24个中外猪种(群)的AFLP多态性及其群体遗传关系

利用 12对AFLP引物组合检测了 19个中国地方猪种 (群 )、1个培育猪种和 4个欧美引进猪种混合基因组DNA的遗传变异 ,根据AFLP分析结果计算了 2 4个猪种 (群 )间的遗传相似系数 ,据此构建了UPGMA聚类关系图。结果表明 ,AFLP标记有着很高的多态检测效率 (Ai) ,平均每个引物组合检测到 17.3个多态标记 ,非常适合于猪种(群 )遗传多样性分析和品种鉴定 ;中国地方猪种与欧美引进猪种间的遗传分化十分明显 ,两类猪群间的亲缘关系较远 ;南昌白猪与大白猪、铅山黑猪与玉山黑猪有着极近的亲缘关系 ,分别与其育成历史、地理分布和RAPD分析结果相一致。另外还对部分猪种 (群 )的聚类分析结果与其形态学。

SARS尸检的肺部病理改变

目的通过研究严重急性呼吸综合征(severeacuterespiratorysyndrome,SARS)患者的尸检肺部标本,总结SARS的肺部病变特点及发病机制。方法详细检查SARS患者肺脏标本的大体特点,并用常规方法研究显微光镜下SARS累及各肺叶的病变特点。结果7例SARS患者的双肺均明显膨胀,镜下表现以弥漫性肺泡损伤(DAD)不同时期的病变为主。7例均有肺水肿及透明膜形成,肺水肿尤以早期明显。病程超过3周者开始有肺泡内机化及肺间质纤维化,造成肺泡的纤维性闭塞。几乎每例都可见到小血管内的微血栓和肺出血、散在的小叶性肺炎、肺泡上皮脱落、增生等病变。2例可见曲霉菌感染,1例累及左全肺及右肺部分区域,1例出现在肺门淋巴结。肺门淋巴结多表现为充血、出血及淋巴组织减少,窦组织细胞增多。结论SARS肺可能为SARS病毒造成的弥漫性肺泡损伤,表现为肺泡上皮及毛细血管的严重损伤导致肺水肿和肺泡及细支气管的纤维素性渗出性炎症,出现透明膜,继而出现肺泡内机化及肺泡间隔的成纤维细胞增生,共同使肺泡萎陷、机化,最终形成纤维化实变。肺门淋巴组织的减少可能是此病影响免疫系统的又一形态学表现。

DNA免疫诱导的抗轮状病毒抗体反应

本文报道了用编码轮状病毒SA11毒株VP4蛋白的野生型和经修饰后的基因，以质粒DNA形式免疫动物后，血清抗轮状病毒抗体反应和体内mRNA的表达。用ELISA和微量中和实验分别测定血清中抗轮状病毒抗体，结果表明，所有动物的血清都有不同水平识别轮状病毒抗原成分和中和全病毒的能力。

Imbalance between expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in invasiveness and metastasis of human gastric carcinoma

AIM: The expressive balance between matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor of metalloproteinase-1 (TIMP-1) plays a critical role in maintaining the degradation and synthesis of extracellular matrix. Loss of such balance is associated with invasion and metastasis of tumors. This study aimed to determine the expression of MMP-9 and TIMP-1 in gastric carcinoma, and the association of the expressive imbalance between MMP9 and TIMP-1 with the invasion and metastasis and prognosis of gastric carcinoma.METHODS: We used immunohistochemistry to determine the expressions of MMP-9, TTMP-1 and proliferating cell nuclear antigen Ki-67 in the gastric specimens taken from 256 patients with primary gastric carcinoma. The patients were followed-up for up to 96 months.RESULTS: No association between the expression of MMP9 and TIMP-1 and patients' sex and age, tumor size and location of gastric carcinoma was observed. The incidence of the positive expression of MMP-9 in cases with tumors invasion to muscularis propria and visceral peritoneum (70.13% and 69.09%, respectively) was significantly higher than that in cases with tumor invasion only to lamina propria or submucosa (42.50 %, P=0.0162). The positive correlation between MMP-9 expression and the depth of tumor invasion was observed (Pearson correlation coefficient=0.2129,P=0.016). Along with the increase of the metastatic station of lymph nodes, the incidence of the MMP-9 expression was increased by degrees; a positive correlation between them was observed (Pearson correlation coefficient=0.2910,P=0.0001). There was also a significant correlation between MMP-9 expression and the TNM stage in gastric carcinoma (Pearson correlation coefficient=0.3027, P＜0.0001). The incidence of MMP-9 expression in stage Ⅱ and Ⅲ/Ⅳ (75.00%and 76.15%, respectively) was significantly higher than those in stage Ⅰ (46.15 %, P＜0.0001). A negative correlation between TIMP-1 immunoreactivity and the depth of invasion,status of lymph node metastasis and TNM stage was observed (Pearson correlation coefficient =-0.1688, -0.3556and -0.3004, P=0.023, ＜0.0001 and ＜0.0001, respectively).Four types of co-expression of MMP-9 and TIMP-1 were observed; i.e. MMP-9 positive but T IMP-1 negative (n=115),both positive (n=52), both negative (n=62) and MMP-9negative but TIMP-1 positive (n=27). The frequency of serosal invasiveness was significant higher in patients with MMP-9 but without TIMP-1 expression than those with other types of the co-expression (P=0.0303). The incidence of lymph node metastasis was highest in patients with MMP-9but without TIMP-1 expression, and lowest in those with TIMP-1 but without MMP-9 expression (P＜0.0001). The survival rate in patients with MMP-9 but without TIMP-1expression was lower than that in those with TIMP-1 but without MMP-9 expression (P=0.0014).CONCLUSION: Our results in gastric carcinoma demonstrated a significant positive association of MMP-9 over-expression with proliferation of tumor cells, the depth of invasiveness,lymph node metastasis and TNM stage, suggesting MMP-9can serve as a molecular marker of tumor invasion and metastasis. We also demonstrate a significant negative relationship of TIMP-1 expression with the depth of invasiveness and lymph node metastasis, which provide a new idea in the tumor biological and genetic treatment.The interaction between MMP-9 and TIMP-1 in the processes of tumor invasion and metastasis is that MMP-9 mainly promotes tumor invasion and metastasis and TIMP-1 inhibits functions of MMP-9. The imbalance between MMP-9 and TIMP-1 expression may suggest the occurrence of tumor invasion and metastasis, predict poor prognosis. For patients with imbalanced MMP-9 and TIMP-1 expression, the optimal treatment scheme needs to be selected.

Therapeutic effects and prognostic factors in three-dimensional conformal radiotherapy combined with transcatheter arterial chemoembolization for hepatocellular carcinoma

AIM: To evaluate the therapeutic efficacy of threedimensional conformal radiotherapy (3D-CRT) combined with transcatheter arterial chernoembolization (TACE) on the patients with hepatocellular carcinoma (HCC).METHODS: Between 1998 and 2001, 94 patients with HCC received 3D-CRT combined with TACE. A total 63 patients had a Okuda stage Ⅰ lesion and 31 patients had stage Ⅱ. The median tumor size was 10.7 cm (range 3.0-18 cm), and liver drrhosis was present in all the patients. There were 43 cases of class A and 51 dass B. TACE was performed using lipiodol,5-fluorouracil, cisplatin, doxorubicin hydrochloride and mitomycin, followed by gelatin sponge cubes. Fifty-nine patients received TACE only one time, while the others 2 to 3 times. 3D-CRT was started 3-4 wk after TACE. All patients were irradiated with a stereotactic body frame and received 4-8 Gy single high-dose radiation for 8-12 times at the isocenter during a period of 17-26 d (median 22 d).RESULTS: The median follow-up was 37 mo (range 10-48 mo) after diagnosis. The response rate was 90.5%. The overallsurvival rate at 1-, 2-, and 3- year was 93.6%, 53.8% and 26.0% respectively, with the median survival of 25 too. On univariate analysis, age (P=-0.026), Child-Pugh classification for cirrhosis of liver (P=0.010), Okuda stage (P=-0.026),tumor size (P=0.000), tumor type (P=0.029), albuminemia (P=0.035), and radiation dose (P=0.000) proved to be significant factors for survival. On multivariate analysis,age (P=-0.024), radiation dose(P=-0.001), and tumor size (P=0.000) were the significant factors.CONCLUSION: 3D-CRT combined with TACE is an effective and feasible approach for HCC. Age, radiation dose and tumor size were found to be significant prognostic factors for survival of patients with HCC treated by 3D-CRT combined with TACE. Further study for HCC is needed to improve the treatment efficacy.

Expression and significance of PTEN, hypoxia-inducible factor-1 alpha in colorectal adenoma and adenocarcinoma

AIM: To investigate the expression and significance of PTEN,hypoxia-inducible factor-1 alpha (HIF-1α), and targeting gene VEGF during colorectal carciogenesis.METHODS: Total 71 cases colorectal neoplasms (9 cases of colorectal adenoma and 62 colorectal adenocarcinoma)were formalin fixed and paraffin-embedded, and all specimens were evaluated for PTEN mRNA, HIF-1α mRNA and VEGF protein expression. PTEN mRNA, HIF-1α mRNA were detected by in situ hybridization. VEGF protein was identified by citrate-microwave SP immunohistochemical method.RESULTS: There were significant differences in PTEN, HIF1α and VEGF expression between colorectal adenomas and colorectal adenocarcinoma (P＜0.05). The level of PTEN expression decreased as the pathologic stage increased.Conversely, HIF-1α and VEGF expression increased with the Dukes stage as follows: stage A (0.1029±0.0457:0.1207± 0.0436), stage B (0.1656±0.0329: 0.1572±0.0514),and stage C+D (0.2335±0.0748: 0.2219±0.0803). For PTEN expression, there was a significant difference among Dukes stage A, B, and C+D, and the level of PTEN expression was found to be significant higher in Dukes stage A or B than that of Dukes stage C or D. For HIF-1α expression,there was a significant difference between Dukes stage A and B, and the level of HIF-1α expression was found to be significantly higher in Dukes stage C+D than that of Dukes stage A or B. The VEGF expression had similar results as HIF-1α expression. In colorectal adenocarcinoma,decreased levels of PTEN were significantly associated with increased expression of HIF-1α mRNA (r=-0.36, P＜0.05)and VEGF protein (r=-0.48, P＜0.05) respectively. The levels of HIF-1 were positively correlated with VEGF expression (r=0.71, P＜0.01).CONCLUSION: Loss of PTEN expression and increased levels of HIF-1α and VEGF may play an important role in carcinogenesis and progression of colorectal adenocarcinoma.

Correlation of N-myc downstream-regulated gene 1 overexpression with progressive growth of colorectal neoplasm

AIM:To study the function of N-myc downstream-regulated gene 1 (NDRG1) in colorectal carcinogenesis and its correlation with tumor lymph node metastasis,METHODS:NDRG1 was detected at its protein level by immunohistochemistry (IHC) and image analysis (IA), and NDRG1 mRNA was detected by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded sections with a total of 190 specimens including 38 normal colorectal mucosae, 31 colorectal adenomas, 45 non-metastatic colorectal carcinomas (CRCs), 38 metastatic primary CRC and subsequently regional lymph nodes respectively. At the same time, the correlations of NDRG1 with sex, age of patients and histological types of colorectal carcinomas were observed.RESULTS: NDRG1 proteins were gradually increased in colorectal carcinogenesis (P<0.05 or P<0.01). There was a significant difference in the expression of NDRG1 between non-metastatic and metastatic CRCs (P<0.05), and the correlation was positive (P<0.01, rs=0.329). However, there was no obvious difference in the expression of NDRG1 between the primary sites of CRCs and that in the metastatic sites of corresponding regional lymph nodes, nor was there an apparent difference in sex, age, and histological types.The expression of NDRG1 mRNA was generally in concordance with that of NDRG1 protein.CONCLUSION: NDRG1 gene may play an important role in colorectal carcinogenesis.In addition, NDRG1 may be a putative tumor metastasis promoter gene and is regarded as one of the molecular biological markers that can forecast early metastasis of CRCs. NDRG1 gene in the metastatic sites of regional lymph nodes may preserve its expression characteristics in the primary sites of CRCs to some extent.The expression of NDRG1 is not affected by sex, age and histological types. The role of NDRG1 in tumor metastatic process can be demonstrated by in vivo and in vitro.

Nitric oxide and calcium ions in apoptotic esophageal carcinoma cells induced by arsenite

AIM: To quantitatively analyze the nitric okide (NO) andCa2+ in apoptosis of esophageal carcinoma cells induced byarsenic trioxide (As2O3).METHODS: The cell line SHEEC1, a malignant esophagealepithelial call induced by HPV in synergy with TPA in ourlaboratory, was cultured in a serum-free medium and treatedwith As2O3. Before and after administration of As2O3, NOproduction in cultured medium was detected quantitativelyusing the Griess Colorimetric method. Intracellular Ca2+ waslabeled using the fluorescent dye Fluo3-AM and detectedunder confocal laser scanning microscope (CLSM), whichwas able to acquire data in real-time enabling Ca2+ dynamicsof individual cells in vitro. The apoptotic cells wareexamined under electron microscopy.RESULTS: Intracellular concentration of Ca2+ increased from1.00 units to 1.09-1.38 units of fluorescent intensity at As2O3treatment and NO products subsequently released fromAs2O3-treated cells increased from 0.98-1.00 × 10-2 mol@ L-1up to 1.48-1.52 × 10-2 mol@ L-1 and maintained in a highlevel contineously. Finally apoptosis of cells occurred,chromatin being agglutinated, cells shrunk, nuclei becameround and mitochondria swelled.CONCLUSION:Ca2+ and NO increased with cell damage andapoptosis in cells treated by As2O3. The Ca2+ is an initialmessenger to the apoptotic pathway. To investigate Ca2+and NO will be a new direction for studying the apoptoticsignaling messenger of the esophageal carcinoma cellsinduced by As2O3.

Maspin expression and its clinicopathological significance in tumorigenesis and progression of gastric cancer

AIM:To investigate maspin expression in tumorigenesis and progression of gastric cancer and to explore its relevant molecular mechanisms.METHODS:Formalin-fixed and paraffin-embedded tissues from normal mucosa (n=182), dysplasia (n=69), cancer (n=l13) of the stomach were studied for maspin expression by immunohistochemistry. Microvessel density (MVD) in gastric cancer was labeled using anti-CD34 antibody. Maspin expression was compared with clinical parameters and MVD of tumors. Caspase-3 expression was also detected in gastric carcinoma by immunohistochemistry. The relationship between Caspase-3 and maspin expression was concerned as well.RESULTS:The positive rates of maspin expression were 79.8% (145/182), 75.4%(52/69) and 50.4%(57/113) in normal mucosa, dysplasia and cancer of the stomach,respectively.Cancer less frequently expressed maspin than normal mucosa and dysplasia (P<0.05).Maspin expression showed a significantly negative association with invasive depth, metastasis, Lauren's and Nakamura's classification (P<0.05),but not with tumor size, Borrmann's classification,growth pattern or TNIVl staging (P>0.05). The positive rate of Caspase-3 was significantly lower in gastric cancer than in normal gastric mucosa (P<0.05,32.7% vs 50.4%). It was noteworthy that maspin expression was negatively correlated with MVD, but positively correlated with expression of Caspase-3 in gastric cancer (P<0.05).CONCLUSION:Down-regulated maspin expression is a late molecular event in gastric carcinogenesis. Reduced expression of maspin contributes to progression of gastric cancer probably by inhibiting cell adhesion, enhandng cell mobility,decreasing cell apoptosis and facilitating angiogenesis.Additionally altered expression of maspin underlies the molecular mechanism of differentiation of gastric cancer and supports the different histogenetic pathways of intestinal and diffuse gastric cancers. Maspin expression can be considered as an effective and objective marker to reveal biological behaviors of gastric cancer.

Hypoxia-inducible factor-1 in tumour angiogenesis

Hypoxia-inducible factor-1 (HIF-1), composed of HIF-α andHIF-13 subunits, is a heteroclimeric transcriptional activator.In response to hypoxia, stimulation of growth factors, andactivation of oncogenes as well as carcinogens, HIF-1α is overexpressed and/or activated and targets those geneswhich are required for angiogenesis, metabolic adaptationto low oxygen and promotes survival. HIF-1 is critical forboth physiological and pathological processes. Several dozensof putative direct HIF-1 target genes have been identifiedon the basis of one or more c/s-acting hypoxia-responseelements that contain an HIF-1 binding site. A variety ofregulators including growth factors, genetic alterations, stressactivators, and some carcinogens have been documentedfor regulation of HIF-1 in which several signaling pathwaysare involved depending on the stimuli and cell types.Activation of HIF-1 in combination with activated signalingpathways and regulators is implicated in tumour progressionand prognosis. This review presents a summary of thestructure and function of HIF-1α, and correlation amongspecific regulators and their signaling pathways.

Prognostic and clinicopathological features of E-cadherin,α-catenin,β-catenin,γ-catenin and cyclin D_1 expression in human esophageal squamous cell carcinoma

AIM: To investigate the expression of E-cadherin, α-catenin,β-catenin, γ-catenin and cyclin D1 in patients with esophageal squamous cell carcinoma (ESCC), and analyze their interrelationship with clinicopathological variables and their effects on prognosis. METHODS: Expression of E-cadherin, α-catenin, β-catenin, γ-catenin and cyclin D1 was determined by EnVision or SABC immunohistochemical technique in patients with ESCC consecutively, their correlation with clinical characteristics was evaluated and analyzed by univariate analysis. RESULTS：The reduced expression rate of E-cadherin, α-catenin, β-catenin and γ-catenin was 88.7%, 69.4%, 35.5% and 53.2%, respectively. Cyclin D1 positive expression ratewas 56.5%. Expression of γ-catenin was inversely correlated with the degree of tumor differentiation and lymph node metastasis (x^2=4.183 and x^2=5.035, respectively, P<0.05), whereas the expression of E-cadherin was correlated only with the degree of differentiation (x^2=5.769, P<0.05). Reduced expression of E-cadherin and γ-catenin was associated with poor differentiation of tumor, reduced expression of γ-catenin was also associated with lymph node metastasis. There obviously existed an inverse correlation between level of E-cadherin and γ-catenin protein and survival. The 3-year survival rates were 100% and 56% in E-cadherin preserved expression group and inreduced expression one and were 78% and 48% in γ-catenin preserved expression group and in reduced expression one, respectively. The differences were both statistically significant. Correlation analysis showed the expression level of α-catenin correlated with that of E-cadherin and β-catenin (P<0.05). CONCLUSION: The reduced expression of E-cadherin and γ-catenin, but not α-catenin, β-catenin and cyclin D1, implies more aggressive malignant behaviors of esophageal carcinoma cells and predicts the poor prognosis of patients.

Diallyl disulfide-induced G2/M arrest of human gastric cancer MGC803 cells involves activation of p38 MAP kinase pathways

AIM: To determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G2/M arrest in human gastric cancer MGC803 cells.METHODS: MGC803 cell growth inhibition was measured by MTT assay. Phase distribution of cell cyde was analyzed by flow cytometry. Expression of Cdc25C, p38, phosphorylation of p38 (pp38) were determined by Western blotting.RESULTS: MT1- assay showed that SB203580, a specific p38 MAPK inhibitor blocked DADS-induced growth inhibition.Flow cytometry analysis revealed that treatment of MGC803 cells with 30 mg/L DADS increased the percentage of cells in the G2/M phase from 9.3% to 39.4% (P<0.05), whereas inhibition of p38 activity by SB203580 abolished induction of G2/M arrest by DADS. Western blotting showed that phosphorylation of p38 was increased 3.52-fold following treatment of MGC803 cells with 30 mg/L DADS for 20 min(P<0.05), whereas Cdc25C was decreased 68% following treatment of MGCS03 cells with 30 mg/L DADS for 24 h(P<0.05). Decreased Cdc25C protein expression by DADS was attenuated by SB203580 (P<0.05).CONCLUSION: DADS-induced G2/M arrest of MGC803 cells involves activation of p38 MAP kinase pathways.Decreased Cdc25C protein expression by p38 MAPK played a crucial role in G2/M arrest after treatment with DADS.

Influence of serum from liver-damaged rats on differentiation tendency of bone marrow-derived stem cells

AIM: Recent studies in both rodents and humans indicated that bone marrow (BM)-derived stem cells were able to home to the liver after they were damaged and demonstrated plasticity in becoming hepatocytes. However, the question remains as to how these stem cells are activated and led to the liver and where the signals initiating the mechanisms of activation and differentiation of stem cells originate. The aim of this study was to investigate the influence of serumfrom liver-damaged rats on differentiation tendency of bone marrow-derived stem cells.METHODS: Serum samples were collected from rats treated with a 2-acetylaminofluorene (2-AAF)/carbon tetrachloride (CCl4) program for varying time points and then used as stimulators of cultured BM stem cells. Expression of M2- and L-type isozymes of rat pyruvate kinase, albumin as well as integrin-β1 were then examined by reverse transcription polymerase chain reaction (RT-PCR) to estimate the differentiation state of BM stem cells.RESULTS: Expression of M2-type isozyme of pyruvate kinase (M2-PK), a marker of immature hepatocytes, was detected in each group stimulated with experimental serum, but not in controls including mature hepatocytes, BM stem cells without serum stimulation, and BM stem cells stimulated with normal control serum. As a marker expressed in the development of liver, the expression signal of integrin-131 was also detectable in each group stimulated with experimental serum. However, expression of L-type isozyme of pyruvate kinase (L-PK) and albumin, marker molecules of mature hepatocytes, was not detected in groups stimulated with experimental serum.CONCLUSION: Under the influence of serum from rats with liver failure, BM stem cells begin to differentiate along a direction to hepatoo/te lineage and to possess some featuresof immature hepatocytes.

Relationship between the expression of human telomerase reverse transcriptase gene and cell cycle regulators in gastric cancer and its significance

AIM: To investigate the expression of human telomerase reverse transcriptase gene (hTRT) in gastric cancer (GC)and its relevance with cell cycle regulators including P16INK4,cyclin and P53.METHODS: In situ hybridization (ISH) for hTRT mRNA was performed in 53 cases of gastric cancer and adjacent cancerous tissues. Immunohistochemical staining (S-Pmethod) for hTRT protein, P16INK4, cyclinD1 and P53 was performed in 53 cases of GC and adjacent cancerous tissues.RESULTS: Of 53 cases of GC, the expression of hTRT mRNA and hTRT protein was significantly higher than the expression of hTRT mRNA and hTRT protein in adjacent canerous tissues (P＜0.01), the positive rates of hTRTmRNA and hTRT protein were 79.2 % and 88.6 %. There was a stastical difference of the expression of hTRT protein among well differentiated adenocarcinoma, poorly differentiated adenocarcinoma and mucoid carcinoma. And there was a highly significant positive correlation between the expression of hTRT mRNA and hTRT protein (r=0.625, P＜0.01). However, the expression of hTRT mRNA and its protein in GC were not related with other clinicopathological parameters including gender, age, location and size of neoplasm, invasion depth, lymph node metastasis and clinical stage. There was a significant positive correlation between the expression of hTRT mRNA and cyclinD1 protein (r=0.350, P＜0.01). There was a significant positive correlation between the expression of cyclinD1 protein and hTRT protein (r=0.549, P＜0.01), so was between P53 and hTRT protein (r=0.319, P＜0.05).CONCLUSION: The expression of hTRT gene is correlated significantly to the specific defects of cell cycle on G1/S check point; telomerase activity may depend on cell cycle in gastric cancer and it is available to clarify the molecular mechanism of telomerase activity regulation. The expression of hTRT mRNA and hTRT protein in GC is significantly different from the expression of hTRT mRNA and hTRT protein in adjacent cancerous tissue which indicates that these targets are correlated closely to the occurrence of GC and can provide important morphologic index for diagnosis of GC.

Hepatic progenitor cells in human liver cirrhosis:Immunohistochemical,electron microscopic and immunofluorencence confocal microscopic findings

AIM:To investigate whether hepatic progenitor cells(HPC), that reveal the features of oval cells in rodents and small epithelial cells(SEC)in certain human liver disease,were also found in human liver cirrhosis(HLC). METHODS:Surgical liver specimens from 20 cases of hepatitis B virus-positive HLC(15 cases containing hepatocellular carcinoma)were investigated by light microscopic immunohistochemistry(LM-IHC).Among them specimens from 15 cases were investigated by electron microscopy(EM) and those from 5 cases by immunofluorencence confocal laser scanning microscopy(ICLSM).Antibodies against cytokeratin 7 and albumin were used and single and/or double labelling were performed respectively. RESULTS:LM-IHC showed that at the margins of regenerating nodules and in the fibrous septae,a small number of cells in the proliferating bile ductules were positive for CK7 and albumin.At the EM level these HPC were morphologically similar to the SEC described previously,and also similar to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size,oval shape,a high nucleus/cytoplasm ratio,a low organelle content in cytoplasm,and existence of tonofilaments and intercellular junctions.ICLSM revealed that HPC expressed both cytokeratin 7 and albumin. CONCLUSION:HPC with ultrastructural and immunophenotypical features of oval cells,i.e.,hepatic stem cell-like cells as noted in other liver diseases,were found in HLC.These findings further support the hypothesis that bipotent hepatic stem cells,that may give rise to biliary epithelial cells and hepatocytes,exist in human livers.

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

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.

Pathobiological behavior and molecular mechanism of signet ring cell carcinoma and mucinous adenocarcinoma of the stomach:A comparative study

AIM:To elucidate the distinctive pathobiological behavior between signet ring cell carcinoma (SRC) and mucinous adenocarcinoma of the stomach.METHODS: Based on the histological growth patterns and cell-functional differentiation classifications of stomach carcinoma, we conducted a series of comparative studies.All paraffin-embedded and frozen blocks were collected from the files of Cancer Institute of China Medical University. On the basis of histopathological observation, we applied enzymatic and mucous histochemistry, immunohistochemistry,flow cytometry (FCM) and molecular biology to compare these two categories of gastric cancers in terms of the DNA ploidy, proliferative kinetics, the expression of gastric carcinoma associated gene product and instabilities of mitochondrial DNA (mtDNA).RESULTS:Gastric SRC was commonly seen in females below 45 years, mostly presenting diffuse growth and ovary or uterine cervix metastasis. The majority of SRC were absorptive and mucus-producing functional differentiation type (AMPFDT), which growth relied on estrogen. Meanwhile,stomach mucinous adenocarcinomas were mostly observed in males over 50 years, prone to massive growth or nest growth and extensive peritoneal infiltration, showing two categories of cell-functional differentiation types: AMPFDT and mucus-secreting functional differentiation type (MSFDT).Expressions of ER, enzyme c-PDE and 67kDaLN-R in SRC were evidently higher than that in mucinous adenocarcinoma,while expressions of LN, CN-Ⅳ, CD44v6, and PTEN protein were obviously lower in SRC than that in mucinous adenocarcinoma (P<0.05).There was no statistic significance in VEGF, ECD and instabilities of mtDNA (P>0.05) between the above two gastric carcinomas.CONCLUSION: Though SRC and mucinous adenocarcinoma were both characterized by abundant mucus-secretion, they were quite different in morphology, ultrastructure, cell-functional differentiation and protein expression, indicating different mechanisms of carcinogenesis. We concluded that combining histological growth patterns, cell-functional differentiation type with tumor related markers might be significant in early diagnosis and prognosis assessment for SRC and mucinous adenocarcinoma of the stomach.

β-catenin up-regulates the expression of cyclinD1, c-myc and MMP-7 in human pancreatic cancer: Relationships with carcinogenesis and metastasis

AIM:To investigate whether abnormal expression of β-catenin in conjunction with overexpression of cyclinD1, c-myc and matrix metalloproteinase-7 (MMP-7) correlated with the carcinogenesis, metastasis and prognosis of pancreatic cancer, and to analyze the relationship of β-catenin expression with cyclinDl, c-myc and MMP-7 expression. METHODS: Using immunohistochemistry,we examined the expression of β-catenin, cyclinD1,c-myc and MMP-7 in 47 pancreatic adenocarcinoma tissues, 12 pancreatic intraepithelial neoplasia (PanIN) and 10 normal pancreases, respectively. Proliferation cell nuclear antigen was also tested as the index of proliferative activity of pancreatic cancer cells. RESULTS: In 10 cases of normal pancreatic tissues, epithelial cells showed equally strong membranous expression of β-catenin protein at the cell-cell boundaries, but the expression of cyclinDl, c-myc and MMP-7 was negative. The expression of β-catenin, cyclinD1, c-myc and MMP-7 in PanIN and pancreatic adenocarcinoma tissues had no significant difference [6/12 and 32/47 (68.1%), 6/12 and 35/47 (74.5%), 5/12 and 33/47 (70.2%), 7/12 and 30/47 (63.8%), respectively]. The abnormal expression of β-catenin was significantly correlated to metastasis and one-year survival rate of pancreatic cancer, but had no relation with size, differentiation and cell proliferation. The expression of cyclinD1 was correlated with cell proliferation and extent of differentiation, but not with size, metastasis and one-year survival rate of the pancreatic cancer. The expression of c-myc was not correlated with size, extent of differentiation, metastasis and 1-year survival rate, but closely with cell proliferation of pancreatic cancer. The overexpression of MMP-7 was significantly associated with metastasis and 1-year survival rate of pancreatic cancer,but not with size, extent of differentiation and cell proliferation.There was a highly significant positive association between abnormal expression of β-catenin and overexpression of cyclinD1, c-myc and MMP-7 not only in PanIN (r= 1.000, 0.845, 0.845), but also in pancreatic cancer (r= 0.437, 0.452, 0.435). CONCLUSION: The abnormal expression of β-catenin plays a key role in the carcinogenesis and progression of human pancreatic carcinoma by up-regulating the expression of cyclinDl, c-myc and MMP-7, resulting in the degradation of extracellular matrix and uncontrolled cell proliferation and differentiation,β-catenin abnormal expression and MMP-7 overexpression may be considered as two useful markers for determining metastasis and prognosis of human pancreatic cancer.

Expression of mucins and E-cadherin in gastric carcinoma and their clinical significance

AIM: To investigate the expression of three types of mucin(MUC1, MUC2, MUC5AC) and E-cadherin in human gastric carcinomas and their clinical significance. METHODS: Ninety-four gastric cancer specimens were classified according to WHO criteria and detected by immunohistochemical assay of expression of mucins and E-cadherin. RESULTS: The positive expression rates of MUC1, MUC2, MUC5AC and E-cadherin were 82% (77/94), 84% (79/94), 40% (38/94) and 56% (53/94) respectively. MUC1 expression was significantly correlated with the types of cancer (the positive rates of MUC1 in well and moderately differentiated tubular adenocardnoma, poorly differentiated adenocardnoma, signet-ring cell carcinoma and mucinous carcinoma were 91%, 87%, 71%, 71%, respectively, P<0.05), age of patients (the positive rates of it among the people who are younger than 40 years, between 40-60 years and over 60 yearwere 74%, 81%, 89%, P<0.05), lymph nodes involvement (the positive rates in the non-interfered group and the interfered group were 78%, 85%, P<0.05) and tumor size (the positive rates in the tumors with the size less than 3 cm, 3-6 cm and larger than 6 cm were 69%, 92%, 69%, P<0.05); MUC2 expression was significantly associated with types of cancers and had the strongest expression in mucinous carcinomas (the posrdve rates of MUC2 in well and moderately differentiated tubular adenocardnoma, poorly differentiated adenocardnoma,signet-ring cell carcinoma and mucinous carcinoma were 94%, 70%, 81%, 100%, P<0.05), but it had no obvious relation to age, gender, tumor location, lymph nodes involvement,depth of invasion and metastasis to extra-gastric organs (P>0.05); MUC5AC expression was not related to any of the characteristics investigated except that it had relation to gender, whereas MUC5AC showed the tendency to higher expression in less invasive lesions and lower expression in advanced stage cancers (P>0.05); No significant difference was found for E-cadherin expression. There were strong positive relationships between the expression of MUC1 and E-cadherin, MUC2 and E-cadherin, MUC1 and MUC2(R=0.33, R=0.22, R=0.32, respectively, P<0.05). According to the COX proportional hazards model, older patients, involvement of lymph nodes, different types of gastri ccancer and MUC2 expression were significantly associated with poorer outcome of gastric carcinoma patients (β=0.08,β=3.94, β=1.33, β=0.75, respectively, P<0.05). CONCLUSION: MUC1 and MUC2 are good markers of different types of gastric cancer. MUC2 is especially a good marker of mucinous carcinoma. MUCl, MUC2 may interfere with the function of E-cadherin in gastric carcinomas, and have synergic effect on progression of gastric cancers.