Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) is thought to be a precursor lesion of infiltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In the present study, we investigated the role of Smad4 in the development of pancreatic tumor, based on PanIN cell line from mice with K-ras G12D mutation in order to investigate the effect of Smad4 silencing on PanIN cells in the development and malignant transformation in nude mice. Methods: Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfeetion with lentiviral-mediated Smad4 RNA interference (RNAi). In xenograft model experiments, BALB/c nude mice were randomly divided into 2 groups (5 mice per group) implanted with PanIN or PanlN-S cells subcutaneously. Two weeks after tumor cells inoculation, tumor volume and weight were estimated. PCNA staining was used to evaluate cell proliferation and CD31 polyclonal antibody was used to assess micro-vessel density (MVD) in tumors. Results: Effect of siRNA of Smad4 gene in PanlN cells was confirmed by RT-PCR and Western blot. Compared with PanlN groups, there was a dramatic increase in tumor volume and weight in PanIN-S groups (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with 'increased tumor cell proliferation (PCNA reactivity) and angiogenesis (micro-vessel density, MVD). The percentage of PCNA-positive cells in the PanlN-S groups were significantly increased than PanIN groups (P<0.05). CD31 staining revealed a significant increase in the PanlN-S groups compared to the PanlN groups (P<0.05). Conclusion: Silencing of Smad4 in PanlN cells with endogenous expression of K-ras G12D, enhanced progression to invasive adenocarcinomas. Cell proliferation and vascularization may be its important mechanisms.

Objective To investigate the value of dynamic three-dimensional contrast-enhanced ultrasound (3D-CEUS) in evaluating therapeutic response of hepatoma treated with radiofrequency ablation (RFA).Methods Totally 48 cases of patients with hepatic carcinoma (48 lesions) admitted in Xiangya Hospital of Central South University from September 2012 to January 2014 were selected.All patients underwent radiofrequency ablation,of which 30 patients were diagnosed by pathology after surgery,18 patients by clinical diagnosis.All patients underwent two-dimensional contrast-enhanced ultrasound (2D-CEUS) and 3D-CEUS 1 month and 3 months after RFA treatment to evaluate the therapeutic response,and the results of contrast-enhanced ultrasound and enhanced computed tomography (CT) [or magnetic resonance imaging (MRI)] were compared.The final diagnostic results of pathologic biopsy or more than two imaging examinations [ultrasonography,CT,MRI,positron emission tomography (PET)],tumor markers,and more than 3 months follow-up of patients were used as the gold standard.The sensitivity,specificity and accuracy of dynamic 3D-CEUS,2D-CEUS,enhanced CT (or MRI) in the diagnosis of tumor inactivation were calculated respectively.Results After radiofrequency ablation,dynamic 3D-CEUS could provide more valuable information in 75.0％ (36/48) lesions,which contribute to assess the efficacy of radiofrequency ablation.While compared with 2D-CEUS,3D-CEUS did not change the diagnosis or clinical management in 12 (25.0％) lesions.40 of 48 lesions were found no-enhancement in entire CEUS procedure suggesting that the tumor completely inactivated,while 8 lesions showed local enhancement on the edge of lesion suggesting that part of the tumors were active.39 of 48 lesions showed no-enhancement and other 9 with irregular enhancement on enhanced CT (or MRI).The sensitivity,specificity and accuracy of CEUS and enhanced CT (or MRI) in detection of residual tumor after radiofrequency ablation were 80.0％,100％,95.8％ and 80.0％,97.4％,93.8％,respectively.Conclusions There was no statistical significance among 3D-CEUS,2D-CEUS and enhanced CT or MRI in evaluating therapeutic response of hepatoma treated with radiofrequency ablation.But 3D-CEUS can provide more valuable information,3D-CEUS has potential usefulness in the evaluation of percutaneous radiofrequency ablation of hepatic tumors.

OBJECTIVETo investigate the effect of PD0332991 (C24H29N7O2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice.

METHODSThe self renewal ability of HSCs was measured by cobblestone forming cell assay (CAFC). The colony-forming cell (CFC) assay was used to quantify the changes of numbers and functions of HPC after the treatment of the compound. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were analyzed by flow cytometry.

RESULTSThere were obvious changes in cell cycle regulation between control and PD0332991 groups. HSCs in G1 phase increased significantly from 76.3% to 89.5% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 20.5% to 7.3% (P<0.05). HPCs in G1 phase also increased from 74% to 87.4% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 25.54% to 11.6% (P<0.05). The apoptotic fractions between control and PD0332991 groups had no statistical difference (P>0.05). After cultured with PD0332991, the expression levels of cell cycle genes CDK1, CyclinA2, CyclinF, p18, p19 and p27 decreased by 58.77%, 66.35%, 56.33%, 62.18%, 32.28% and 36.53% respectively, while expression of CDK7 increased by 27.27% (P<0.05). No visible expression difference was observed in apoptosis and self-renew related genes. After treatment of PD0332991, the self-renewal ability of HSC decreased significantly. There were almost no CFCs in PD0332991 group in CAFC assay. Similarly, the frequency of CFCs was dramatically lower in PD0332991 group.

CONCLUSIONThese results suggested that PD0332991 affected HSC/HPC from mice mainly through inhibiting the cell cycle rather than apoptosis. It also suggested that CDK4/6 might play a key role in the regulation of HSC/HPC.

Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Piperazines ; pharmacology ; Pyridines ; pharmacology