Toll-like receptor 3 (TLR3) is a member of the Toll-like receptors which recognize pathogen-associated molecular patterns leading to the activation of the innate immune response. Recent reports have strongly indicated that they play important roles in cancer cells. Since TLR3 has been recently suggested as a possible therapeutic target in certain types of cancers, in the present study, TLR3 expression and its function were explored in hepatocellular carcinoma (HCC) and human umbilical vein endothelial cells (HUVECs). The expression of TLR3 in various HCC cell lines and HUVECs was detected using quantitative real-time PCR (qRT-PCR) and immunocytochemistry. TLR3 activity was determined by Luciferase reporter assays. The effects of TLR3 double-stranded RNA (dsRNA) agonists on angiogenesis were tested by aortic ring assay and HUVEC tube formation experiments. After dsRNA treatment, cell apoptosis was assessed by Annexin V and PI staining through FACS, and the migration ability was measured by a migration assay. The results showed that TLR3 was expressed in HCC cell lines and HUVECs at the mRNA and protein level. Luciferase reporter assays demonstrated that TLR3 was activated by the dsRNA analog BM-06 or poly(I:C). Rat aortic ring outgrowth and endothelial cell tube formation were suppressed after treatment with dsRNA. In addition, dsRNA triggered apoptosis in MHCC97H, SMMC-7721 and HUVEC cell lines and inhibited cell migration. In conclusion, TLR3 agonists not only affect tumor microenvironment by suppressing angiogenesis but also directly induce tumor cell apoptosis and inhibit tumor cell migration. TLR3 may be a new target for HCC therapy.

Objective To investigate the modification of purifying and culturing methods and conservation for human endometrial cells(EECs) and endometrial stromal cells(ESCs) from different menstrual cycles of normal endometrial tissues. Methods The normal endometrium tissues(8 samples in proliferative phase and 8 samples in secretory phase) were doublely digested by 0.1% collagenase I.EECs and ESCs were isolated with 100-and 400-mesh cell sieves and purified with selective wall-pasting according to the different menstrual cycles.EECs were co-cultured with ESCs by 0.4μm pore size culture plate. The purity of ESCs and EECs was determined by morphology and immunefluorescencel staining with specific markers.One-passage ESCs were frozen for 3 months and thawed thenafter. Results The EECs and ESCs of 15 samples from 16 cases were cultured successfully and the purity of EECs and ESCs was more than 95%.The co-culture pproloned spontaneous apoptosis of EECs.ESCs could be passaged stably in vitro and freeze-stored did not affect its vitality and purity. Conclusion Doublely digesting,screening filtration and different selective wall-pasting time technique can highly purity and isolate EECs and ESCs and ESCs can be freeze-stored and thawed.

OBJECTIVE:To investigate the method of establishing damaged endometrial stromal cells (ESC) model in vitro.METHODS:(1) From June to December 2011 ESC from normal endometrim at proliferation phase (n = 8) and secretory phase (n = 8) were isolated, cultured and identified in vitro. (2) ESC was treated with different concentrations of mifepristone or withdrawal of mifepristone at different time point. The proliferation inhibition percent was measured by cell counting kit-8 (CCK-8). (3) 0 µmol/L(control group)and 60 µmol/L (experimental group) concentration of mifepristone was added into ESC for 48 hours, then withdrew of mifepristone, continued to be cultured for 48 hours. The morphological changes were observed and apoptosis of ESC in different menstrual cycle were detected by flow cytometry. The mRNA and protein level of vascular endothelial growth factor (VEGF), caspase-3, 8, and 9 were determined by one-step quantitative real-time PCR (Q-PCR) and western blot.RESULTS:(1) ESC from 16 specimens of endometrium were all isolated and cultured successfully. (2) The proliferation inhibition rate of ESC was correlated with concentration and duration of mifepristone positively. The proliferation of ESC could be recovered at a range of time after withdrawal of mifepristone. However, when the concentration of mifepristone was 100 µmol/L, the growth of ESC recovered very hardly. (3) The damaged ESC spacing increased, the spindle shape and vacuolization in the cytoplasm were observed in experimental group; the rate of apoptosis of these damaged cells was significantly increased compared with control groups, which were (52 ± 12)% vs. (13 ± 5)% at the proliferative phase and (53 ± 6)% vs. (32 ± 3)% at the secretory phase (all P < 0.05). The relative mRNA level of VEGF was 0.52 ± 0.12 in experimental group and 1.00 ± 0.17 in control group at proliferation phase (P < 0.05). And the relative mRNA level of VEGF was 0.19 ± 0.03 in experimental group and 0.81 ± 0.07 in control group at secretory phase (P < 0.05). The relative level of VEGF protein in the experimental group were both decreased 1.98 and 2.79 folds at the proliferation phase and the secretory phase when compared with those in control group, respectively (P < 0.05). While the relative levels of caspase-3, 8, 9 mRNA were 5.62 ± 0.65, 5.41 ± 0.53, 7.22 ± 0.51 in the experimental group and 1.00 ± 0.44, 1.00 ± 0.21, 1.00 ± 0.32 in control group at the proliferative phase. In the mean time, the relative levels of caspase-3, 8, 9 mRNA were 10.22 ± 0.72, 25.3 ± 1.72, 9.48 ± 1.89 in experimental group and 1.42 ± 0.14, 1.14 ± 0.28, 1.16 ± 0.12 in control group at the secretory phase, respectively (P < 0.05). Compared with the control group, the levels of caspase protein in the experimental group were increased 2.04 and 1.60 folds in caspase-3, 4.23 and 1.49 folds in caspase-8, 2.65 and 3.5 folds in caspase-9 at the proliferative phase and at the secretory phase, respectively (P < 0.05).CONCLUSION:The damaged model of ESC can be established after 48 hours by the withdrawal of 60 µmol/L mifepristone in treatment of ESC for 48 hours.

Objective To prepare a hybridoma secreting stable monoclonal antibody against rat activation transcription factor 3(ATF3).Methods Peptides(168-181 amino acids of rat ATF3 protein) were synthesized according to predicted antigenic peptides sequence and coupled to keyhole limpet hemocyanin(peptide-KLH).Then mAb against peptide-KLH was prepared by hybridoma technique.A commercial mouse mAb isotyping kit was used to identify the isotype of this mAb.Meanwhile,ELISA,Western blot and IHC were used to determine whether the mAb could bind rat ATF3 protein.Results One hybridoma secreting stable mAb against rat ATF3 was produced.Immunoglobulin subclass determination identified this mAb to be the IgG1/κ subtype.Western blot result revealed that the mAb could specifically recognize rat ATF3 protein in glomerular mesangial cells(GMC).Immunohistochemical staining showed that the mAb could react with ATF3 expressed both in the cytoplam and the nuclei of GMC in rats with Thy-1 nephritis while the expression of ATF3 in the nuclei was more significant.Conclusion A mouse mAb against rat ATF3 protein was prepared successfully,which provides a useful tool for studying ATF3 biological function in the future.

Objective To investigate the expression of nucleostemin gene(NS)respectively in tissues and in cell lines of liver cancers.Explore the significance for the development of liver cancer.Methods According to the sequence of NS gene in GenBank(Accession:AY825265),the primers of a segment and the complete CDS were designed.The segment and the complete sequence were cloned from the cDNA of liver cancer tissue.The PCR products were ligated into the pMD-18 plasmid by T4 DNA Ligase system and sequenced.The expressions were detected in 40 specimens of liver cancer and matched paracancer tissues by PCR.The expressions of NS in 3 kinds of liver cancer cell lines were also detected.Results The authors got the cDNA clones of a short segment and a long segment,which were respectively proved to be a 450 bp segment and the NS gene.Electrophoresis analysis showed that the expression of NS in liver cancer tissues was higher than that in matched paracancer tissues.In the test of liver cancer cell lines,the NS expression in HepG2(with the lowest ability of transfer)was the lowest among the 3 liver cancer cell lines,while the expression in MHCC(with the highest ability of transfer)was the highest.Conclusion Nucleostemin should actively participate in the proliferation of liver cancer cells.