[Expression of Foxa2 and its early alarm value of cancerous in gastric polyps].

OBJECTIVE: To explore the expression of Foxa2 in different pathological types of gastric polyps and examine the correlation with cancerous risk. METHODS: According to computerize random number, a total of 2000 patients were selected to receive endoscopic biopsy during November 2011 to October 2012. Tissues were harvested from 170 with gastric polyps and suspicious cancerous lesions and their histological types detected. There were hyperplastic polyps(n = 35), adenomatous polyps(n = 31), fundic gland polyps(n = 42), advanced gastric cancer tissues (n = 32)and normal gastric mucosa tissues (n = 30). ABC immunohistochemical staining and reverse transcription(RT)-PCR were employed to detect the expression of Foxa2 in these different types of tissues. Imagepro plus was used for quantitative and statistical analyses. RESULTS: A low-level expression of Foxa2 was 3.6% ± 1.3% in normal gastric mucosa group. And its expreesion gradually higher in proliferative inflammatory polyp group(33.1% ± 8.0%), adenomatous polyp group (71.4% ± 1.7%) and gastric cancer group(96.3% ± 0.9%, all P < 0.05). Its expression was 35.6% ± 5.6% in fundic gland polyps, similar to that of proliferative inflammatory polyp group (P > 0.05), it was markedly lower than the gastric cancer group (P < 0.05) and higher than the normal gastric mucosa group (P < 0.05). Correlation analyses of clinicopathological parameters showed that no significant correlation existed between its expression and patient gender, age, predilection, Helicobacter. pylori infection or proton pump inhibitor used (all P > 0.05). However, the size of polyps was correlated with Foxa2 (rs = 0.69, P < 0.05). CONCLUSION: The expression level of Foxa2 in different types of gastric polyps may be used as a clinical predicator of polyps risk.

Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal-regulated kinase/matrix metalloproteinase-9 axis.

UNLABELLED: The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.

[Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro].

OBJECTIVE: To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. METHODS: hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. RESULTS: The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. CONCLUSION: hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.

[Experimental study of transplanting human mesenchymal stem cells via portal vein to treat acute liver injury of mice induced with acetaminophen].

OBJECTIVE: To evaluate the effects of human mesenchymal stem cells (hMSCs) transplantation via portal vein to treat acute liver injury in mice induced with acetaminophen. METHODS: A model of acute liver injury was established by acetaminophen gavage with a dose of 500 mg/kg. Twenty severe combined immune deficient mice (SCID mice) were randomly divided into 2 groups; one with hMSCs transplantation via their portal veins, the other group served as controls and only saline was infused into their veins. Liver function tests, fluorescein staining and reticular fiber staining of liver histological preparations and fluorescence- and light-microscopy were applied to observe the biochemical and pathological changes in the mice before and after the transplantation of hMSCs. RESULTS: Liver function of the hMSCs group was significantly better than that of the controls (P less than 0.05). Fluorescence microscopy revealed that the hMSCs appeared in the areas of the periportal veins at first and then extended to the central vein areas; the reticular fiber staining indicated that hMSCs could repair the architecture of the hepatic acini. No prominent fibrosis and pseudolobules were found. CONCLUSIONS: hMSCs transplantation via portal vein to SCID mice with acute liver injury induced by acetaminophen can improve their liver function effectively; hMSCs growth in their livers and acinus reconstruction can be affected. We think it is a good method to treat acute liver injury.

The androgen receptor directly targets the cellular Fas/FasL-associated death domain protein-like inhibitory protein gene to promote the androgen-independent growth of prostate cancer cells.

Androgens provide survival signals to prostate epithelial cells, and androgen ablation induces apoptosis in the prostate gland. However, the molecular mechanisms of actions of the androgen-signaling pathway in these processes are not fully understood. Here, we report that androgens induced expression of the cellular Fas/FasL-associated death domain protein-like inhibitory protein (c-FLIP) gene, which is a potent inhibitor of Fas/FasL-mediated apoptosis. The androgen receptor was recruited to the promoter of the c-FLIP gene in the presence of androgens. We found that c-FLIP promoter contained multiple functional androgen response elements. In addition, we show that c-FLIP overexpression accelerated progression to androgen independence by inhibiting apoptosis in LNCaP prostate tumors implanted in nude mice. Our results suggest that the androgen receptor affects survival and apoptosis of prostate cells through regulation of the c-FLIP gene in response to androgens.

[Sustainment of hepatocyte function with mixed cellular co-encapsulation].

OBJECTIVE: To evaluate the effects of mixed microcapsules of hepatocytes mixed with hepatocytes, transgenic hepatic stellate cell strain (HGF/CFSC), and/or bone marrow derived Thy-1(+) beta(2)M(-) cells (BDTCs) to sustain liver function. METHODS: Three kinds of microcapsules containing hepatocytes, hepatocytes + CFSC/HGFs, or hepatocyte + CFSC/HGF + BDTC were prepared and cultured in conditioned culture fluids. The morphology of the microcapsules and the encapsulated cells were observed by microscopy. The supernatant was collected regularly to detect the secretion of albumin and urea. Forty Wistar rats underwent 90% hepatectomization to establish acute liver failure model. Six hours after the operation the rats were randomly divided into 4 groups to be intraperitoneally injected with one of the 3 kinds of microcapsules containing 3.5 x 10(7) hepatocytes as experimental groups (Groups II, III, and IV) or injected with blank microcapsule as control group. The behaviors of the rats were observed daily. Blood was collected from the eyeball at different time points to detect relevant biochemical indicators. Twenty-one and 42 days after the operation the rats were killed. Abdominal lavage was performed to collect the microcapsules to undergo microscopy. Liver specimens were colleted to undergo pathological examination. RESULTS: Severe liver failure occurred in the rats transplanted with blank microcapsules. Most of the rats in Groups II, III, and IV began to eat within 20 hours after hepatectomization. Nine of the 10 rats in the control group died within 48 hours after hepatectomization. Nine of the 10 rats of Group II survived a long time. All the rats in Groups III and IV survived till the end of experiment. In comparison with the supernatant of Group II, the contents of albumin and urea in the supernatants of Groups III and IV were significantly higher (all P < 0.01). The liver function indicators, ALT, AST, lactic dehydrogenase, and albumin worsened since one day after the operation. Five days after the transplantation of microcapsule, the above indicators showed remarkable improvement, and recovered to normal 7 days after. Twenty-one and 42 days after the transplantation regeneration was seen and edema was reduced in the livers in Groups II, III, and IV. Twenty-one days after the transplantation most of the microcapsules were still free in the peritoneal cavity in Group II. In Group III, most of the microcapsules aggregated around the portal vein, fibrosis at the surface of microcapsule to a certain degree was seen and surviving hepatocytes could be found inside the capsules 21 days after. Forty-two days after, vascularization of microcapsules was seen in Groups III and IV, especially in the latter group. CONCLUSION: Mature hepatocyte, transgenic liver nonparenchymal cells and/or BMSCs co-encapsulate transplantation effectively improve acute liver failure. The microenvironment created by CFSC/HGF and/or BDTC is propitious to the maintenance of capsulated hepatocytes' function and longevity.

[Autologus bone marrow-derived mesenchymal stem cells intracoronary delivery after acute myocardial infarction in miniature pig].

OBJECTIVE: To evaluate the possibility that using intracoronary delivery of autologus bone marrow-derived mesenchymal stem cells (MSCs) to improve the cardiac function after acute myocardial infarction (AMI) in miniature pig. METHODS: MSCs were cultured in Dulbecco's modified Eagle's medium-F12 (DMEM/F12) medium. AMI model was made by blocking the blood stream of the first diagonal branch in miniature pig, and released the branch after 90 minutes. After 10-14 days, (4-6) x 10(7) culture-expanded autologus 4', 6-diamidino-2-phenylindole (DAPI)-labelled MSCs were transplanted into each host heart's AMI area through intracoronary way. Ultrasonic cardiography (UCG) was performed to observe the left ventricular function at 3 months after transplantation. The cellular transplanted hearts were harvested and investigated by immunohistochemical analysis. RESULTS: Left ventricular function of the MSCs group was improved significantly 3 months later compared with the control group [(54.65 +/- 3.39) vs (43.98 +/- 4.21)%, (P < 0.01)]. Exogenous MSCs survived and site-differentiated into cardiomyocytes in infracted hearts. CONCLUSION: MSCs can play a benificial role to repair damaged heart. Heart function can be improved after MSCs transplantation in porcine myocardial infarction model.

[In vitro differentiation of human bone marrow-derived mesenchymal stem cells into blood vessel endothelial cells].

OBJECTIVE: To study differentiation of human bone marrow-derived mesenchymal stem cells (BDMSC) into blood vessel endothelial cells for ideal cell origin of complex organ tissue engineering vascularization and injured tissue repairing by cell transplantation. METHOD: After different days of induction with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 3D fibrin-gels and matrigel, BDMSC and angiogenesis were determined by the utilization of morphological observation, tissue section and CD34, CD31, vascular endothelial growth factor receptor-1 (VEGFR-1, Flt-1), VEGFR-2 (Flk-1), and vWF that were special for blood vessel endothelial cells. RESULT: After 3D-cultured and induced with VEGF and bFGF in vitro in fibrin-gels and matrigel for 3-21 days, BDMSC expressed CD34, CD31, Flt-1, Flk-1, and vWF came into vessel-like configuration. CONCLUSION: VEGF, bFGF as well as Flt-1 and Flk-1, expressed by BDMSC, may form a feasible microenviroment after induction and play an important role during processes of blood vessel endothelial cell differentiation and vessel-like configuration forming of BDMSC. Mesenchymal stem cells may be applied to tissue engineering vascularization and injured tissue repairing by cell transplantation.

[Sustaining effect of gene-transferring hepatic stellate cell strain CFSC/HGF on hepatocytes development].

OBJECTIVE: To investigate the sustaining effects of gene-transferring hepatic stellate cell strain CFSC/HGF on the development of hepatocytes. METHODS: A CFSC/HGF strain, expressing HGF steadily and effectively was established by recombined retroviral vector pMSCV-HGF infection. Morphology and ultra structure of hepatocytes, albumin and urea production, as well as ICG uptake and excretion were studied continuously following the hepatocytes cultured on the CFSC/HGF feeder layers. Parallel group of collagen-dependent hepatocytes culturing and hepatocytes culturing on CFSC were also conducted. Semi-quantitative RT-PCR analysis was made to evaluate the expression of HGF receptor c-Met. RESULTS: The hepatocytes cocultured on CFSC/HGF feeder layers had a higher survival rate, and the functions of albumin secretion and urea syntheses and ICG uptake and excretion, were superior to the other two culture methods. The result of RT-PCR indicated that the c-Met expressed on the CFSC/HGF coculturing hepatocytes was up-regulated 2.23 times. CONCLUSION: Gene-transferring hepatic stellate cell strain CFSC/HGF exhibited a remarkable sustaining effect on the hepatocytes development. The up-regulation of c-Met expressed on the surfaces of the hepatocytes induced by CFSC/HGF might play some part in this function.

[Differentiation of bone marrow derived Thy-1+ beta2M- cells into liver cells in AA induced liver injury micro-environment].

OBJECTIVE: To investigate the differentiation of bone marrow derived Thy-1+ beta2M- cells (BDTCs) into liver cells in allyl alcohol (AA) induced liver injury micro-environment. METHODS: BDTCs of male F344 rats were isolated by two-step magnetic separation system (MACS) technique, and infused intraportally into female recipients after labeling with PKH26. Thirty recipients were divided randomly into 3 groups: (1) AA-injured liver + BDTCs infusion, (2) normal liver + BDTCs infusion and (3) AA-injured liver + NS infusion (control). Blood biochemical examination, fluorescence labeled cellular localization, Y-chromosome sry gene in-situ hybridization and immunohistochemistry were carried out to evaluate BDTCs distribution, differentiation and proliferation in recipients's livers after different intervals. RESULTS: Fluoromicroscopy and in situ hybridization suggested that BDTCs of donors were interspersed in pieces and cords among the necro-periportals induced by AA; immunohistochemistry indicated that those implanted cells expressed OV-6, AFP, CK19 and albumin successively, while positive cells were hardly seen in the normal liver + BDTCs infusion group. Compared with the controls, the blood biochemical restitution was more rapid in group (1), (9.8 d +/- 3.1 d vs. 13.7 d +/- 4.2 d). CONCLUSION: The injury micro-environment induced by AA facilitates BDTCs integration with hepatic cell plates and differentiation into mature liver cells. BDTCs differentiation into liver cells might accelerate endogenous liver cell regeneration and reparation.

Change of hs-CRP, sVCAM-1, NT-proBNP levels in patients with pregnancy-induced hypertension after therapy with magnesium sulfate and nifedipine.

OBJECTIVE: To investigate the change of the hs-CRP, sVCAM-1, NT-proBNP levels of the patients with pregnancy-induced hypertension (PIH) syndrome. METHODS: A total of 200 patients with PIH were divided into mild, moderate and severe group, and 50 healthy pregnancy patients served as the control group. The serum sVCAM-1 levels were detected by enzyme-linked immunosorbent assay, hs-CRP were detected by immunity transmission turbidity, and NT-proBNP levels were determined by the colloidal gold method. Patients were treated with magnesium sulfate and nifedipine and the contrastive analysis was performed before and after treatment. And the pathological changes in placental of PIH patients were detected by hematoxylin-eosin staining at the same time. RESULTS: The hs-CRP, sVCAM-1, NT-proBNP levels of patients in the mild, moderate and severe PIH group were significantly higher than that in the control group (P<0.05). The hs-CRP, sVCAM-1, NT-proBNP levels in the severe group were significantly higher than the mild group and the moderate group, the difference was statistically significant (P<0.05). The hs-CRP, sVCAM-1, NT-proBNP of the moderate group were significantly higher than the mild group (P<0.05). There was a positive correlation between hs-CRP, sVCAM-1, NT-proBNP expression levels and the degree of the PIH. The expression of hs-CRP, sVCAM-1, NT-proBNP levels of the moderate and the severe group were significantly decreased (P<0.05). The number of placental villi and interstitial blood vessel in the moderate and severe PIH group were significantly less than the control group (P<0.05). CONCLUSIONS: The increased levels of serum hs-CRP, sVCAM-1, NT-proBNP may be involved in the process of vascular endothelial cell injury of the PIH, and the hs-CRP, sVCAM-1, NT-proBNP can be used as the auxiliary index for diagnosis of PIH and determination of PIH severity.

Association of ghrelin Leu72Met polymorphism with type 2 diabetes mellitus in Chinese population.

BACKGROUND: Ghrelin, a novel endogenous ligand for the growth hormone secretagogue receptor, is considered to implicate the development of the type 2 diabetes mellitus (T2DM). The Leu72Met (+408C>A) polymorphism of the preproghrelin, has been linked to obesity, insulin resistance and diabetes. OBJECTIVE: To investigate the distribution of ghrelin gene Leu72Met polymorphism and its association with the type 2 diabetes mellitus in Chinese population. METHODS: We conducted a case-control study on 877 patients with T2DM and 864 controls, which were genotyped by the polymerase chain reaction (PCR) technique, denaturing high performance liquid chromatography (DHPLC) and DNA sequence analysis. Laboratory analyses were carried out in the hospital laboratory. RESULTS: No significant difference in the Leu72Met genotype distributions and allele frequency was observed between type 2 diabetes mellitus and controls (both P>0.05). The polymorphism was not associated with T2DM. However, among the T2DM group, the patients carrying Leu72Leu genotype had significantly increased levels of FPG and serum creatinine compared with variant genotypes (Leu72Met and Met72Met) (P<0.05). In the control group, the subjects with variant genotypes had significantly increased levels of FINS, HOMA-IR compared with Leu72Leu genotype (P<0.05). CONCLUSION: The Leu72Met polymorphism of the preproghrelin gene was not associated with T2DM in Chinese population. However, it may have some roles in the etiology of insulin resistance.

Validation of the association between PSMA6 -8 C/G polymorphism and type 2 diabetes mellitus in Chinese Dongxiang and Han populations.

OBJECTIVE: The aim of our study was to validate association between -8 C/G variant of PSMA6 gene and T2DM in Chinese Dongxiang and Han populations. METHOD: We genotyped PSMA6 gene -8 C/G polymorphism in the control groups and T2DM groups in two populations from China using PCR-RFLP technique. Phenotypes and biochemical indicators were measured by biochemical technique. RESULT: The frequencies of CG+GG genotype were observably different from CC genotype in the T2DM groups and control groups (for Dongxiang population: OR = 1.341, 95% CI: 1.101-1.632, P = 0.004; for Han population: OR = 1.313, 95% CI: 1.085-1.569, P = 0.006 after adjusting for gender, age, and BMI, respectively). In the Dongxiang population, the FPG, HOMA-IR, SBP and TG levels of CG+GG genotype were markedly higher than those of the CC genotype in control group (all P < 0.05). However, in the Han population, we only found that the FPI level of the CC genotype was significantly higher than that of the CG+GG genotype in control group (P < 0.05). CONCLUSION: Our investigation suggests that -8 C/G variant of PSMA6 gene may be associated with T2DM and diabetes-related metabolic traits in Chinese Dongxiang and Han populations.

Association of P213S polymorphism of the L-selectin gene with type 2 diabetes and insulin resistance in Chinese population.

AIMS: L-selectin belongs to selectin family of adhesion molecule and participates in the generation and development of type 2 diabetes (T2D). In this study, we evaluated the relationship between the P213S polymorphism of L-selectin gene and T2D and insulin resistance in the Chinese population. METHODS: We genotyped P213S polymorphism in 801 patients with T2D and 834 healthy controls in the Chinese population using polymerase chain reaction-ligase detection reaction (PCR-LDR) technique. Plasma glucose, insulin, lipid, blood urea nitrogen, creatinine and uric acid levels were measured by biochemical technique. RESULTS: The frequency of 213PP genotype and P allele of the L-selectin gene in patients with T2D was significantly higher than that in controls (P=0.007; P=0.019, respectively). The relative risk of allele P suffered from T2D was 1.191 times higher than that of allele S. Moreover, the levels of FPG and HOMA-IR of PP and PS genotype carriers were significantly higher than those of SS genotype carriers in the T2D group (P<0.05). CONCLUSION: These findings indicated that the P213S polymorphism of L-selectin gene may contribute to susceptibility to T2D and insulin resistance in the Chinese population, and P allele appears to be a risk factor for T2D.

Cells extract from fetal liver promotes the hematopoietic differentiation of human embryonic stem cells.

Here, we have now developed a new inducing system to promote the differentiation of human stem cells (hESCs) toward hematopoietic lineages by the treatment with cells extract of human fetal liver tissue (hFLT). The embryoid bodies (EBs) obtained from human H1 embryonic stem cells were exposed to buffer, hFLT cells extract, heated hFLT cell extract, and cell extract of human liver cells lines-LO2. Then, the feature of EBs in different groups was characterized by real-time RT-PCR and colony-forming assays. The results showed the treatment by hFLT cells extract could activate the hematopoietic genes expression and improve the capacity for hematopoietic progenitor development of hEBs. After that, we cocultured hFLT extract treated hEBs on the hFLSCs (human fetal liver stromal cells) feeder to differentiate them into hematopoietic cells. As a control, untreated hEBs were cocultured on hFLSCs feeder with cytokines. The feature of induced cells from hEBs was characterized by flow cytometry, Wright-Giemsa staining, and colony-forming assays. The results demonstrated that hFLT cells extract was capable of inducing hEBs into hematopoietic cells and combing it with hFLSCs feeder could largely promote hematopoietic differentiation of hESCs. This method may supply a new way to substitute the cytokines required in hematopoietic induction of hESCs.

[Differentiation of rat bone marrow derived Thy-1+ beta2M- cells into hepatocytes].

OBJECTIVE: To investigate the differentiation of bone marrow derived Thy-1(+)beta(2)M(-) cells (BDTC) into mature and functional liver cells and its mechanism. METHODS: BDTC were cocultured with allyl alcohol (AA)-injured hepatocytes and cultured alone in conditional medium containing HGF and bFGF, respectively. BDTC morphologic transformation was observed with phase-contrast and electron-microscopy. Hepatocyte-specific gene expression in cultured BDTC was identified by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Indocyanine green (ICG) ingestion/excretion and urea, albumin production were carried out to evaluate hepatocyte-related function. RESULTS: Some BDTC derived hepatocyte-like cells with high nuclear to cytoplasmic ratio containing mono- or multi-nuclei and abundant mitochondria, endoplasmic reticulum and glycogenic granules appeared after 7-day culture in both the two culture systems. These cells expressed hepatocyte-specific genes (AFP, OV-6, CK18, etc.), and possessed functions of ICG uptake, albumin production and ammonium metabolism. CONCLUSION: Rat BDTCs could differentiate into mature and functional liver cells in special stimulation systems. Moreover, these differentiations were realized by "transdifferentiation", and might dispense with "cell fusion".

[Inhibiting effect of arsenic trioxide on telomerase activity of NB4 and Jurkat cell lines].

To investigate the inhibiting effect of arsenic trioxide (As(2)O(3)) on the telomerase activity of leukemia cell lines NB4 and Jurkat cells, MTT assay, electrophoresis of genomic DNA, protein/DNA dual parameter flow cytometry as well as a semi-quantitative telomeric repeat amplification protocol (TRAP) assay and RT-PCR were used to examine the effect of As(2)O(3) on cell proliferation, telomerase activity and expression of cell cycle regulatory proteins. The results showed that cell proliferation and telomerase activity were significantly inhibited and apoptosis was induced in these cells after exposure to As(2)O(3). Furthermore, the expression of some cell cycle and apoptosis related proteins, such as Bcl-2, Rb, P16, caspase-3, cyclin A and cyclin E, was altered in As(2)O(3) treated NB4 cells. Cell cycle was arrested at G(1) and G(2)/M phases in both cells. It is concluded that the change of cell cycle regulatory proteins plays an important role in decline of the telomerase activity during the proliferation inhibition and apoptosis of NB4 and Jurkat cells induced by As(2)O(3).