Objective To examine the expression of VEGF,MMP-1,and CXCR4 in breast cancer stem cells and its significance.Methods Flow cytometry was employed to separate breast cancer stem cells from MCF-7 cell.Then the expression of VEGF,MMP-1,and CXCR4 was detected by PCR in different subsets of cells.ResultsCompared with the non-stem cells of breast cancer,the expression of VEGF,MMP-1,and CXCR4 in breast cancer stem cells was significantly higher,but the expression of MMP-1 was not different.ConclusionsThe breast cancer stem cells could achieve increased metastasis ability by enhanced expression of VEGF and CXCR4,which are the key factors in metastasis of breast cancer.

Objective To investigate the expression of BCORL1 and E-cadherin and their correlation analysis in gastric carcino-ma .Methods We freshly collected 58 samples of surgically resected paired gastric carcinoma and normal tumor-adjacent tissues and detected BCORL1 and E-cadherin expression in the samples using immunohistochemical staining .The correlation between BCORL1 and E-cadherin protein expression was analysed .Results The protein expression of BCORL1 in gastric carcinoma tissues was sig-nificantly upregulated compared to those of the normal tumor-adjacent tissues(60 .3% vs .17 .2% ,P=0 .000) ,but expression of E-cadherin in gastric carcinoma tissues was significantly lower than those in the normal tumor-adjacent tissues (27 .6% vs .63 .8% , P=0 .000) .Clinicopathological analysis suggested that EphA2 and E-cadherin protein expression were associated with histopatho-logical differentiation ,depth of invasion ,lymph node metastasis and TNM stage(P<0 .05) .BCORL1 was significantly negative cor-related with E-cadherin protein in gastric carcinoma(r= -0 .571 ,P=0 .002) .Conclusion The high-expression of BCORL1 is cor-related with malignant clinicopathological characteristics ,and BCORL1 is negative associated with E-cadherin ,suggesting that BCORL1 promotes tumor progression and metastasis through transcriptional regulating E-cadherin in gastric carcinoma .

Background and purpose:Over-expression of Hypoxia-inducible factor-1? in tumors was known to be associated with resistance to radiation,chemotherapy and with the more malignant tumor phenotypes relative to increased invasiveness,metastatic potential.Furthermore,research has shown that HIF-1? over-expression is associated with aberrant P53 accumulation in human tumors.So we investigated the significance of HIF-1? expression and the relationships among expression of HIF-1?,P53 and P-gp in gastric carcinoma.Methods:The expressions of HIF-1?,P53 and P-gp were investigated by immunohistochemistry in 74 specimens of gastric carcinoma.Results:The positive percentage of P53 protein was higher in the group with HIF-1? positive than the one with HIF-1? negative(70.45% vs 30.0%).The positive rate of P-gp protein was also higher in HIF-1? positive group than in HIF-1? negative group(61.36% vs 36.67%),The positive expression of HIF-1? was significantly related to expression of P-53 and P-gp(r_(s)=0.372,0.256).The positive rate of P-gp protein was higher in P53 positive group than in P53 negative group.Spearman rank correlation test showed a positive correlation between P53 expression and P-gp(r_(s)=0.0283,P

BACKGROUND: In vitro differentiation of embryonic stem cells into hepatocytes has been successfully reported to a certain degree; however, whether embryonic stem cells are able to effectively enter hepatic plate of host after intrahepatic transplantation, whether embryonic stem cells can further differentiate into hepatocytes and express hepatocyte function, and risk factors for neoplastic formation are still unclear at present. OBJECTIVE: To study the intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation models, and to investigate the liver tissue replacement, growth and differentiation in vivo, and neoplastic formation.DESIGN: Randomized controlled animal study.SETTING: Department of Pediatric Surgery, the Second Hospital affiliated to Sun Yat-sen University. MATERIALS: Twenty-four BALB/c mice, 6-8 weeks old, weighing 20-35 g, irrespective of gender, were provided by Guangzhou Experimental Animal Center. Embryonic stem cells-derived hepatic stem cells were differentiated from embryonic stem cells. E14 was provided by Stem cell Center of our hospital. METHODS: This study was performed at the Stem Cell Center, the Second Hospital affiliated to Sun Yat-sen University from July 2006 to June 2007. Twenty-four mice were randomly divided into a liver repopulation model + stem cell transplantation group (group A) and a liver resection + stem cell transplantation group (group B), with 12 mice in each group. Mice in the group A were intraperitoneally injected with 50 mg/kg retrorsine once every two weeks for totally twice. Four weeks after the second injection, about 70% liver was resected. And then, the embryonic stem cells-derived hepatic stem cells, labeled by 1×105 carboxy fluoresce in diacetate succinimidyl ester (CFDA-SE), were transplanted into mouse liver through portal vein. On the other hand, 70% liver of mice in the group B was resected and embryonic stem cells-derived hepatic stem cells were transplanted into mouse liver. MAIN OUTCOME MEASURES: The distribution, incorporation, and proliferation of transplanted cells were observed under fluorescent microscopy. Two weeks later, hepatic function was stained with albumin fluorescence immunoassay (double fluorescence staining) and assayed by level of serum albumin. Embryonic stem cells-derived hepatic stem cells were poured into liver of remedial liver regeneration mice, and undifferentiated embryonic stem cells were transplanted into subcutaneous tissue in axillary region as the controls to observe neoplastic formation in embryonic stem cells-derived hepatic stem cells. RESULTS: ① Growth of hepatic stem cells in recipient mice: One week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, some scattered region was green under fluorescent microscopy. The area of green region increased apparently in 2 weeks, and cord-like structure could be observed. ② Liver function: Immunofluorescent staining of albumin (double fluorescence staining) demonstrated that labeled cells expressed positive albumin (yellow fluorescence) in liver tissue of recipient mice, but there was not significant difference in serum albumin level between group A and group B (P > 0.05). ③ Reliability of hepatic stem cell transplantation: Teratoma did not form over 6 months; however, transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks. CONCLUSION: The transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation model mice can effectively and further grow and differentiate, or even partially express hepatocyte function; in particular, the transplantation is safe.

BACKGROUND: Recently, little attention has been paid to how to induce and identify the functions of differentiated cells in the methods for embryonic stem (ES) cells differentiation into hepatocytes. Whether the differentiated cells express functional characteristics of hepatocytes should be one of the markers to identify the hepatic differentiation of ES cells.OBJECTIVE: To direct mouse embryonic stem cells in vitro differentiation into functional hepatocytes by introduction of murine cholestatic serum in hepatocyte growth factor (HGF)-induced system.DESIGN: A controlled observation and in vitro cytological trial.SETTING: Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Sun Yat-sen University.MATERIALS: The experiment was carried out in the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from October 2004 to February 2007. The mouse E14 ES cell line was kindly provided by the Stem Cell and Tissue Engineering Center of Sun Yat-sen University. Twenty male SD rats, aged 2 weeks, were purchased from the Experimental Animal Center of Sun Yat-sen University. All animal experimental procedures were abided by the rules of animal ethnics.METHODS: The SD rats were undergone common bile duct ligation to induce cholestasis. Ten days after the operation, the whole blood of rats was collected to prepare cholestatic serum. The ES cells were cultured using hanging-drop method for 5-7 days to develop embryonic bodies (EBs). The dissociated EBs cells were then induced hepatic differentiation with spontaneous system, HGF (20 μg/L) system and cholestatic serum (5%) plus HGF (20 μg/L) system, respectively.MAIN OUTCOME MEASURES: The cellular morphologic changes were observed using transverse microscopy dynamically. (2) The cell staining for albumin, α-fetoprotein, CK18/19, glycogen, indocyanine green (ICG) and fluorescein diacetate (FDA) was done after 4 weeks differentiation. (3) The hepatocyte-specific metabolic functions of synthesizing albumin, triacylglycerol and urea nitrogen were assayed at 3 days interval.RESULTS: (1) The differentiation of ES cells cultured in spontaneous system was uncontrolled and the cells could grow into a wide range of three-germ cells. The HGF could promote ES cells differentiation into endoderm and mesoderm (myocardium). But the differentiated cells only expressed low levels of hepatic specific functions in these two induced systems. (2) Under cholestatic serum plus HGF system, the ES cells could differentiate into polygonal cells with very uniform morphology which were positive in glycogen, ICG and FDA staining and showed higher capabilities of synthesizing albumin, triacylglycerol and urea nitrogen than the differentiated cells in the other systems (P＜0.05-0.01).CONCLUSION: The cholestatic serum, a mimic pathological microenvironment in vitro, could effectively promote ES cells-derived hepatocytes induced by HGF to express high level of liver-specific metabolism functions.

AIM: To explore mechanism by which sodium butyrate induces mouse embryonic stem cells(ES) to differentiate into hepatocytes in vitro.METHODS: E14 mouse ES cells were cultivated in a routine way,and then cultivated in suspension to form embryonic bodies(EBs).EBs were transferred into 6-well culture dishes and 3 mmol/L sodium butyrate was added into the culture medium.Morphological changes were investigated by phase contrast microscopy.?-fetoprotein(AFP),albumin(ALB) and cytokeratin 18(CK18) were examined by immunofluorescence staining.AFP,ALB,?_1-antitrypsin(AAT) and TTR mRNA were assayed by RT-PCR.Proportion of ALB positive cells was analyzed by flow cytometry.Periodic acid Schiff(PAS) reaction and indocyanine green(ICG) uptake assay were performed to assess the characteristic hepatocyte function of the differentiated cells.RESULTS: In the presence of sodium butyrate,parts of ES cells differentiated into a population with epithelial morphology similar to mouse hepatocytes.AFP and TTR mRNA expression were observed at 7 d,and ALB and AAT mRNA expressed at 14 d.Hepatocytes specific markers,ALB,AFP and CK18 were positive expression in immunofluorescence staining at 14 d.PAS reaction and ICG uptake were positive for the hepatocyte-like cells.CONCLUSION: Mouse ES cells can be induced into hepatocyte-like cells by sodium butyrate efficiently,and these ES cells-derived hepatocytes possess characteristic hepatocytic function.

0.05).No teratoma was formed in the experimental group,while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION:The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure,proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.

AIM: To investigate whether a pathological micro-environmental culture system consisting of cholestatic sera induces embryonic stem cells (ESC) to differentiate into hepatocyte-like cells in vitro, and select hepatic stem cells from differentiating embryonic stem cells. METHODS: Mouse ESC, E14 cell line, were cultured in Dulbecco's modified Eagle's medium containing 106 U/L recombinant mouse leukemia inhibitory factor (rmLIF) and 10% FCS. After embryonic bodies formed by the hanging drop culture method, they were exposed to fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF) for one week, and then placed to a pathological micro-environmental culture system consisting of 5% cholestatic sera and cultured for 2 weeks. Morphological examination, immunocytochemical staining of albumin and CK8/18 were carried out, and mRNA level of albumin and transthyretin were detected by RT-PCR. Glycogen storage and urea synthesis of the cells were tested with PAS staining and colorimetric assay, respectively. RESULTS: The proliferation of cells was inhibited at the early stage when cultured in a pathological micro-environmental culture system consisting of 5% cholestatic sera, but 2 weeks later, a large number of epithelial-like cell colonies were observed, which exhibited hepatocellular phenotype, expressing albumin and CK8/18, transcribing mRNA of albumin and transthyretin and synthesizing glycogen and urea. CONCLUSION: A pathological micro- environmental culture system consisting of 5% cholestatic sera could not only induce embryonic stem cells to differentiate into hepatocyte-like cells, but select hepatic stem cells from differentiating embryonic stem cells initially induced by FGF-4 and HGF in vitro as well.

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro . METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.

Objective To investigate status of macrolide resistance and determine molecular mechanisms in Mycoplasma pneumoniae.Nethods All of 370 throat swab specimens were cultured to isolate Mycoplasma pneumoniae.Mycoplasma pneumoniae isolates were identified by nested PCR for specific 16SrRNA gene.Antibiotic susceptibility test was done to identify acrolide resistant strains.23SrRNA gene wag amplified by nested PCR followed by direct automatic sequencing method.The DNA sequences were compared to the sequence of Mycoplasma pneumoniae M129(accession no.X68422)to find molecular mechanisms of drug resistance.Results Fifty clinical strains were isolated from 370 specimens.Of 50 strains.4 strains were susceptible to macrulide,46 strains were macrolide resistant with the percentage of 92%.MICs of resistant strains to erythromycin.Azithromycin and josamycin were elevated.The sequence of 23SrRNA gene in 4 Susceptible strains and the reference strain FH was identical to Mycoplagma pneumoniae gene in GenBank.46 resistant strains arbored a point mutation respectively,among them,40 strains had all A to G transition at position 2063.1 strain had an A to C transition at position 2063,the other five strains showed an A to G transition at position 2064.Conclusions Macrolide resistance in Mycoplasma pneumoniae iS very serious health conceru.The point mutation in 23SrRNA.Xpecailly predominant position 2063 mutation contributed to the macrolide resistance in Mycoplagma pneumoniae.The MICs of resistant strains to erythromycin,azithromycin and iosamycin are much higher than Mycoplasma pneumoniae reference strain FH.