Postoperative adjuvant arterial chemoembolization improves survival of hepatocellular carcinoma patients with risk factors for residual tumor:A retrospective control study

AIM: To evaluate the effect of postoperative adjuvant transcatheter arterial chemoembolization (TACE) on the prognosis of hepatocellular carcinoma (HCC) patients with or without risk factors for the residual tumor. METHODS: From January 1995 to December 1998, 549 consecutive HCC patients undergoing surgical resection were included in this research. There were 185 patients who underwent surgical resection with adjuvant TACE and 364 patients who underwent surgical resection only. Tumors with a diameter more than 5 cm, multiple nodules, and vascular invasion were defined as risk factors for residual tumor and used for patient stratification. Kaplan-Meier method was used to analyze survival curve and Cox proportional hazard model was used to evaluate the prognostic significance of adjuvant TACE.RESULTS: In the patients without any risk factors for the residual tumor, the 1-, 3-, 5-year survival rates were 93.48%,75.85%, 62.39% in the control group and 97.39%, 70.37%,50.85% in the adjuvant TACE group, respectively. There was no significant difference in the survival between two groups (P = 0.3956). However, in the patients with risk factors for residual tumor, postoperative adjuvant TACE significantly prolonged the patients' survival. There was a statistically significant difference in survival between two groups (P= 0.0216). The 1-, 3-, 5-year survival rates were 69.95%, 49.86%, 37.40% in the control group and 89.67%,61.28%, 44.36% in the adjuvant TACE group, respectively. Cox proportional hazard model showed that tumor diameter and cirrhosis, but not the adjuvant TACE, were the significantly independent prognostic factors in the patients without risk factors for residual tumor. However, in the patients with risk factors for residual tumor adjuvant TACE, and also tumor diameter, AFP level, vascular invasion, were the significantly independent factors associated with the decreasing risk for patients' death from HCC. CONCLUSION: Postoperative adjuvant TACE can prolong the survival of patients with risk factors for residual tumor,but can not prolong the survival of patients without risk factors for residual tumor.

Blockage of transforming growth factor β receptors prevents progression of pig serum-induced rat liver fibrosis

AIM: To test the hypothesis that introduction of antisense TβR Ⅰ and TβR Ⅱ eukaryotic expressing plasmids into a rat model of immunologically induced liver fibrosis might block the action of TGF-β1 and halt the progression of liver fibrosis. METHODS: RT-Nest-PCR and gene recombination techniques were used to construct rat antisense TβR Ⅰ and TβR Ⅱ recombinant plasmids which could be expressed in eukaryotic cells. The recombinant plasmids and empty vector (pcDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model. Expression of exogenously transfected gene was assessed by Northern blot, and hepatic expressions of TβR Ⅰ and TβR Ⅱ were evaluated by RT-PCR and Western blot.We also performed ELISA for serum TGF-β1, hydroxyproline of hepatic tissues, immunohistochemistry for collagen types Ⅰ and Ⅲ, and VG staining for pathological study of the liver tissues. RESULTS: The exogenous antisense TβR Ⅰ and TβR Ⅱ plasmids could be well expressed in vivo, and block mRNA and protein expression of TβR Ⅰ and TβR Ⅱ in the fibrotic liver at the level of mRNA respectively. These exogenous plasmid expressions reduced the level of TGF-β1 (antisense TβR Ⅰ group 23.998+3.045 ng/mL, antisense TβR Ⅱ group 23.156+3.131 ng/mL, disease control group 32.960+3.789 ng/mL; F-=-38.19, 36.73, P<0.01). Compared with disease control group, the contents of hepatic hydroxyproline (antisense TβR Ⅰ group 0.169+0.015 mg/g liver, antisense TβR Ⅱ group 0.167+0.009 mg/g liver, disease control group 0.296+0.026 mg/g liver; F=14.39, 15.48, P<0.01) and the deposition of collagen types Ⅰ and Ⅲ decreased in the two antisense treatment groups(antisense TβR Ⅰ group, collagen type Ⅰ 669.90+50.67, collagen type Ⅲ 657.29+49.48; antisense TβR Ⅱ group, collagen type Ⅰ 650.26+51.51, collagen type Ⅲ 661.58+55.28; disease control group, collagen type I 1209.44+116.60, collagen type Ⅲ 1175.14+121.44; F=15.48 to 74.89, P<0.01). Their expression also improved the pathologic classification of liver fibrosis models (compared with disease control group, X^2=17.14, 17.24, P<0.01). No difference was found in the level of TGF-β1, the contents of hepatic hydroxyproline and collagen types Ⅰ and Ⅲ and pathologic grade between pcDNA3 control group and disease control group or between the two antisense treatment groups (F=0.11 to1.06, X^2=0.13 to 0.16, P>0.05). CONCLUSION: Antisense TβR Ⅰ and TβR Ⅱ recombinant plasmids have certain reverse effects on liver fibrosis and can be used as possible candidates for gene therapy.

Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis

AIM: To establish the methylation profile of the promoter CpG islands of 31 genes that might play etiological roles in colon carcinogenesis.METHODS: The methylation specific PCR in conjunction of sequendng verification was used to establish the methylationprofile of the promoter CpG islands of 31 genes in colorectal cancer (n = 65), the neighboring non-cancerous tissues (n = 5), colorectal adenoma (n = 8), and normal mucosa (n = 1). Immunohistochemically, expression of 10 genes was assessed on the home-made tissue microarrays of tissues from 58 patients. The correlation of tumor specific changes with each of clinical-pathologic features was scrutinized with relevant statistic tools.RESULTS: In comparison with the normal mucosa of the non-cancer patients, the following 14 genes displayed no tumor associated changes: breast cancer 1, early onset (BRCA1), cadherin 1, type 1, E-cadherin (epithelial) (CDH1),death-associated protein kinase 1 (DAPK1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), melanoma antigen, family A, 1 (directs expression of antigen MZ2-E) (MAGEA1), tumor suppressor candidate 3 (N33), cyclin-dependent kinase inhibitor 1A (p21, Cipl) (p21^WAF1), cyclin-dependent kinase inhibitor 1B (p27, 10pl) (p27^WAF1), phosphatase and tensin hornolog (mutated in multiple advanced cancers 1) (PTEN), retinoic acid receptor, beta (RAR-, Ras association (RaIGDS/AF-6) domain family 1 C (RASSFIC), secreted frizzled-related protein 1 (SFRP1), tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinfiammatory) (TIMP3),and von HippeI-Lindau syndrome (VHL). The rest 17 targets exhibited to various extents the tumor associated changes.As changes in methylation of the following genes occurred marginally, their impact on the formation of colorectal cancer were trivial: adenomatous polyposis coli (APC) (8%, 5165),Ras association (RaIGDS/AF-6) domain family 1A (RASSFIA) (3%, 2/65) and cyclin-dependent kinase inhibitor 2A,alternated reading frame Co14~) (6%, 4/65). The following genes exhibited moderate changes in rnethylation: O-6rnethylguanine-DNA rnethyltransferase (MGMT) (20%, 13/65),rnutL hornolog 1, colon cancer, nonpolyposis type 2 (E. coli) (hMLH1) (18%, 12/65), cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) P16^NK4a) (10%, 10/65),rnethylated in tumor 1 (MINT1) (15%, 10/65), methylated in tumor 31 (MINT31) (11%, 7/65). The rest changed greatly in the rnethylation pattern in colorectal cancer (CRC): cyclin A1 (cyclin al) (100%, 65/65), caudal type homeobox transcriptdon factor 1 (CDX1) (100%, 65/65), RAR(85%, 55/65), myogenic factor 3 (MYOD1) (69%, 45/65),cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)(p15^INK4b) (68%, 44/65), prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) (COX2) (72%, 47/65), cadherin 13, H-cadherin (heart) (CDH13) (65%, 42/65), CAAX box 1 (OO~/) (58%, 38/65),tumor protein p73 (p73) (63%, 41/65) and Wilrns tumor 1 (WT/) (58%, 38/65). However, no significant correlation of changes in rnethylation with any given clinical-pathological features was detected. Furthermore, the frequent changes in rnethylation appeared to be an early phase event of colon carcinogenesis. The in situ expression of 10 genes was assessed by the irnrnunohistochernical approach at the protein level: CDH1, CDH13, COX2, cyclin A1, hMLH1,MGMT, p14^ARF, p73, RAR-, and TIMP3 genes in the context of the rnethylation status in colorectal cancer. No clear correlation between the hyperrnethylation of the promoter CpG islands and the negative expression of the genes was established.CONCLUSION: The methylation profile of 31 genes was established in patients with colon cancer and colorectal adenornas, which provides new insights into the DNA rnethylation mediated mechanisms underlying the carcinogenesis of colorectal cancer and may be of prognostic values for colorectal cancer.

Efficacy of different treatment strategies for hepatocellular carcinoma with portal vein tumor thrombosis

AIM: To evaluate the efficacy of different treatment strategies for hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) and investigate factors influencing prognosis.METHODS: One hundred and seventy-nine HCC patients with macroscopic PVTT were enrolled in this study. They were divided into four groups and underwent different treatments: conservative treatment group (n = 18),chemotherapy group (n = 53), surgical resection group (n = 24) and surgical resection with postoperative chemotherapy group (n = 84). Survival rates of the patients were analyzed by the Kaplan-Meier method. A log-rank analysis was performed to identify group differences. Cox's proportional hazards model was used to analyze variables associated with survival.RESULTS: The mean survival periods of the patients in four groups were 3.6, 7.3, 10.1, and 15.1 mo respectively.There were significant differences in the survival rates among the groups. The survival rates at 0.5-, 1-, 2-, and 3-year in surgical resection with postoperative chemotherapy group were 55.8%, 39.3%, 30.4%, and 15.6% respectively, which were significantly higher than those of other groups (P＜0.001). Multivariate analysis revealed that the strategy of treatment (P＜0.001) and the number of chemotherapy cycles (P = 0.012) were independent survival predictors for patients with HCC and PVTT.CONCLUSION: Surgical resection of HCC and PVTT combined with postoperative chemotherapy or chemoembolization is the most effective therapeutic strategy for the patients who can tolerate operation.Multiple chemotherapeutic courses should be given postoperatively to the patients with good hepatic function reserve.

Glutamine supplemented parenteral nutrition prevents intestinal ischemia-reperfusion injury in rats

AIM: To examine whether glutamine prevents the injury to the intestinal mucosa after intestinal ischemia-reperfusion (I/R) in rats.METHODS: Thirty male Sprague-Dawley rats were randomly divided into 3 groups: a standard parenteral nutrition (PN) group (n = 10); an I/R-PN group (n = 10); an I/R-glutamine enriched PN (I/R-Gln) group (n = 10). The superior rnesenteric artery (SMA) was clamped. After 60 min of ischemia,reperfusion was initiated and infusion was started. All rats received isocaloric and isonitrogenous nutritional support for 48 h. Spleen, liver, mesenteric lymph nodes (MLN), and intestinal segments were removed for morphological and biochemical analyses, and blood samples were collected for bacterial culture and measurement of endotoxin levels.The permeability of intestinnal mucosa was assayed by measurement of D-(-)-Iactate levels in plasma.RESULTS: In I/R-PN group, extensive epithelial atrophy was observed, mucosal thickness, villous height, crypt depth and villous surface area were decreased significantly compared with PN group, whereas these findings did not occur in the I/R-GIn group. The incidence of intestinal bacterial translocation to spleen, liver, MLN, and blood was significantly higher in I/R-PN group than that in other groups.Plasma endotoxin levels significantly increased in the I/R-PN group compared with the I/R-GIn group. Remarkably higher values of D-(-)-Iactate were also detected in PN group compared with that in I/R-GIn group.CONCLUSION: Glutamine protects the morphology and function of intestinal mucosa from injury after I/R in rats.

CT perfusion at early stage of hepatic diffuse disease

AIM: To determine the validity of the non-invasive method of CT perfusion (CTP) in rat model of hepatic diffuse disease. METHODS: Twenty-eight Wistar rats were divided into two groups. Liver diffuse lesions were induced by diethyln-itrosamine in 14 rats of test group. Rats in control group were bred with pure water. From the 1st to 12th wk after the test group was intervened, both groups were studied every week with CTP. CTP parameters of liver parenchyma in different periods and pathologic changes in two groups were compared and analyzed. RESULTS: The process of hepatic diffuse lesions in test groups was classified into three stages or periods according to the pathologic alterations, namely hepatitis, hepatic fibrosis, and cirrhosis. During this period, hepatic artery flow (HAF) of control group declined slightly, mean transit time (MTT), blood flow (BF) and volume (BV) increased, but there were no significant differences between different periods. In test group, HAF tended to increase gradually, MTT prolonged obviously, BV and BF decreased at the same time. The results of statistical analysis revealed that the difference in the HAF ratio of test group to control group was significant. The ratio of BV and BF in test group to control group in stage of hepatitis and hepatic cirrhosis, hepatic fibrosis and early stage of hepatic cirrhosis was significantly different, but there was no significant difference between hepatitis and hepatic fibrosis. The main pathological changes in stage of hepatitis were swelling of hepatic cells, while sinusoid capillarization and deposition of collagen aggravated gradually in the extravascular Disse's spaces in stage of fibrosis and early stage of cirrhosis. CONCLUSION: The technique could reflect some early changes of hepatic blood perfusion in rat with liver diffuse disease and is valuable for their early diagnosis.

Nitrogen Losses from Flooded Rice Field

A field microplot experiment was conducted during the tillering stage of paddy rice to investigate nitrogen(N) Iosses from flooded rice fields following fertilizer application. After application of ammonium bicarbonate,most of nitrogen in the floodwater was present as NH4-N and its concentration varied widely with time.Concentrations of both NO3-N and NO2-N in the floodwater were low due to the weakened nitrification.Under flooded anaerobic reducing conditions, soil solution concentrations of NO3-N and NH4-N were nothigh, ranging from 0.6 mg L-1 to 4.8 mg L-1, and decreased with soil depth. However, the groundwater wasstill contaminated with NO3-N and NH4-N. Rainfall simulation tests showed that the N losses via runoff inrice fields were closely related to the time intervals between fertilizer applications and rainfall events. Whena large rain fell for a short period after fertilizer application, the N losses via runoff could be large, whichcould have a considerable effect on surface water quality. Both irrigation and N fertilizer application mustbe controlled and managed with great care to minimize N losses via runoff from agricultural land.

Prevalence of nonalcoholic fatty liver among administrative officers in Shanghai:an epidemiological survey

AIM: To determine the prevalence of nonalcoholic fatty liver in a specific population in Shanghai by an epidemiological survey, and to analyze risk factors of fatty liver.METHODS: Total 4009 administrative officers who denied regular alcohol drinking participated in the survey, and underwent physical examination and laboratory tests. The important parameters were body mass index (BMI), waist hip circumferences ratio (WHR) and levels of serum lipids.Diagnosis of fatty liver was based on established real-time ultrasonographic criteria, the presence of an ultrasonographic pattern consistent with 'bright liver', with evident ultrasonographic contrast between hepatic and renal parenchyma, vessel blurring, and narrowing of the lumen of the hepatic veins. Analysis of data was performed through SPSS for Windows statistical package.RESULTS: The overall prevalence of fatty liver was 12.9 %,15.8 % in males and 7.5 % in females, and the prevalence of fatty liver in males younger than 50 years old, was significantly higher (13.3 %) than that of in females (2.7 %).But the difference between the sexes became less significant in people older than 50 years (19.1% vs 18.1%). The prevalence of fatty liver was increased with age; this was markedly presented in females younger than 50 years.Multiple variant regression analysis demonstrated that the prevalence of fatty liver was positively correlated to several risk factors, including male, aging (＞50yr), hyperlipidemia,impaired glucose tolerance/diabetes mellitus, hypertension and overweight/obesity.CONCLUSION: There is a high prevalence of nonalcoholic fatty liver among certain population in Shanghai, to which overweight and hyperlipidemia are closely relevant.

Expression of survivin in human gastric carcinoma and gastric carcinoma model of rats

AIM: To study the expression of survivin, an inhibitor of apoptosis protein, in human gastric carcinomas and gastric carcinoma models of rats.METHODS: With the method of immunohistochemical staining, we studied the expression of survivin in 20 cases of chronic gastritis and 56 cases of gastric carcinomas. We used N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and high dose sodium-chloride diet to induce rat gastric carcinomas. Survivin expression was studied in glandular stomachs of normal rats, adenocarcinomas and tissues adjacent to the tumor, as well as in rats during the induction period.RESULTS: Survivin was expressed in 27 of 56 (48.2 %)cases of human gastric carcinoma tissues and 1 of 20 (5 %)cases of chronic gastritis. It was found that the expression of survivin had no relation with the elements of age, tumor depth, tumor size, and disease stage, but was significantly related to histological type. The positive rate of survivin expression in cases of intestinal type was significantly higher than that in cases of diffuse type (P＜0.05). In animal experiments, survivin expression in glandular stomachs of normal rats, of rats in middle induction period, in adenocarcinomas and tissues adjacent to tumor were 0,40.0 %, 78.3 % and 38.9 %, respectively. Compared with the survivin expression in normal rats, the differences were significant.CONCLUSION: These data imply that survivin plays an important role in the onset of gastric carcinoma and that high survivin expression is an early event of gastric carcinoma.

Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

AIM: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC)and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism. METHODS: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCI4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and α-smooth muscle actin (α-SMA) in the livers of rats was detected by immunohisto-chemical assay. Liver pathology was determined by H&E staining and reticulum staining. RESULTS: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of α-SMA between CCl4-injured rats with or without OM treatment (F= 8.99, P>0.05). CONCLUSION: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.

siRNA-mediated inhibition of HBV replication and expression

AIM: RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, we investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. METHODS: pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR.RESULTS: Vector-based siRNA could potently reduce hepatitis 13 virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vectorbased siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. CONCLUSION: Our results indicate that RNA interference may be a potential tool to control HBV infection.

The expression of core fucosylated E-cadherin in cancer cells and lung cancer patients: prognostic implications

It is well documented that the glycosylation of E-cadherin is correlated with cancer metastasis, but whether E- cadherin could be core fucosylated remains largely unknown. We found that E-cadherin was core fucosylated in highly metastatic lung cancer cells while absent in lowly metastatic lung cancer cells. Since α-1,6 Fucosyltransferase (α-1,6 FucT) is known to catalyze the reaction of core fucosylation, we investigated the biological function of core fucosylation on E-cadherin by α-1,6 FucT targeted RNAi and transfecting α-1,6 FucT expression vector. As a result, calcium dependent cell-cell adhesion mediated by E-cadherin was strengthened with the reduction of core fucosylation on E- cadherin after RNAi and was weakened with the elevated core fucosylation on E-cadherin after α-1,6 FucT over expression. Our data indicated that α-1,6 FucT could regulate E-cadherin mediated cell adhesion and thus play an important role in cancer development and progression. Computer modeling showed that core fucosylation on E-cadherin could significantly impair three-dimensional conformation of N-glycan on E-cadherin and produce conformational asym- metry so as to suppress the function of E-cadherin. Furthermore, the relationship between the expression of core fucosylated E-cadherin and clinicopathological background of lung cancer patients was explored in lung cancer tissue of patients. It turns out to demonstrate that core fucosylated E-cadherin could serve as a promising prognostic indicator for lung cancer patients.

Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis

AIM: To investigate the in vitro anti-hepatocellular carcinoma (HCC) activity of docetaxel against SMMC-7721 HCC cells and its possible mechanism.METHODS: The HCC cells were given different concentrations of docetaxel and their growth was measured by colony forming assay. Cell cycle and apoptosis were analyzed by flow cytometry and fluorescence microscopy (acridine orange/ethidium bromide double staining, AO/EB), as well as electronic microscopy. The SMMC-7721 HCC cell reactive oxygen species (ROS) and glutathione (GSH) were measured after given docetaxel.RESULTS: Docetaxel inhibited the hepatocellular carcinoma cells growth in a concentration dependent manner with IC505×10-10 M. Marked cell apoptosis and G2/M phase arrest were observed after treatment with docetaxel ≥10-8M.Docetaxel promoted SMMC-7721 HCC cells ROS generation and GSH deletion.CONCLUSION: Docetaxel suppressed the growth of SMMC7721 HCC cells in vitro by causing apoptosis and G2/M phase arrest of the human hepatoma cells, and ROS and GSH may play a key role in the inhibition of growth and induction of apoptosis.

Ultrastructure and molecular biological changes of chronic gastritis,gastric cancer and gastric precancerous lesions:a comparative study

AIM: To carry out a comparative study on ultrastructure and molecular biological changes of chronic gastritis (CG),gastric cancer (GC) aand gastric precancerous lesions.METHODS: By the use of histochemical staining, SEM with EDAX, TEM with EDAX, image analysis technique, RIA and chemiluminescence method, gastric mucosa of 168 patients were synchronously analyzed in morphology, trace elements, DNA, cAMP, SOD, 3H-TdR LCT and serum LPO were also done.RESULTS: The incidence of epithelial nucleoplasmic ratio ＞1, lobulated nuclei, inter-chromatin aggregation of granules, nucleolar hypertrophy, and the content of DNA,Zn, Cu in nuclei and serum LPO of each group were showed as belows: normal control group (0.0, 0.0, 6.7, 0.0, 12.6±2.7,7.6±0.4, 58.4±0.3, 2.6±0.6), CSG group (5.7, 2.9, 7.4, 2.9,15.2±3.1, 8.1±0.5, 58.9±0.5, 4.2±0.7), CAG group (31.3,29.7, 45.3, 42.2, 16.5±3.1, 8.6±0.4, 59.3±0.5, 4.5±0.6), CA group (100.0, 100.0, 72.2, 50.0, 30.7±8.2, 8.8±0.3, 59.5±0.4,6.8±1.6), ATP++group (61.5, 38.5, 23.1, 38.5, 23.5±8.9,8.3±0.4, 59.1±0.4, 5.1±1.2), IM+++ ATP++group (77.8, 55.5,33.3, 44.4, 25.1±7.2, 8.4±0.5, 59.5±0.4, 6.5±1.1),IM++++ATP++ group (100.0, 100.0, 75.0, 62.5, 28.5±9.1,8.9±0.5, 59.7±0.4, 7.6±0.7), IMⅡb group (100.0, 62.5, 75.0,50.0, 27.3±10.3, 8.6±0.3, 59.5±0.4, 6.1±0.9); whereas the content of Zn, Cu in mitochondria and cAMP, SOD in gastric mucosa, and 3H-TdR LCT of each group were showen as belows: normal control group (9.2±0.5, 58.3±0.3, 15.9±1.5,170.5±6.1, 1079.7±227.4), CSG group (8.6±0.5, 57.8±0.3,14.6±1.8, 163.3±5.6, 867.3±240.5), CAG group (8.3±0.4,57.5±0.3, 13.4±1.8, 161.2±4.3, 800.9±221.8), CA group (8.9±0.4, 57.1±0.3, 10.2±3.9, 152.2±3.8, 325.7±186.8),ATP++ group (9.1±0.4, 57.0±0.3, 12.4±1.8, 161.5±3.8,642.9±174.3), IM+++ ATP++ group (8.6±0.4, 56.9±0.3,12.0±2.3, 152.2±2.5, 326.3±160.3), IM++++ATP++ group (8.5±0.3, 56.8±0.2, 10.4±0.9, 147.4±2.6, 316.1±170.7),IMⅡb group (8.6±0.3, 56.9±0.3, 11.9±1.9, 150.0±2.8,318.9±145.8), there were significant differences between groups (P＜0.05-0.01).CONCLUSION: There was a significant difference between CG and GC in their ultrastructure and molecular biology.Only on the condition of changes of internal environment in combination with the harmful effect of external environment, chronic atrophic gastritis can then develop into gastric cancer. Hence it might have similar epithelial cell ultrastructure and molecular biological changes in ATP++, IMⅡb and cancer, hence there were similar patterns of occurrence, development and transformation.Recognition of this trend might help to explore problems of prevention and cure.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent.Here,we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme pu-trescine N-methyltransferase(PMT)and the downstream enzyme hyoscyamine6β-hydroxylase(H6H)of scopolamine biosynthesis in transgenic henbane(Hyoscyamus niger)hairy root cultures.Transgenic hairy root lines expressing both pmt and h6h produced significantly higher(P<0.05)levels of scopolamine compared with the wild-type and transgenic lines har-boring a single gene(pmt or h6h).The best line(T 3 )produced411mg/liter scopolamine,which was over nine times more than that in the wild type(43mg/liter)and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184mg/liter).To our knowledge,this is the highest scopolamine content achieved through genetic engi-neering of a plant.We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield,which was more efficient than plants harboring only one of the two genes.It seems that the pulling force of the downstream enzyme(the faucet enzyme)H6H plays a more important role in stimulating scopolamine accumulation in H.niger whereas the functioning of the upstream enzyme PMT is increased propor-tionally.This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Transfection of mEpo gene to intestinal epithelium in vivo mediated by oral delivery of chitosan-DNA nanoparticles

AIM: To prepare the chitosan-pmEpo nanoparticles and to study their ability for transcellular and paracellular transport across intestinal epithelia by oral administration. METHODS: ICR mice were fed with recombinant plasmid AAV-tetO-CMV-mEpo (containing mEpo gene) or pCMVβ(containing LacZ gene), whether it was wrapped by chitosan or no. Its size and shape were observed by transmission electron microscopy. Agarose gel electrophoresis was used to assess the efficiency of encapsulation and stability against nuclease digestion. Before and after oral treatmant, blood samples were collected by retro-orbital puncture, and hematocrits were used to show the physiological effect of mEpo. RESULTS: Chitosan was able to successfully wrap the plasmid and to protect it from DNase degradation. Transmission electron microscopy showed that freshly prepared particles were approximately 70-150 nm in size and fairly spherical.Three days after fed the chitosan-pCMVβ complex was fed,the mice were killed and most of the stomach and 30% of the small intestine were stained. Hematocrit was not modified in naive and ‘naked' mEpo-fed mice, a rapid increase of hematocrit was observed during the first 4 days of treatment in chitosan-mEpo-fed animals, reaching 60.9±1.2% (P<0.01),and sustained for a week. The second feed (6 days after the first feed) was still able to promote a second hematocrit increase in chitosan-mEpo-fed animals, reaching 65.9±1.4%(P<0.01), while the second hematocrit increase did not appearin the ‘naked' mEpo-second-fed mice. CONCLUSION: Oral chitosan-DNA nanoparticles can efficiently deliver genes to enterocytes, and may be used as a useful tool for gene transfer.

Modulation of Isoflavones on Bone-nodule Formation in Rat Calvaria Osteoblasts in vitro

Objective To observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro. Methods Osteoblasts obtained from newborn Sprague-dawley rat calvarias were cultured for several generations. The second generation cells were cultured in Minimum Essential Medium supplemented with ascorbic acid and Na-beta-glycerophosphate for several days, in the presence of daidzein and genistein, with or without the estrogen receptor antagonist ICI 182780. Number of nodules was counted at the end of the incubation period (day 20) by staining with Alizarin Red S calcium stain. The release of osteocalcin, as a marker of osteoblast activity, was also determined on day 7 and day 12 during the incubation period. Results Compared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein. 17β-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation than daidzein and genistein. The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17β-estradiol on day 7 and day 12 (day 12 were higher). The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17β-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts. However, the effects induced by daidzein could not be inhibited by ICI 182780. Conclusion These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts. The effects, like those induced by 17β-estradiol, are mediated by the estrogen receptor dependent pathway. Daidzein also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts, but it is not, at least not merely, mediated by the estrogen receptor dependent pathway.

Epithelioid angiomyolipoma of the liver:Cross-sectional imaging findings of 10 immunohistochemically-verified cases

AIM： To retrospectively evaluate the computed tomography （CT）/magnetic resonance imaging （MRI） imaging features of epithelioid angiomyolipoma of the liver （Epi-HAML）, with pathology as a reference. METHODS： The CT/MRI findings （number, diameter, lobar location, and appearance of lesions） in a series of 10 patients with 12 pathologically proven epithelioid angiomyolipomas of the liver were retrospectively analyzed. The imaging features, including attenuation/ signal intensity characteristics, presence of fat, hypervascular, outer rim, and vessels within lesion, were evaluated and compared with that of non-Epi- HAML in 11 patients （13 lesions）. The Fisher exact test was used to compare difference in probability of imaging features between the two types. RESULTS： For 21 patients, CT images of 15 patients and MR images of six patients were available. No patient underwent two examinations. For the 15 patients with a CT scan, all HAML lesions in the two groups （10 Epi-HAML and seven non-Epi-HAML） manifested as hypoattenuation. For the six patients with MRI, all lesions （two Epi-HAML and six non-Epi- HAML） were hypointense on TlWI （fat suppression） and hyperintense on T2WI. There were 10 non-Epi-HAML, but only two Epi-HAML lesions showed the presence of fat, which significantly different between the two types （P = 0.005）. On the dynamic contrast enhancement （DCE） imaging, eight Epi-HAML, and 13 non-Epi lesions manifested as hypervascular. Punctate or curved vessels were displayed in 10 Epi-HAML as well as in nine non- Epi lesions and outer rim enhancement could be found with eight Epi-HAML as well as six non-Epi lesions. CONCLUSION： Little or no presence of adipose tissue was found to be an imaging feature of Epi- HAML, compared with the non-Epi type. In addition, hypervascularity with opacification of central punctiform or filiform vessels on DCE would be a characteristic enhancement pattern for Epi-HAML.

TGF-β1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-b 1 (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of α5β1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment. Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-β1-enhanced cell migration.

Role of hepatitis B virus infection in pathogenesis of IgA nephropathy

AIM: To investigate the role of hepatitis B virus (HBV) in the pathogenesis of IgA nephropathy (IgAN).METHODS: HBV antigens (HBAg, or HBsAg, HBcAg, and HBeAg) in renal tissues with IgAN were detected by immunohistochemical technique. The distribution and localization of HBV DNA were observed by using in situ hybridization. Southern blot analysis was performed to reveal the state of renal HBV DNA.RESULTS: Among 100 patients with IgAN, HBs antigenemia was detected in 18 patients (18.00 %). HBAg in renal tissues was detected in 31 patients (31.00 %), the positive rate of HBAg, HBsAg and HBcAg was 64.52 % (20/31), 32.26 %(10/31), 32.26 % (10/31), respectively in glomeruli. HBcAg was also found in tubular epithelia and interstitia, which was 45.16 % (14/31) and 6.45 % (2/31), respectively. Five out of six cases with positive HBV DNA by in situ hybridization were proved to be HBV DNA positive by Southern blot analysis, and all were of the integrated form. Eight specimens were demonstrated to be HBV DNA positive by in situ hybridization, which was localized in the nuclei of tubular epithelial cells and glomerular mesangial cells as well as in infiltrated interstitial lymphocytes.CONCLUSION: There is a relationship between HBV infection and IgAN. In addition to the humoral immune damage mediated by HBAg-HBAb immune complex, the cellular mechanism mediated by HBV originating from renal cells in situ may be also involved in the pathogenesis of IgAN.