Ultrasonic-Assisted Electrochemical Nanoimprint Lithography: Forcing Mass Transfer to Enhance the Localized Etching Rate of GaAs.

Electrochemical nanoimprint lithography (ECNL) has emerged as a promising technique for fabricating three-dimensional micro/nano-structures (3D-MNSs) directly on semiconductor wafers. This technique is based on a localized corrosion reaction induced by the contact potential across the metal/semiconductor boundaries. The anodic etching of semiconductor and the cathodic reduction of electron acceptors occur at the metal/semiconductor/electrolyte interface and the Pt mold surface, respectively. However, the etching rate is limited by the mass transfer of species in the ultrathin electrolyte layer between the mold and the workpiece. To overcome this challenge, we introduce the ultrasonics effect into the ECNL process to facilitate the mass exchange between the ultrathin electrolyte layer and the bulk solution, thereby improving the imprinting efficiency. Experimental investigations demonstrate a positive linear relationship between the reciprocal of the area duty ratio of the mold and the imprinting efficiency. Furthermore, the introduction of ultrasonics improves the imprinting efficiency by approximately 80 %, irrespective of the area duty ratio. The enhanced imprinting efficiency enables the fabrication of 3D-MNSs with higher aspect ratios, resulting in a stronger light trapping effect. These results indicate the prospective applications of ECNL in semiconductor functional devices, such as photoelectric detection and photovoltaics.

Regulation of heterogeneous electron transfer reactivity by defect engineering through electrochemically induced brominating addition.

Enhancing the electrochemical activity of graphene holds great significance for expanding its applications in various electrochemistry fields. In this study, we have demonstrated a facile and quantitative approach for modulating the defect density of single-layer graphene (SLG) via an electrochemically induced bromination process facilitated by cyclic voltammetry. This controlled defect engineering directly impacts the heterogeneous electron transfer (HET) rate of SLG. By utilizing Raman spectroscopy and scanning electrochemical microscopy (SECM), we have established a correlation between the HET kinetics and both the defect density (nD) and mean distance between defects (LD) of SLG. The variation of the HET rate (k0) with the defect density manifested a distinctive three-stage behavior. Initially, k0 increased slightly with the increasing nD, and then it experienced a rapid increase as nD further increased. However, once the defect density surpassed a critical value of about 1.8 × 1012 cm-2 (LD < 4.2 nm), k0 decreased rapidly. Notably, the results revealed a remarkable 35-fold enhancement of k0 under the optimal defect density conditions compared to pristine SLG. This research paves the way for controllable defect engineering as a powerful strategy to enhance the electrochemical activity of graphene, opening up new possibilities for its utilization in a wide range of electrochemical applications.

[Expression of epidermal fatty acid-binding protein in cross-species hepatocellular carcinoma].

OBJECTIVE: To evaluate the utility of the cross-species screening strategy for investigating key molecule(s) involved in onset and progression of hepatocellular carcinoma (HCC). METHODS: HCC-related molecule data from our previous studies and in the literature were collected to establish a cross-species dataset. Tissue samples of HCC, non-HCC surrounding liver (para-HCC), and normal liver that were collected from humans, tree shrews and rats. The genes reported to have the most differential expression in HCC were verified by analyzing the mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. RESULTS: The cross-species dataset of HCC-related molecules included four genes: epidermal fatty acid-binding protein (E-FABP), liver (L)-FABP, tyrosine a-ketoglutarate transaminase (TKT), and cytokeratin (CK8). In humans, E-FABP mRNA expression was significantly higher (P less than 0.05) in HCC (0.87+/-0.14 vs. para-HCC: 0.64+/-0.12 and normal liver: 0.67+/-0.07; F=20.910). Similar results were obtained in tree shrew (HCC: 0.87 +/- 0.25 vs. para-HCC: 0.73 +/- 0.19 and normal liver: 0.68+/-0.19; F=3.807) and rat (HCC: 0.97+/-0.22 vs. para-HCC: 0.78+/-0.16 and normal liver: 0.80 +/- 0.13; F=4.482). The Western blotting analyses revealed a similar statistically significant trend. CONCLUSION: The cross-species screening strategy for tumor genes may represent a feasible and convenient process of identifying key molecule(s) for human HCC. E-FABP may be a particularly crucial molecule for hepatocarcinogenesis.

[Factors influencing long-term hepatitis B virus infection of the tree shrew (Tupaia belangeri chinensis) as an in vivo model of chronic hepatitis B].

OBJECTIVE: To determine the methods for establishing an in vivo model of long-term hepatitis B virus (HBV) infection in the Chinese tree shrew (Tupaia belangeri chinensis). METHODS: Seventy-seven neonate (1-3 days old) and 49 young adult (2 weeks to 1 year old) tree shrews were inoculated with different HBV sources (chronic hepatitis B (CHB) human patient serum, single or pooled; HBV-infected tree shrew serum, single only; HBV-infected HepG2.2.15 cells' culture medium supernatant; HBV genome-transfected HepG2.2.15 cells' culture medium supernatant) through various routes of injection (subcutaneous, intraperitoneal, and direct liver via abdominal skin; adults also received intravenous and indirect liver via spleen). Serum and liver biopsies were collected from the animals at various time points post-inoculation for detection of HBV markers by fluorescence quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, time-resolved immunofluorescence, Southern blotting, dot blotting, immunohistochemistry, and microscopy. RESULTS: Among the neonatal group of tree shrews, six (7.8%) were confirmed as HBV-infected for more than 72 (up to 228) weeks after inoculation and another seven (9.1%) were suspected of persistent infections. None of the young adult tree shrews developed persistent infection. Inoculation with single-source serum from either CHB humans or tree shrews were responsible for the most cases of infections, and the subcutaneous injection produced more infections than the other inoculation routes. The most reliable methods of determining HBV infection status were detection of serum HBV immunoreactive markers and intrahepatic HBV DNA. CONCLUSION: In order to establish an in vivo model of CHB in the tree shrew, the animals should be inoculated in the neonatal period using subcutaneous injection.

Electrochemically-driven large amplitude pH cycling for acid-base driven DNA denaturation and renaturation.

In this paper, we present an electrochemically driven large amplitude pH alteration method based on a serial electrolytic cell involving a hydrogen permeable bifacial working electrode such as Pd thin foil. The method allows solution pH to be changed periodically up to ±4~5 units without additional alteration of concentration and/or composition of the system. Application to the acid-base driven cyclic denaturation and renaturation of 290 bp DNA fragments is successfully demonstrated with in situ real-time UV spectroscopic characterization. Electrophoretic analysis confirms that the denaturation and renaturation processes are reversible without degradation of the DNA. The serial electrolytic cell based electrochemical pH alteration method presented in this work would promote investigations of a wide variety of potential-dependent processes and techniques.

[Effects of AKR1B10 gene silence on the growth and gene expression of HCC cell line MHCC97H].

OBJECTIVE: To explore the biological function and possible underlying mechanism of aldo-keto reductase family 1 member B10 (AKR1B10) gene during hepatocarcinogenesis. METHODS: A pair of chemically synthesized small interfering RNA (siRNA) targeting on AKR1B10 was transfected into liver cancer cell line MHCC97H by LipofectamineTM 2000. After confirming the interfering effects of AKR1B10-siRNAs through Quant SYBR Green polymerase chain reaction (Real-time PCR), Western blot and enzymatic activity assay, the capabilities of proliferation and apoptosis of the transfected cells were observed by CCK-8 assay and flow cytometry analysis, and the expressions of a group of tumor-related gene such as c-myc, c-fos, N-ras were observed through Real-time PCR. RESULTS: The expressions of AKR1B10 and the enzymatic activity were down-regulated significantly in AKR1B10-siRNA-transfected cells. Compared with mock and blank control groups, cell growth in AKR1B10-siRNA-transfected group was inhibited by 26.6%+/-3.1% at 72h after transfection. The ratio of apoptotic cells was 37.3%+/-1.0% in AKR1B10-siRNA-transfected group, which was significantly higher than that in mock and blank control groups (P < 0.01). Real-time PCR showed that the expressions of oncogene c-myc, c-fos and N-ras, and the proliferation-associated gene ki-67 were down-regulated in AKR1B10-siRNA-transfected cells, while the expressions of apoptosis-promoting gene caspas-3 and bax were up-regulated. CONCLUSIONS: AKR1B10 might promote proliferation, inhibit apoptosis and then induce malignant transformation of hepatocytes by regulating the expression level of some tumor-related genes.

[Differentially expressed proteins in the precancerous stage of rat hepatocarcinogenesis induced by diethylnitrosamine].

OBJECTIVE: To screen the differentially expressed proteins especially at the precancerous stage of diethylnitrosamine (DEN) induced hepatocarcinogenesis by comparative proteome research. METHODS: Rats were divided into normal and DEN groups and sacrificed periodically. The liver samples were stained with gamma-glutamyl transpeptidase (GGT) and HE to distinguish the preneoplastic lesion (pre-HCC) from the normal and HCC tissues. The two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) were then applied to analyze the differentially expressed protein between pre-HCC and normal tissues, pre-HCC and HCC, as well as HCC and normal tissues. A few of the candidate proteins such as laminin receptor 1 (67LR) and agmatinase were validated by Western blot and RT-PCR. RESULTS: Totally, there were 82 proteins that differentially expressed two fold or more in one kind of tissues sample than the other, 47 of which occurred in the pre-HCC tissues. Eight proteins including 67LR were consistently up-regulated from normal tissue to pre-HCC and then to HCC tissues, while 22 proteins including agmatinase showed progressively down-regulated in these tissues samples. CONCLUSION: The protein expression profiles are different during the process of hepatocarcinogenesis. Further study on the differentially expressed protein, especially these upregulated in the precancerous stage such as 67LR and agmatinase, might contribute to prevention and early diagnosis of human HCC.

[Long-term observation of hepatitis B virus (HBV) replication in new-born tree shrews inoculated with HBV].

OBJECTIVE: To observe the hepatitis B virus (HBV) replication in the tree shrews that were inoculated with HBV at neonatal period. METHODS: Six new-born tree shrews were inoculated with human HBV positive serum. Blood samples and liver biopsies were collected at different time points after inoculation. The HBV infection markers were tested by nested polymerase chain reaction (nPCR), fluorescence quantitative polymerase chain reaction (FQ-PCR), Southern blot, ELISA and immunohistochemistry staining. The liver tissues were observed under electron and light microscope. RESULTS: 48 weeks after inoculation, HBV DNA and HBV cccDNA were detected in the serum and liver samples of three animals (number 1, 2 and 6) by nPCR. The copy-numbers of HBV DNA detected by FQ-PCR in their serum and liver samples were 103 and-104/ml respectively,and the total DNA in 1microg liver tissue was 107-108. Southern blot indicated that HBV replication intermediates such as HBV cccDNA and HBV ssDNA was detectable in liver tissues. HBsAg was detected by ELISA, and immunohistochemical staining showed a gradual increase of HBsAg-positive liver cells. High copy number of HBV DNA and suspected HBV EM particles could be detected in the liver samples from one of the three animals that have survived more than 2 years after inoculation. The other three animals showed low HBV DNA copy number, and the rest of the signs of HBV infection were negative or transiently positive. CONCLUSIONS: Neonatal tree shrews can be infected with human HBV. HBV can replicate inside the liver cells of tree shrew.

[Ginkgo biloba extracts (EGb761) inhibits aflatoxin B1-induced hepatocarcinogenesis in Wistar rats].

OBJECTIVE: To investigate the effect of Ginkgo biloba extracts (EGb761) on aflatoxin B1 (AFB1)-induced hepatocarcinogenesis and its antioxidant activity in Wistar rats. METHODS: 71 Wistar rats were randomly divided into three groups: AFB1 (group A); AFB1 +EGb761 (group B), Control (group C). Rats in gurop A and B were injected with AFB, through abdomen and the doses were 100-200 microg/kg, one to three times a week. Liver biopsy were performed in all rats during 14th w, 28th w, 42th w and 55th w, and were executed at 64th w. Gammaglutamyl transpeptidase-positive hyperplastic cell foci (gamma-GT foci) and histopathology of the liver tissue were observed. The levels of malondialdehyde (MDA), as well as the activity of Glutathione peroxidase (GSH-Px) was examined. RESULTS: At 42th w and 55th w, the gamma-GT focus area (mm2/focus) and general area of foci (mm2/cm2) of group B were significantly smaller than that in group A (P = 0.000). The incidence of hepatocelluiar carcinoma (HCC) in group B (26.92%) was significantly lower than that in group A(76%) (P = 0.000). Group C didnt have HCC development. EGb 761 markedly increased GSH-Px activity, reduced MDA levels (P < 0.05). CONCLUSION: EGB761 shows effective inhibition to hepatocarcinogenesis induced by AFB1 in rats, which may be related to its antioxidant activity.

[The biological function of peroxiredoxin II on Hep3B cells and its underlying mechanism].

OBJECTIVES: To study the biological function and its possible underlying mechanism of peroxiredoxin II (PrxII) in liver cancer cell line Hep3B. METHODS: Two pairs of double-stranded small interfering RNA (siRNA) targeted on PrxII gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive oxygen species (ROS) and malondialdehyde (MDA), were applied to explore whether the antioxidant mechanism was involved in the effects of PrxII functioning on Hep3B cell. RESULTS: The two pairs of siRNA significantly inhibited PrxII mRNA and protein expression. Compared to the mock and blank control groups, the two PrxII-silent groups showed decreased rates of cell growth and clone formation and increased rates of cell apoptosis. The numbers of the formed colonies were 42.0+/-2.8 and 40.5+/-0.7 respectively in the two PrxII-silent groups, while they were 121.5+/-2.1 and 130.0+/-1.4 in the mock and blank control groups (P less than 0.05). The levels of endogenous ROS and MDA were significantly higher in the two PrxII-silent groups than those in the mock and blank control groups (P less than 0.05). CONCLUSION: PrxII might play an important role in the hepatocarcinogenesis, possibly through an antioxidant function which may provide a favorable microenvironment for cancer cell survival and progression.

[Dynamic expression of survivin during hepatocarcinogenesis in rats].

OBJECTIVE: To determine the dynamic expression of survivin gene in hepatocarcinogenesis of rats induced by aflatoxin B1 (AFB1). METHODS: 78 Sprague-Dawley rats were used in this study. Hepatocellular carcinoma was induced in the rats by aflatoxin B1. Liver and HCC tissues were examined by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The earliest hepatocellular carcinoma occurred at 46th week after AFB1 treatment. The HCC incidence was 54.9% (28/51) at 46th week and 64.9% (24/37) at 58th week. The positive rates of survivin protein expression in 24 HCC, para-cancerous liver tissues of experimental group were 41.7% and 54.2%, respectively, with no significant difference between them (P > 0.05). No survivin expression was detected in the experimental group before 46th week, neither in the rats without HCC occurrence nor the normal controls. The level of survivin mRNA expression in HCC at 58th week was significantly higher than that in pre-HCC, no-HCC and normal liver tissues in the control group (P < 0.01). The level of survivin mRNA expression in para-carcinoma tissues was also significantly higher than that in no-HCC and normal liver tissues of the control (P < 0.01). The level of survivin mRNA in pre-HCC at 12th, 20th, 36th, 46th weeks were significantly higher than those in normal liver tissues taken from control group during the same periods (P < 0.01). CONCLUSION: The over-expression of survivin gene is related to the occurrence of HCC and may play an important role in the carcinogenesis of HCC.

[The expression of peroxiredoxin II in hepatocellular carcinoma and its significance].

OBJECTIVE: To evaluate the mRNA and protein expressions of peroxiredoxin II (PrxII) in hepatocellular carcinoma (HCC) and their significance. METHODS: HCC was induced by aflatoxin B1 (AFB1) in 6 tree shrews (Tupaia belangeri chinensis). The expression levels of PrxII mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot on HCC tissues and on their surrounding liver tissues (para-HCC). Biopsied liver tissues were taken before the HCC induction (pre-HCC) from the same animals and from a group of blank controlled animals that served as controls. Liver biopsy specimens from 18 cases of human HCC and from 17 healthy human volunteers were studied using the same methods. RESULTS: The mRNA and protein expressions of PrxII in tree shrew HCC tissues were significantly higher than those in para-HCC and pre-HCC tissues, and also higher than those in the liver tissues from the control animals (all P < 0.05). The expression levels of PrxII mRNA and protein in human HCC tissues were also significantly higher than those in their para-HCC tissues and in the human normal liver tissues (P < 0.05). CONCLUSION: PrxII might play an important role in hepatocarcinogenesis and might be used as a molecular target for HCC prevention and treatment.

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis.

AIM: To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma (HCC), the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS: Tree shrew HCC was induced with aflatoxin B1 (AFB1), and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30th-, 60th-, and 90th-wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique. RESULTS: The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bcl-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION: Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.

The tree shrews: useful animal models for the viral hepatitis and hepatocellular carcinoma.

Hepatitis B virus (HBV)-induced hepatitis and hepatocellular carcinoma (HCC) are major diseases worldwide. HBV infection and chemical carcinogens such as aflatoxin B1 are known to be two key factors in the development of HCC. Animal models for hepatitis and HCC are very useful in the in vivo studies of mechanism involved in the development and prevention of these diseases and the pre-clinical research of drugs for the treatment of these diseases. Now, several animals, such as woodchucks, ground squirrels, chimpanzees, ducks and tree shrews, have been used to establish hepatitis and HCC models. HCC occurs in some woodchucks and ground squirrels that are infected with their own hepatitis viruses and exposed to carcinogens. Chimpanzees and ducks can be infected with human and duck hepatitis B viruses, respectively, but HCC is rarely observed in these animals. The tree shrews are non-rodent, small animals and close to primates in evolution. This review focuses on the establishment of human HBV-induced hepatitis and human HBV-associated HCC in tree shrews and their applications in the study of HCC development.

[A cDNA microarray study of the differential expression of genes in signal transduction pathway during hepatocarcinogenesis in tree shrews].

OBJECTIVES: To study the differential expression of genes in signal transduction pathway (STP) during the hepatocarcinogenesis in tree shrews induced by AFB1 and/or HBV and to elucidate the molecular mechanism of hepatocellular carcinoma (HCC) development. METHODS: Adult tree shrews were divided into three groups: Group A was fed AFB1 only, Group B was infected firstly with HBV then fed AFB1 as in Group A, Group C served as the normal control. Liver biopsies were obtained at the 30th, 60th and 90th week of the experiment or until HCC occurred and the animals were sacrificed. Tree shrew-specific cDNA microarray was applied for detecting the differential expression of corresponding genes in each group at different time points during the experiment, and real time RT PCR was applied to verify the results of the cDNA microarray. RESULTS: Genes of IGF-II, C-rel, and NF-kappaB2 were differentially expressed between para-cancerous tissues and HCC tissues in both group A and group B, and the differential expression of bcl-2, cyclin A and CNTF was only seen in group B. Between the experimental groups A and B and the control group C, there were differential expressions of CNTF and cyclin A in the early 30th week and middle 60th week stage of hepatocarcinogenesis in tree shrews. Real time RT PCR results showed that the expression level of IGF-II and C-Rel in group A and of IGF-II in group B in HCC tissues were significantly lower than that in the adjacent non-cancerous tissues and in the biopsies taken at the 30th and 60th week of the experiment. Nevertheless, there were no significant differences between the para-cancerous tissues and the cancer tissues at the 30th and 60th week. These results were consistent with the cDNA microarray assay. The expression levels of C-Rel and CNTF in group B were not obviously altered in the para-cancerous tissues, HCC and at the 60th week, but they were significantly lower in these tissues than that in the tissues at the 30th week. In group A, the expression levels of CNTF in adjacent liver and HCC tissues were higher than that in para-cancerous lesions, but the difference did not reach a statistically significant level. In group C, the expression level of IGF-II, C-Rel and CNTF at different stages showed no significant differences, which was consistent with the cDNA microarray results. CONCLUSIONS: To apply the tree shrew-specific cDNA microarray to detect the differential expression of genes related to signal transduction pathway during tree shrew hepatocarcinogenesis could be a valuable utility for further comprehending the mechanism of HCC. IGF-II, NF-kappaB2, C-rel, Bcl-2, and cyclin A. CNTF may be involved in the occurrence and progress of HCC in tree shrews.

Alteration of p53 and p21 during hepatocarcinogenesis in tree shrews.

AIM: To investigate p53 mutation and p21 expression in hepatocarcinogenesis induced by hepatitis B virus (HBV) and aflatoxin B(1) (AFB(1)) in tree shrews, and to reveal the role of these genes in hepatocarcinogenesis. METHODS: Tree shrews were divided into four groups: group A, those infected with HBV and fed with AFB(1) (n = 39); group B, those infected with HBV alone (n = 28); group C, those fed with AFB(1) alone (n = 29); and group D, normal controls (n = 20). The tree shrews underwent liver biopsies once every 15 wk. Expression of p53 and p21 proteins and genes in the biopsies and tumor tissues of the experimental tree shrews was detected, respectively, by immunohistochemistry, and by Southern blotting and reverse transcription-polymerase chain reaction and sequencing. RESULTS: The incidence of hepatocellular carcinomas (HCC) was higher in group A (66.7%) than that in group B (3.57%) and C (30%). The time of HCC occurrence was also earlier in group A than that in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, respectively, P<0.01). p53 protein was not detected by immunohistochemistry in all groups before the 75(th) wk of the experiment. At the 105(th) wk, the positive rates fo p53 were 78.6%, 60% and 71.4% in groups A, B and C, respectively, which were significantly higher than that in group D (10%) (all P<0.05). An abnormal band of p53 gene was observed in groups A and C. The mutation points of p53 gene in tree shrews with HCC were at codons 275, 78 and 13. The nucleotide sequence and amino acid sequence of tree shrew's wild-type p53 showed 91.7% and 93.4% homologies with those of human p53, respectively. The immunopositivity for p21 was found before HCC development. The incidence of HCC was significantly higher in tree shrews that were positive for p21 than those negative for p21 (80.0% vs 11.0%, P<0.001). The incidence of HCC in p21 positive animals in group A was significantly higher than those positive for p21 in group C (P<0.05). CONCLUSION: A remarkable synergistic effect on HCC development exists between HBV and AFB(1). p53 mutation promotes the development of HCC. HBV and AFB(1) may synergistically induce p53 gene mutation, and stimulate ras gene expression. ras gene is activated at the earlier stage during hepatocarcinogenesis. p21 protein may be an early marker, and the alterations of p53 may be a late event in the development of HCC.

Differential expression of genes during aflatoxin B(1)-induced hepatocarcinogenesis in tree shrews.

AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B(1) (AFB(1)), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB(1) for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of Atlas(TM) cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as "important genes" because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could change their expressing levels both in HCC and in HCC-surrounding non-cancerous liver tissues. A few modular genes were up-regulated only in HCC but not in surrounding liver tissues, while some apoptosis-related genes were down-regulated in HCC and up-regulated in surrounding liver tissues. To compare gene-expressing levels among the liver tissues taken at different time points during hepatocarcinogenesis may be helpful to locate the responsible gene (s) and understand the mechanism for AFB(1) induced liver cancer.

Alteration of the p53 gene during tree shrews' hepatocarcinogenesis.

OBJECTIVE: To detect the expression and variation of the p53 gene in hepatocarcinogenesis of tree shrews induced by hepatitis B virus (HBV) and aflatoxin B1 (AFB1). METHODS: Tree shrews were divided into four groups: group A, infected with HBV and fed with AFB1; group B, only infected with HBV; group C, fed with AFB1 alone; and group D normal control. The tree shrews underwent liver biopsy every 15 weeks. Liver and tumor tissues were detected by immunohistochemistry and molecular biotechnologies. RESULTS: The incidence of hepatocellular carcinoma (HCC) was higher in group A (66.7%) than in groups B (0) and C (30%). HCC occurrence was earlier in group A than in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, t=3.336, P<0.01). Mutated p53 protein was not found in all groups before 75 weeks of experiment. At the 105th week, the expression rates of mutated p53 protein were 78.6%, 60.0% and 71.4% in groups A, B and C respectively, which were significantly higher than that in group D (10%) (chi2> or =5.03, P<0.05). An abnormal band of the p53 gene was detected in groups A and C. The mutational points of the p53 gene in liver cancer of tree shrews were at codon 275, 78 and 13. Nucleotide sequence and amino acids sequence of tree shrew's wild-type p53 were 91.7% and 93.4% in homology compared with those of human p53, respectively. CONCLUSIONS: Remarkable synergistic effect on HCC exists between HBV and AFB1. Mutated p53 protein expressed before occurrence of HCC promotes the development of HCC. HBV and AFB1 may synergistically induce p53 gene mutation.

[Different gene expression during hepatocarcinogenesis in tree shrew induced by aflatoxin B1].

OBJECTIVE: To understand the molecular mechanism and find out the responsible genes for liver cancer by exploring the regulation of gene expression during hepatocarcinogenesis in tree shrew induced by aflatoxin B1 (AFB1). METHODS: The tissues from tree shrew of different stages during the pathogenesis and development of hepatocellular carcinoma (HCC), liver cancer tissue, para-cancerous tissues, pre-cancerous liver tissues, liver tissues of the same stage from normal controls and the liver tissues taken before AFB1-treatment were analyzed for gene expression by cDNA array. RESULTS: Four patterns of gene expression were observed during AFB1-induced hepatocarcinogenesis. They were: genes up-regulated in HCC tissue and para-cancerous tissue, especially in HCC tissues; genes with similar expressing level in both HCC tissue and para-cancerous tissue, but higher than that in pre-cancerous tissue; genes down-regulated in HCC tissue; genes up-regulated before HCC appeared but down-regulated after HCC appeared. CONCLUSION: Dynamic observation of gene expression will be beneficial to elucidate the mechanisms of AFB1- induced hepatocarcinogenesis and locate the responsible genes.

[Expression of tumor-specific cancer/testis antigens in hepatocellular carcinoma].

OBJECTIVE: (1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression. METHODS: The cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes. RESULTS: The MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05). CONCLUSIONS: With a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.