete responseto TACE therapy showed significantly less pre-treatmentP-VEGF than those nonresponders (P = 0.025). A high pre-therapeutic P-VEGF level was associated with poor responseto treatment (P = 0.018).CONCLUSION: A high pre-treatment P-VEGF level is auseful marker for tumor nroeression, esBeciallv for vascularinvasion. TACE increases the level of P-VEGF onlytemporarily which may be associated with tumor ischemia.P-VEGF may be useful in predicting treatment response,monitoring disease course after TACE and judging the effectof different TACE regimens.

Combined interventional therapies of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most commonmalignancies in the world, responsible for an estimated one million deaths annually. It has a poor prognosis due to its rapid infiltrating growth and complicating liver cirrhosis.Surgical resection, liver transplantation and cryosurgery are considered the best curative options, achieving a high rate of complete response, especially in patients with small HCC and good residual liver function. In nonsurgery, regional interventional therapies have led to a major breakthrough in the management of unresectable HCC, which include transarterial chemoembolization (TACE), percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), microwave coagulation therapy (MCT), laser-induced thermotherapy (LITT), etc. As a result of the technical development of locoregional approaches for HCC during the recent decades,the range of combined interventional therapies has been continuously extended. Most combined multimodal interventional therapies reveal their enormous advantages as compared with any single therapeutic regimen alone,and play more important roles in treating unresectable HCC.

Expression of IGF-II in early experimental hepatocellular carcinomas and its significance in early diagnosis

AIM: To investigate the serum level and expression of insulin growth factor II (IGF-II) in liver tissues of rats with early experimental hepatocellular carcinomas (HCC) and its significance in early diagnosis.METHODS: Early experimental hepatocellular carcinomas were induced by diethylnitrosamine (DENA) in 180 male SD rats. Another 20 male SD rats served as control. The IGF-IIserum level was measured by ELISA. Immunohistochemistry and electron microscopic immunohistochemistry were usedto observe the expression of IGF-II in normal and tumor liver tissues and its ultrastructural location in malignant hepatocytes. The expressions of IGF-II in human hepatoma cell lines HepG2, SMMC7721 and human embryonic liver cell line L-02 were measured by immunocytochemistry. IGF-II mRNA level was studied by in situ hybridization.RESULTS: IGF-II was expressed in the cytoplasm of both sinusoidal cells in paracancerous cirrhotic liver tissue and malignant hepatocytes in early experimental HCC tissues.Gold particles were seen on the rough endoplasrnic reticulum and the mitochondrion in malignant hepatocytes. IGF-II was expressed in the human hepatoma cell lines. The mRNA level of IGF-II was higher in rat liver tumor tissue than in normal rat liver tissue. The serum IGF-II level of the early experimental HCC group was 34.67±10.53 ng.ml^-1 and that of the control group was 11.75±5.84 ng.ml^-1. The rank sum test was used for statistical analysis. There was a significant difference between the two groups (P<0.01).CONCLUSION: During the induction of early experimental HCC by DENA, IGF-II may promote hepatocytic proliferation via a paracrine mechanism in the pre-cancerous stage. When hepatocytes are transformed into malignant cells, they may secrete IGF-II and promote malignant cell proliferation by an autocrine mechanism. IGF-II may be a possible biological marker in the early diagnosis of HCC.

Antiangiogenic effect of somatostatin receptor subtype 2 on pancreatic cancer cell line:Inhibition of vascular endothelial growth factor and matrix metalloproteinase-2 expression in vitro

AIM:To investigate the anti-angiogenic effect of somatostatin receptor subtype 2 (SSTR2) gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect.METHODS: The full length human SSTR2 cDNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection. Positive clones were screened by G418 and stable expression of SSTR2 was detected by immunohistochemistry SABC methods and RT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect vascular endothelial growth factor (VEGF) levels in the cell culture supernatants of SSTR2-expressing cells, vector control and mock control cells. Furthermore, the expressions of VEGF and matrix metalloproteinase-2 (MMP-2) were detected by immunohistochemistry SABC methods and RT-PCR in these cells.RESULTS: VEGF levels in the cell culture supernatants were significantly reduced in the SSTR2-expressing cells (first week,172.63±21.2ng/L and after two months, 198.85±26.44ng/L)compared with the vector control (first week, 790.39±86.52ng/L and after two months, 795.69±72.35ng/L) and mock control (first week, 786.42±90.62ng/L and after two months,805.32±84.36ng/L) (P<0.05).The immunohistochemical assay showed a significant reduction of the integral optical density of VEGF and MMP-2 in the SSTR2-expressing cells (42.25±8.6 and 70.5±6.25, respectively) compared with the vector control (85.75±12.9 and 110.52±13.5, respectively) and mock control (82.6±9.28 and 113.56±9.62,respectively) (P<0.05).Conversely, the average gray value of VEGF and MMP-2 was significantly increased in the SSTR2-expressing cells (121.56±8.43 and 134.46±19.95, respectively) compared with the vector control (55.72±5.6 and 62.26±12.68,respectively) and mock control cells (58.48±6.2 and 65.49±9.16, respectively) (P<0.05). Moreover, the expressions of VEGF mRNA and MMP-2 mRNA were significantly reduced in the SSTR2-expressing cells (0.1384±0.017 and 0.2343±0.070, respectively) compared with the vector control (1.024±0.117 and 0.806±0.119,respectively) and mock control (1.085±0.105 and 0.714±0.079,respectively) (P<0.05).CONCLUSION: The expression of reintroduced human SSTR2 gene exerts its antiangiogenic effects by downregulating the expressions of the factors involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a new strategy of gene therapy for pancreatic cancer.

Effects of carbon dioxide and nitrogen on adhesive growth and expressions of E-cadherin and VEGF of human colon cancer cell CCL-228

AIM: To study the effects of carbon dioxide on the metastatic capability of cancer cells, and to compare them with that of nitrogen.METHODS: The colon cancer cell CCL-228 was treated with 100 % carbon dioxide or nitrogen at different time points and then cultured under normal condition. Twelve hours after the treatment, the survival rates of suspension cells and the expressions of e-cadherin and VEGF were examined.RESULTS: After 60 min of carbon dioxide and longer time of nitrogen treatment, the suspended cells increased and the expression of e-cadherin decreased while the expression of VEGF was enhanced significantly. And the effects of nitrogen were similar to, but weaker than, those of carbon dioxide.CONCLUSION: Carbon dioxide may improve the metastatic capability of cancer cells and its effects are significantly stronger than that of nitrogen. A sequential use of carbon dioxide and nitrogen in pneumoperitoneum may take the advantage of both gases.

Involvement of extracellular signal-regulated kinase/mitogenactivated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

AIM: To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes: multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.METHODS: A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure.RESULTS: Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold).Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the transfected cells decreased to some extent. Compared with controls, a significant decrease in MDR-1 mRNA (53.3%), MRP-1 mRNA (59.7%) as well as LRP mRNA (56.4%) was observed in the UO126 treated transfected cells after 12 h. Western blot also demonstrated that the protein expression of these MDR associated genes slightly reduced after treated with U0126 for 12 h (MDR-1 40.1%, MRP-1 29.4%, LRP35.7%). This change was accompanied with the rise of cell apoptosis ratio confirmed by Annexin V-PI detection. The apoptosis index of UO126treated cells increased by 1.28 fold, compared with that of transfected cells. Obviously, the MDR phenotype of these cells was obviously related with increased activities of the ERK/MAPK pathway.CONCLUSION: HBx protein might be one of the causes for the occurrence of MDR in HCC, and ERK/MAPK pathway might be involved in this change.

Clinical study on nutrition support in patients with severe acute pancreatitis

AIM: To investigate the effect of nutritional support therapy on severe acute pancreatitis (SAP).METHODS: A total of 96 patients with severe acute pancreatitis were divided randomly into control and treatment groups.The former group received total parenteral nutrition (TPN)via central venous infusion, while parenteral nutrition (PN)and enteral nutrition (EN) therapies were applied in different phases for the latter group. The nutrition status, acute phase responses, pancreas lesions, enteric mucosa penetrability and immune functions were monitored.RESULTS: Body weight and prealbumin concentration were increased in treatment group, compared to those in the control group, but albumin concentration did not change significantly.Acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ)scores decreased after 7 d of treatment, whereas the scores of the control group decreased on the 11th day. Concentrations of tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and serum C reactive protein (CRP) dropped earlier in the treatment group (on the 4th day) than that in the control group (on the 7th day). No difference was observed in pancreatic lesions between the control and treatment groups.Concentration of endotoxin and lactulose/manicol (L:M) ratio of urine did not change in treatment group, but those in the control group were elevated markedly. Compared with the treatment group, CD4:CD8 T cells ratio and immunoglobulin G (IgG) concentration in the control group decreased significantly.CONCLUSION: Compared to TPN, the combined therapy of EN and PN could improve the nutrition status and moderate the acute phase response obviously. Moreover, the integrity of enteric mucosa and immune function were protected more effectively in treatment group than in the control one. On the other hand, EN did not simulate the excretion of pancreas and avoid exaggerating the inflammation of pancreas. Thus,appropriate application of PN and EN appears to be more effective for patients with SAP.

Combined transarterial chemoembolization and arterial administration of Bletilla striata in treatment of liver tumor in rats

AIM: To evaluate and compare the effect of combined transarterial chemoembolization (TACE) and arterial administration of Bletilla striata (a Chinese traditionalmedicine against liver tumor) versusTACE alone for the treatment of hepatocellular carcinoma (HCC) in ACI rats.METHODS: Subcapsular implantation of a solid Morris hepatoma 3 924A (2 mm3) in the liver was carried out in 30 male ACI rats. Tumor volume (V1) was measured by magnetic resonance imaging (MRI) on day 13 after implantation. The following different agents of interventional treatment were injected after retrograde catheterization via gastroduodenal artery (on day 14), namely, (A) TACE (0.1 mg mitomycin +0.1 mi Lipiodol) + Bletilla striata (1.0 mg) (n= 10); (B) TACE + Blebilla stnata(1.0 mg) + ligation of hepatic artery (n=10),(C) TACE alone (control group, n=10). Tumor volume (V2)was assessed by MRI (on day 13 after treatment) and the tumor growth ratio (V2/V1) was calculated.RESULTS: The mean tumor volume before (V1) and after (V2) treatment was 0.0355 cm3 and 0.2248 cm3 in group A,0.0374 cm3 and 0.0573 cm3 in group B, 0.0380 cm3 and 0.3674 cm3 in group C, respectively. The mean ratio (V2/V1)was 6.2791 in group A, 1.5324 in group B and 9.1382 in group C. Compared with the control group (group C), group B showed significant inhibition of tumor growth (P＜0.01),while group A did not (P＞0.05). None of the animals died during implantation or in the postoperative period.CONCLUSION: Combination of TACE and arterial administration of Bletilla striata plus ligation of hepatic artery is more effective than TACE alone in the treatment of HCC in rats.

Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

Assessment of hepatocellular carcinoma vascularity before and after transcatheter arterial chemoembolization by using first pass perfusion weighted MR imaging

AIM:To assess the vascularity of hepatocellular carcinoma (HCC) before and after transcatheter arterial chemoembolization (TACE) with the quantitative parameters obtained by first pass perfusion weighted MR imaging (FP-MRI). METHODS:Seventeen consecutive patients with one to three lesions in liver underwent FP-MRI before treatment. FP-MRI was also performed one,three,six,nine months, and one year after TACE.The baseline signal intensity (SO) of pre-TACE and one month after TACE was analyzed,the vascularity of HCC assessed by steepest slope of the signal intensity versus time curves (SS) was blindly correlated with their DSA feature and clinical outcome. RESULT:No significant difference was found on baseline signal intensity (S0) between pre-TACE and one month after TACE (F=0.309,P=0.583),The SS (mean,32% per second) of lesion one month after TACE was lower than that of pre-TACE (mean,69% per second),but with no statistical significance (F=3.067,P=0.092).When local recurrence occurred,the time intensity curves became steeper.The vascularity of HCC before and after TACE graded by SS closely correlated with that by DSA (K=0.453,P<0.05). CONCLUSION:FP-MRI is a useful criterion for selecting effective interventional treatment for patients with HCC in their initial treatment and during follow up.

Identification of effective siRNA against K-ras in human pancreatic cancer cell line MiaPaCa-2 by siRNA expression cassette

AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. METHODS: Three different sites of SECs were constructed by PCR. K1/siRNA,K2/siRNA and K3/siRNA are located at sites 194,491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. RESULTS: Introduction of the Kl/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the Kl/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. CONCLUSION: Kl/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that Kl/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

Changes of tumor microcirculation after transcatheter arterial chemoembolization:First pass perfusion MR imaging and Chinese ink casting in a rabbit model

AIM: To observe the change of tumor microcirculation after transcatheter arterial chemoembolization (TACE) with bletilla microspheres by using first pass perfusion MR imaging (FP) and Chinese ink casting.METHODS: VX2 carcinoma cells were surgically implanted into the left and right lobes of liver of 30 New Zealand white rabbits, which were divided into 3 groups at random. Emulsion of lipiodol mixed with mitomydn C, and 5-FU bletilla microspheres were injected into the hepatic artery respectively, and saline was used as control agent. MR imaging was performed with turbo-flash sequence 14 d after tumor implantation and 7 d after interventional therapy. The steepest slopes (SS) of the signal intensity versus time curves were created for quantitative analysis, 7.5% Chinese ink gelatin solution was injected through ascending artery (17 cases) or portal vein(2 cases) for lesion microvessel area (MVA) measurement after the last MRI examination.The correlation between perfusion imaging and MVA was studied blindly.RESULTS: The SS values at the rim of tumor in lipiodol group (mean, 49% per second) and bletilla group (mean,35% per second) were significantly decreased (P<0.05) as compared with control group (mean, 124% per second), no difference was found between lipiodol and bletilla groups(P>0.05). In lipiodol group, the MVAs (24 974±11 836μm^2) in the center of the tumor were significantly smaller than those of the control group (35 510±15 675 μm^2) (P<0.05),while the MVAs (80 031±22 745 μm^2) around the tumor were significantly increased because small and dense plexuses appeared around the tumor which correlated to intense reaction of granulation tissue. None of the vessels was seen in the tumor in bletilla group, the peripheral MVAs of the tumor were significantly smaller than those of the control group (P<0.05) and lipiodol group (P<0.05). There was a good correlation between SS and MVAs in control group (rsl, 0.985, P<0.0001) and bletilla group (rsl, 0.743,P<0.05), the correlation was not significant in lipiodol group(rsl, 0.527, P>0.05).CONCLUSION: TACE with bletilla microspheres may enhance its anti-tumor effect by inhibiting the angiogenesis,and FP-MRI provides useful information to assess the TACE effect by depicting tumor vascularization and perfusion,

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

Effects of pentoxifylline on the hepatic content of TGF-β1 and collagen in Schistosomiasis japonica mice with liver fibrosis

AIM: To study the effects of pentoxifylline (PTX) on thecontent of hepatic TGF-β1, type Ⅰ and type Ⅲ collagen inschistosomiasis japonica mice with liver fibrosis and itsmechanism of anti-fibrosis.METHODS: Forty mice with schistosomiasis were dividedinto four groups: one group as control without anytreatment, other three were treated with Praziquantel 500mg/(kg.d)for 2 d, high dose PTX 360 mg/(kg.d) for 8 wk,and low dose PTX 180 mg/(kg.d) for 8 wk respectively.Immunohistochemical technique and multimedia colorpathographic analysis system were applied to observe thecontent change of hepatic TGF-β1, type Ⅰ and type Ⅲcollagen in schistosomiasis japonica mice with liver fibrosisbefore and after PTX treatment.RESULTS: Effects of PTX on the content change of hepaticTGF-β1, type Ⅰ and type Ⅲ collagen in schistosomiasis japonicamice with liver fibrosis were related to the dosage of PTX,high dose PTX treated group could significantly reduce thecontent of TGF-β1 (0.709±0.111), type Ⅰ (0.644±0.108) andtype Ⅲ (0.654±0.152) collagen compared with those ofcontrol group (0.883±0.140, 0.771±0.156, 0.822±0.129)with statistical significance (P＜0.05). Low dose PTX couldalso reduce the hepatic content of TGF-β1 (0.752±0.152),type Ⅰ (0.733±0.117) and type Ⅲ (0.788±0.147) collagen,but without statistical significance (P＞0.05). Both high doseand low dose PTX groups have significant differences onthe content of TGF-β1, type Ⅰ and type Ⅲ collagen (P＜0.05,P＜0.05, P＜ 0.01,respectively).CONCLUSION: High dose of PTX treatment could reducethe content of hepatic TGF-β1, type Ⅰ and type Ⅲ collagensignificantly in schistosomiasis japonica mice with liverfibrosis, and thus plays its role of antifibrosis.

Angiogenesis in rabbit hepatic tumor after transcatheter arterial embolization

AIM: To investigate the effect of transcatheter arterial embolization (TAE) on angiogenesis of hepatic tumor.METHODS: Twenty New Zealand White rabbits were randomly divided into two groups of 10 each and VX2 carcinoma was implanted in the left medial lobes of the livers. Fourteen days later, a silicon catheter was inserted into the left hepatic artery of rabbit with VX2 hepatic tumor and infusion was performed via the hepatic artery using Lipiodol (the TAE group) or saline (the control group). Rabbits were sacrificed 7 d after treatment and tumor tissues were excised. Expression of vascular endothelial growth factor (VEGF) protein and microvessel density (MVD) of tumors were examined using immunohistochemistry. The staining intensity of VEGF was evaluated with a computer-assisted image-analyzer. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the VEGF mRNA expression of tumors.RESULTS: MVD was higher in the TAE group compared with the control group (28.6±10.6 vs 16.3±6.9, P<0.01).Expression of VEGF protein was enhanced after TAE. The staining intensity of VEGF in the TAE group was 0.162±0.018,significantly higher than in the control group (0.142±0.01,P<0.01). At mRNA level, VEGF165 mRNA was significantly higher in the TAE group compared with the control group (2.58±0.42 vsl.99±0.21, P<0.001). MVD was well correlated to VEGF expression in both the TAE group (r=0.69, P<0.05) and the control group (r=-0.72, P<0.05).CONCLUSION: TAE promotes the development of neovascularization of residual tumors through up-regulation of VEGF expression, possibly due to hypoxic insult.

ROS-related Enzyme Expressions in Endothelial Cells Regulated by Tea Polyphenols

Objective Elevation of reactive oxygen species (ROS), especially the level of superoxide is a key event in many forms of cardiovascular diseases. To study the mechanism of tea polyphenols against cardiovascular diseases, we observed the expressions of ROS-related enzymes in endothelial cells. Methods Tea polyphenols were co-incubated with bovine carotid artery endothelial cells (BCAECs) in vitro and intracellular NADPH oxidase subunits p22phox and p67phox, SOD-1, and catalase protein were detected using Western blot method. Results Tea polyphenols of 0.4 ug/mL and 4.0 ug/mL (from either green tea or black tea) down-regulated NADPH oxidase p22phox and p67phox expressions in a dose-negative manner (P<0.05), and up-regulated the expressions of catalase (P<0.05). Conclusions Tea polyphenols regulate the enzymes involved in ROS production and elimination in endothelial cells, and may be beneficial to the prevention of endothelial cell dysfunction and the development of cardiovascular diseases.

Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro

Aim: The effects of certain uropathogenic microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermidis and Mycobacterium tuberculosis) on human sperm motility characteristics were studied in vitro. Methods: In 10 healthy fertile men, ejaculates were aseptically obtained by masturbation and With a swim-up technique, a sperm suspension of high motility and purity was obtained. Several uropathogenic bacteria were obtained from outpatients with genitourinary tract infections. The sperm suspension was incubated with the pathogens at a bacteria: sperm ratio of 50:1 at 37℃. The sperm mobility parameters were estimated with a computerassisted sperm analyzer (CASA) provided with a multiple-exposure photography system (Madi Corp., Zhejiang, China). Measurements were carried out at 0, 2 and 4 hours of incubation. Results: Staphylococcus aureus significantly decreased the sperm motility and viability, but Staphylococcus epidermidis, Mycobacterium tuberculosis and Neisseria gonorrhoeae did not. Conclusion: Staphylococcus aureus has an inhibitory effect on human sperm motility in vitro.

Inactivation of RASSF1A, the tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma

AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSFIA. RASSFIA mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3. Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSFIA promoter were evaluated by methylation specific PCR (MS-PCR). RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSFIA mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939 cell lines (X2= 14.270, P= 0.001<0.01) showed RASSFIA express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%. CONCLUSION: The data we present suggest that RASSFIA which we have been searching for at 3p21.3 may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSFIA.

Vascular endothelial growth factor antisense oligodeoxynucleotides with lipiodol in arterial embolization of liver cancer in rats

AIM:Transcatheter arterial embolization (TAE) of the hepatic artery has been accepted as an effective treatment for unresectable hepatocellular carcinoma (HCC). However,embolized vessel recanalization and collateral circulation formation are the main factors of HCC growth and recurrence and metastasis alter TAE. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis.This study was to explore the inhibitory effect of VEGF antisense oligodeoxynucleotides (ODNs) on VEGF expression in cultured Walker-256 cells and to observe the anti-tumor effect of intra-arterial infusion of antisense ODNs mixed with lipiodol on rat liver cancer.METHODS: VEGF antisense ODNs and sense ODNs were added to the media of non-serum cultured Walker-256 cells.Forty-eight hours later, VEGF concentrations of supernatants were detected by EUSA. Endothelial cell line ECV-304 cells were cultured in the supernatants. Seventy-two hours later,growth of ECV-304 cells was analyzed by NTT method. Thirty Walker-256 cell implanted rat liver tumor models were divided into 3 groups.0.2 mL lipiodol (LP group, n=10), 3OD antisense ODNs mixed with 0.2 mL lipiodol (LP+ODNs group, n=10) and 0.2 mL normal saline (control group, n=10) were infused into the hepatic artery. Volumes of tumors were measured by MRI before and 7 d alter the treatment.VEGF mRNA in cancerous and peri-cancerous tissues was detected by RT-PCR. Microvessel density (MVD) and VEGF expression were observed by immunohistochemistry.RESULTS: Antisense ODNs inhibited Walker-256 cells' VEGF expression, The tumor growth rate was significantly lower in LP+ODNs group than that in LP and control groups (140.1±33.8%, 177.9±64.9% and 403.9±69.4% respectively, F=60.019,P<0.01).VEGF mRNAs in cancerous and peri-cancerous tissues were expressed highest in LP group and lowest in LP+ODNs group. The VEGF positive rates showed no significant difference among LP, control and LP+ODNs groups (90%,70% and 50%, H=3.731, P>0.05).The MVD in LP+ODNs group (53.1±18.4) was significantly less than that in control group (73.2±20.4) and LP group(80.3±18.5) (F=5.44, P<0.05).CONCLUSION: VEGF antisense ODNs can inhibit VEGF expression of Walker-256 cells.It may be an antiangiogenesis therapy agent for malignant tumors. VEGF antisense ODNs mixed with lipiodol embolizing liver cancer is better in inhibiting liver cancer growth, VEGF expression and microvessel density than lipiodol alone.

Nude mice model of human hepatocellular carcinoma via orthotopic implantation of histologically intact tissue

AIM: To establish a nude mice model of human hepatocellular carcinoma (HCC) via orthotopic implantation of histologically intact tissue, in order to study biologic features of HCC in vivo and to direct clinical treatment respectively.METHODS: Histologically intact fresh specimens of HCC were orthotopically implanted in nude mice (BALB/c, nu/nu). Survival rate and growth curve were investigated with Bultrasound. Morphological characteristics of pathology and spontaneous metastatic rates were detected with microscopy. Expression of multidrug resistance genes studied with immunohistochemical method and RT-PCR, and other biologic features of implanted tumor were observed and compared with human HCC specimens. RESULTS: Out of the specimens from two patients with HCC, only one specimen survived in nude mice. The orthotopic implantation tumor survival rate, spontaneous intrahepatic metastatic rate, pulmonary metastatic rate and bone metastases rate were 100%, 75.0%, 37.5% and 37.5% respectively in the first passage. AFP was kept on secreting and increasing with the size of the tumor. The morphological characteristics and biologic features were similar to the donor's, the protein and mRNA of MDR1 and LRP were expressed in tumors of the model and the donor, and there was no significant difference between them (P>0.05). CONCLUTION: The model of nude mice with orthotopic implantation of histologically intact HCC tissue is an ideal model to study biologic features of HCC in vivo and to direct clinical treatment.