OBJECTIVEHepatic stellate cells (HSC) in hepatocellular carcinoma (HCC) transdifferentiate into extracellular matrix-producing myofibroblasts. Activated HSC can promote invasion and metastasis of HCC. To understand the differences of HSC in normal liver and HCC, we compared the gene expression patterns in HCC cell induction-activated and culture-activated rat HSC.

METHODSHSC were isolated by density centrifugation and exposed to conditioned medium from rat HCC cell line C5F. Expression of 22 012 genes in quiescent HSC, culture-activated HSC and HCC induction-activated HSC was analyzed by cDNA microarray and confirmed by real-time RT-PCR and Western blot.

RESULTS1672 genes were differentially expressed in culture-activated HSC, including proinflammatory factors, cell adhesion molecules, cell surface receptors, signaling transduction molecules and immune factors. 711 genes were differentially expressed in HCC induction-activated HSC. Some of them were identical to those in culture-activated HSC. HCC Induction-activated HSC showed specific gene expression patterns, including Raf1, Rac2, Adam17, Wnt6, MMP-9 and TNF, suggesting that HCC cells can specifically induce HSC activation.

CONCLUSIONThe gene expression patterns in HCC induction-activated HSC are different from those in culture-activated HSC. HCC induction-activated HSC may play a major role in the invasion and metastasis of HCC. In vivo activation should be considered as the standard for the study of HSC biology. HCC induction-activated HSC should be considered as the standard for HSC biology studies.

Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Hepatic Stellate Cells ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Male ; Oligonucleotide Array Sequence Analysis ; Rats ; Rats, Inbred F344

Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.

Objects To explore the association of ulcerative colitis (UC) with the imbalance between Th1,Th 2 and Th17 cells in the colonic tissues.Methods A total of 41 UC patients and 52 controls was recruited in the present study.The real-time fluorescent quantitative PCR was applied for detecting the mRNA levels of Thl,Th2 and Th 17 cells-associated transcription factors T-bet,GATA-3 and RORγt and cytokines IFN-γ,IL-4 and IL-17A in the colonic tissues.Simultaneously,the expressions of IFN-γ,IL-4 and IL-17A in the colonic tissues were also examined by an immunohistochemical staining method.Results Compared with the controls,the mRNA expressions of GATA-3,RORγt and IL-17A were more significantly enhanced in UC patients (0.84 ± 0.24 vs.0.69 ± 0.22,P=0.002;0.99 ± 0.29 vs.0.83 ± 0.23,P=0.004;1.59 ± 0.65 vs.1.35 ± 0.43,P=0.035).According to the ＂Truelove and Witts Severity Index＂,those patients were divided into different subgroups.The mRNA expressions of GATA-3,RORγt,and IL-17A were shown to be higher in patients with moderate and severe UC than in those with mild UC (0.90 ± 0.18 vs.0.78 ± 0.16,P=0.030;1.11 ± 0.31 vs.0.87 ± 0.26,P=0.011;1.83 ± 0.64 vs.1.34 ± 0.66,P=0.020).Moreover,the immunohistochemistry results demonstrated that the IL-17A positive cells were positioned mainly in the intestinal epithelial layer and lamina propria.Compared to the controls,the mean integral optic density of IL-17A was significantly increased in the colonic tissues of UC patients (0.25 ± 0.07 vs.0.13 ± 0.03,P＜0.001).The similar results were obtained for IL-17A in patients with moderate and severe UC when compared to those with mild UC (0.31 ± 0.07 vs.0.19 ± 0.06,P＜0.001).In contrast to the controls,the mRNA ratios ofGATA-3/T-bet,RORγt/ T-bet and RORγt/GATA-3 were significantly higher in the tissues of colonic UC patients (1.12 ± 0.30 vs.0.96 ± 0.31,P=0.014;1.33 ± 0.37 vs.1.15 ± 0.33,P=0.015;1.44 ± 0.45 vs.1.20 ± 0.42,P=0.009),and in the patients,the mRNA ratios for GATA-3/T-bet,RORγt/T-bet and RORγt/GATA-3 were significantly higher in the patients with moderate and severe UC than in those with mild UC (1.27 ± 0.35 vs.1.00 ± 0.32,P＜0.001;1.45 ± 0.37 vs.1.19 ± 0.36,P=0.028;1.59 ± 0.43 vs.1.28 ± 0.46,P=0.031).Conclusions These findings suggest that the imbalance between Thl,Th2 and Th17 cells in the colonic tissues may be implicated in UC.

Objective To investigate the effects of chronic hypobaric hypoxia on COX1 protein and oxidative stress in lung tissue. Methods Rats were randomly divided into normoxia group (1 500 m)and hypoxia group (4 300 m). The rats in hypoxia group were sampled after exposure to hypoxia for 30 days. The COX1 protein in lung tissue of rats was determined by Western blot method; HIF-1αlevel in lung tissue and serum, ROS in plas-ma and SOD, CAT enzyme in lung tissue were determined by ELISA method; PAP of rats were determined by physiological signal acquisition system. Results HIF-1 protein express in serum and lung tissue of rats in hypoxia group was significantly higher than that of the normoxia group (P<0.01), the content of ROS was significantly lower than that of the normoxia group (P<0.01), the expression of COX1 protein in lung tissues of hypoxia group was significantly decreased(P<0.01),serum total antioxidant capacity was elevated in hypoxia group(P<0.01).Conclusions The effects of chronic hypobaric hypoxia on lung tissue may be caused by direct injury, not only by oxidative stress.

Objective To investigate the effects of chronic hypobaric hypoxia on COX1 protein and oxidative stress in lung tissue. Methods Rats were randomly divided into normoxia group (1 500 m)and hypoxia group (4 300 m). The rats in hypoxia group were sampled after exposure to hypoxia for 30 days. The COX1 protein in lung tissue of rats was determined by Western blot method; HIF-1αlevel in lung tissue and serum, ROS in plas-ma and SOD, CAT enzyme in lung tissue were determined by ELISA method; PAP of rats were determined by physiological signal acquisition system. Results HIF-1 protein express in serum and lung tissue of rats in hypoxia group was significantly higher than that of the normoxia group (P<0.01), the content of ROS was significantly lower than that of the normoxia group (P<0.01), the expression of COX1 protein in lung tissues of hypoxia group was significantly decreased(P<0.01),serum total antioxidant capacity was elevated in hypoxia group(P<0.01).Conclusions The effects of chronic hypobaric hypoxia on lung tissue may be caused by direct injury, not only by oxidative stress.

OBJECTIVETo compare different expression profiles of all known chemokine receptors in human hepatocellular carcinoma (HCC) cell lines with different metastasis potentials.

METHODSEighteen pairs of chemokine receptor primers were designed using Premier software. Expression profiles of the 18 chemokine receptors on four HCC cell lines of lower to higher potentials of metastasis (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) were analyzed by RT-PCR. Expression of CXCR4 was detected by RT-PCR.

RESULTSExpression profiles of chemokine receptors on four HCC cell lines with different metastatic potentials had significant differences (P < 0.01), in which CCR10, CXCR4 and CXCR6 expressions decreased gradually as the metastatic potential of the cell lines increased. The expressions of CCR3, CCR4, CCR10, CCR12 and XCR1 on HCCLM6 were significantly reduced compared with SMMC-7721 (P < 0.01), whereas the expressions of CXCR1 (P = 0.006) and CXCR5 (P = 0.003) exceeded that of SMMC-7721. Except for CXCR2, CXCR6 and XCR1, most of chemokine receptors on MHCC97-H were expressed differently compared with MHCC97-L (P < 0.05), in which expressions of CCR1 (P = 0.002), CCR2 (P = 0.004) and CCR5 (P = 0.046) exceeded MHCC97-L. CXCR4 was detected only on the positive controls and SMMC-7721 when the template of total RNA was reduced one-half in RT-PCR.

CONCLUSIONChemokine receptors are expressed very differently at mRNA level on HCC cell lines with different metastatic potentials. The different profiles of chemokine receptors in tumor microenvironment and the function of CXCR4 in HCC should be further studied. Our findings have important implications in understanding the relationship between chemokine receptors and the metastatic potential of HCC.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Receptors, Chemokine ; metabolism

OBJECTIVEAs our previous comparative proteomics study on high and low metastasis human hepatocellular carcinoma (HCC) cell strains revealed that cytokeratin 19 (CK19) was related to higher metastasis potential, we further investigated the relationship between serum CK19 level in HCC patients and their clinico-pathologic characteristics.

METHODSSerum CK19 levels of 101 normal controls and 108 pathology-proven HCC patients were determined using radioimmunoassay, and the their correlation with clinico-pathologic parameters were studied.

RESULTSThe upper limit of one-side 98% confidence interval of normal serum CK19 level was 2.3 microg/L. Among 108 HCC patients, 24 (22.2%) had increased serum CK19 level, ranging from 2.4 to 45.5 microg/L. There were 12 patients (11.1%) with increased CK19 level but normal AFP level. The percentage of poor differentiated tumor was higher in CK19 increased cases (37.5%, 9/24) than in CK19 normal cases (20.2%, 17/84). Moreover, the presence of portal vein tumor emboli was significantly higher in CK19 increased cases (25.0%, 6/24) than in CK19 normal cases (6.0%, 5/84). (Chi-square = 7.403, P < 0.01) In addition, the percentage of TNM stage III/IV tumor was significantly higher in CK19 increased patients (54.2%, 13/24) than in CK19 normal cases. (chi-square = 13.300, P < 0.005)

CONCLUSIONSome HCC patients do have increased serum CK19 level, which could be related to portal vein tumor emboli, poor tumor differentiation and advanced tumor stages.

Adult ; Aged ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; pathology ; Female ; Humans ; Keratins ; blood ; Liver Neoplasms ; blood ; pathology ; Male ; Middle Aged ; Neoplasm Proteins ; blood ; Peptide Fragments ; blood ; genetics ; Proteome ; analysis

Humans ; Male ; Middle Aged ; Scalp ; pathology ; Skin Neoplasms ; diagnosis

Ultrafine powder and cell wall-broken powder of herbal medicine lack of the morphological characters and microscopic identification features. This makes it hard to identify herb's authenticity with traditional methods. We tested ITS2 sequence as DNA barcode in identification of herbal medicine in ultrafine powder and cell wall-broken powder in this study. We extracted genomic DNAs of 93 samples of 31 representative herbal medicines (28 species), which include whole plant, roots and bulbs, stems, leaves, flowers, fruits and seeds. The ITS2 sequences were amplified and sequenced bidirectionally. The ITS2 sequences were identified using Basic Local Alignment Search Tool (BLAST) method in the GenBank database and DNA barcoding system to identify the herbal medicine. The genetic distance was analyzed using the Kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0. The results showed that DNA can be extracted successfully from 93 samples and high quality ITS2 sequences can be amplified. All 31 herbal medicines can get correct identification via BLAST method. The ITS2 sequences of raw material medicines, ultrafine powder and cell wall-broken powder have same sequence in 26 herbal medicines, while the ITS2 sequences in other 5 herbal medicines exhibited variation. The maximum intraspecific genetic-distances of each species were all less than the minimum interspecific genetic distances. ITS2 sequences of each species are all converged to their standard DNA barcodes using NJ method. Therefore, using ITS2 barcode can accurately and effectively distinguish ultrafine powder and cell wall-broken powder of herbal medicine. It provides a new molecular method to identify ultrafine powder and cell wall-broken powder of herbal medicine in the quality control and market supervision.
Cell Wall ; DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; analysis ; Phylogeny ; Plants, Medicinal ; classification ; genetics ; Powders ; Quality Control

OBJECTIVETo isolate and identify the cancer stem cells from primary human ovarian cancer tissues.

METHODSFresh tumor tissues from five cases of pathologically diagnosed ovarian cancers were taken, minced and then digested with collagenase and hyaluronidase to obtain single cell suspension. The erythrocytes were removed with ACK Lysis buffer. The suspensions were sorted by magnetic activated cell sorting (MACS) using CD133-binding microbeads. Then the sorted CD133(+) cells were verified by flow cytometry. The cells were cultured in serum-free medium supplemented with EGF, bFGF, insulin and BSA, and grew into spheroids. Immunofluorescence, differentiation and tumor formation tests of the cells were performed to characterize the properties of cancer stem cells.

RESULTSThe ovarian cancer stem cells were successfully isolated from primary human ovarian tumors, which formed typical spheroids in serum-free medium and had stronger ability of tumorigenesis. The results of related experiments verified that CD133 positive cells owned the properties of cancer stem cells.

CONCLUSIONSThe ovarian cancer stem cells presenting the characteristics of stemness in vitro and in vivo, have been successfully isolated from primary human ovarian tumor tissues by MACS. The isolated ovarian cancer stem cells could be used in future researches of their biological functions.

AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Cell Differentiation ; Cell Separation ; methods ; Female ; Flow Cytometry ; methods ; Glycoproteins ; metabolism ; Humans ; Immunomagnetic Separation ; methods ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Transplantation ; Neoplastic Stem Cells ; metabolism ; pathology ; Ovarian Neoplasms ; metabolism ; pathology ; Peptides ; metabolism