The hUC-MSCs cell line CCRC-1 represents a novel, safe and high-yielding HDCs for the production of human viral vaccines.

MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.

[Experimental study of repairing femoral bone defects with nHA/RHLC/PLA scaffold composite with endothelial cells and osteoblasts in canines].

OBJECTIVE: To explore whether a tissue-engineered construct composed of autogenous endothelial cells, osteoblasts and a new bioresorbable nano-hydroxyapatite/recombinant human-like collagen/polylactic acid (nHA/RHLC/PLA) would enhance bone regeneration and repair femoral head defects in canine models. METHODS: The bone marrow stem cells (BMSCs) were isolated from bone marrow of canine ilium and cultured in Dulbecco's modified eagle medium:nutrient mixture F-12 culture media for 1 week and the second-generation BMSCs were further induced by osteogenic medium (1×10(-8) mol/L dexamethasone, 10 mmol/L B-sodium glycerophosphate and 50 µg/ml vitamin C) and by endothelial cell grow medium (vascular endothelial growth factor and basic fibroblast growth factor) for 14 days in vitro. Thus BMSCs were induced into ECs and OBs. After the second passage, cells were digested and collected.And cell density was adjusted to 1.0×10(6)/ml.The cells and nHA/RHLC/PLA scaffold were co-cultured for 2-4 hours then nHA/RHLC/PLA scaffold composites prepared. Cavity defects of 8 mm in diameter and 10 mm in height were made in femoral heads.The nHA/RHLC/PLA scaffold composited with ECs and osteoblasts (OBs) (group A) and composited with OBs (group B) were inserted into different defects while cell-free nHA/RHLC/PLA scaffold served as controls (group C). New bone formation and defect repair were evaluated at 3 and 6 months by radiographic examination, histology and bone histomorphometry. RESULTS: New bone formation was evident as early as 3 months in groups A, B and C.At 6 months, abundant bone tissue within defects was observed in group A. The control animals with cell-free scaffold showed less bone formation at both timepoints.The scaffold of nHA/RHLC/PLA was degraded and absorbed gradually with the formation of new bone tissues.Histology and bone histomorphometry further revealed significantly increased trabecular bones in group A compared with groups B and C at 6 months postimplantation (P < 0.01). CONCLUSION: More abundant new bone tissue may be found in the bone defect areas implanted with osteoblast-endotheliocyte composite than osteoblasts composite and scaffold materials only.ECs and osteoblasts derived from BMSC are ideal seed cells for repairing femoral head defects.

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer.

The aim of this study was to determine the expression of miR-21, miR-31, miR-96 and miR-135b in 52 paired colorectal cancer (CRC) tissues and to analyze the correlation between microRNAs (miRNAs) and clinicopathological features. We developed a quantification method that relies on a standard plot, constructed from known concentrations of standards, in order to measure the number of miRNAs. In addition to this, we analyzed the expression levels of miR-21, miR-31, miR-96 and miR-135b in 52 cases of primary CRC and corresponding normal mucosal tissue using real-time PCR with SYBR-Green I. An independent sample t-test was used to compare the differential expression between tumor tissues and normal mucosal tissues. The Mann-Whitney U and Kruskall-Wallis tests were used to compare the correlation between miRNA expression levels and clinicopathological features. The expression of miR-21, miR-31, miR-96 and miR-135b was upregulated in the CRC tissues compared to normal mucosal tissues (P<0.05). Furthermore, miR-21 and miR-135b were positively correlated with the clinical stage (P=0.048 and P=0.029, respectively), while miR-96 and miR-135b were correlated with liver metastasis (P=0.006 and P=0.013, respectively). Our results suggest that miR-21, miR-31, miR-96 and miR-135b may function in the process of CRC development and progression. miR-135b levels in particular may correlate with the degree of malignancy.

[Immunosuppression of human amniotic mesenchymal cells on allogeneic peripheral blood lymphocytes].

OBJECTIVE: To explore the immunoregulatory effects of human amniotic mesenchymal cells (hAMCs) on allogeneic peripheral blood lymphocytes. METHODS: The hAMCs were isolated from abandoned human amnion. Peripheral blood mononuclear lymphocytes (PBMLs) were separated from healthy donors by density gradient centrifugation. Then, PBMLs were treated with phytohemagglutinin (PHA) and different concentrations of hAMCs. Proliferation effect of PBMLs was tested using MTS assay, and production of IFN-gamma and TNF-alpha by PBMLs was detected by ELISA. RESULTS: hAMCs could remarkably inhibit the lymphocytes proliferation. When the ratios of hAMCs to PBMLs were 0.05: 1, 0.10 :1, 0.20: 1, the inhibitory rates of PBMLs proliferation were 16.91%, 20.83% and 28.19%, respectively. HAMCs also decreased the production of IFN-gamma and TNF-alpha by PBMLs in a dose-dependent manner (P<0.01). CONCLUSIONS: HAMCs could inhibit the proliferation of allogeneic lymphocytes and reduce secretion of IFN-gamma and TNF-alpha, which might be one of the mechanism for prevention and remission of transplant rejection.

Synthesis of N-substituted acridinediones and polyhydroquinoline derivatives in refluxing water.

Acridinediones were synthesized by the one-pot Hantzsch condensation of an aromatic aldehyde, 5,5-dimethyl-1,3-cyclohexanedione, and aniline/4-methylaniline in refluxing water. This method has then been extended to the four-component reaction of an aromatic aldehyde, 5,5-dimethyl-1,3-cyclohexanedione, ethyl acetoacetate and ammonium acetate for the synthesis of polyhydroquinoline derivatives. This is an environmentally friendly and efficient procedure providing  good to excellent yields.

DNA alterations of microsatellite DNA, p53, APC and K-ras in Chinese colorectal cancer patients.

BACKGROUND: Colorectal cancer is one of the most rapidly increasing cancers in the world, and accumulation of alterations in oncogenes, tumour suppressor genes and mismatch repair (MMR) genes contributes to colorectal tumorigenesis. Thus, we investigated the alterations of 14 microsatellite loci adjacent to MMR genes, p53, adenomatous polyposis coli (APC) and K-ras in 52 Chinese patients with colorectal cancer. MATERIALS AND METHODS: We performed fluorescent polymerase chain reaction and capillary electrophoresis to analyse microsatellite instability (MSI) and loss of heterozygosity (LOH) in microsatellite loci, which included a panel of nine dinucleotide repeats and the Bethesda consensus panel. Additionally, we screened for mutations in exons 4-9 of p53 and the mutation cluster region (MCR) in APC by DHPLC. Codons 12, 13 and 61 in K-ras were analysed using direct sequencing. All variations were confirmed using clone sequencing. RESULTS: The alteration frequency of microsatellite DNA was 55·8% (29/52). Among the microsatellites, five loci exhibited MSI and another nine loci exhibited LOH. The mutation rates of p53, APC and K-ras were 42·3%, 38·5% and 36·5%, respectively. All patients (n = 7) with liver metastasis had a mutation in p53, APC or K-ras. APC mutation was correlated with clinical stage and the presence of lymph node metastasis (P = 0·001 and P = 0·006, respectively). CONCLUSIONS> A total of 80·8% of Chinese patients with colorectal cancer show variations in microsatellite DNA, p53, APC or K-ras. It appears that these microsatellite DNA alterations could be a new biomarker for colorectal cancer.

Expressions of cell cycle associated factors geminin and cdt1 in patients with acute leukemia.

The purpose of this study was to detect the expression levels of geminin and cdt1 in peripheral blood and bone marrow from patients with newly diagnosed acute leukemia (AL), and further explore effects of them in the pathogenesis of AL. mRNA expression of geminin and cdt1 in peripheral blood and bone marrow of newly diagnosed AL patients was detected by SYBR Green real-time quantitative reverse transcription polymerase chain reaction(SYBR-RT-PCR). The results showed that mRNA expressions of both geminin and cdt1 in peripheral blood were positive in 10 out of 13 newly diagnosed ALL patients (76.92%) and in 9 out of 14 newly diagnosed AML patients (64.29%), while no positive expression of these 2 genes was detected in 10 normal controls; mRNA expression levels of geminin and cdt1 in bone marrow of newly diagnosed ALL and AML patients were 108.06 ± 67.34 and 52.37 ± 35.16, 62.66 ± 58.69 and 26.68 ± 22.29, respectively, which were higher than those in normal controls (11.81 ± 2.83 and 7.32 ± 5.77), there were significant differences (p < 0.01 and p < 0.05). mRNA expression of geminin was significantly positive related to mRNA expression of cdt1 in bone marrow of 34 newly diagnosed AL patients (r = 0.55, p < 0.01). It is concluded that mRNA expressions of geminin and cdt1 are enhanced and significantly positively related between them in bone marrow of AL patients. The over-expression of geminin and cdt1 mRNA may play an important role in pathogenesis of AL.

[Experimental study on the induced differentiation of human amnion mesenchymal cells into osteoblasts].

OBJECTIVE: To investigate the feasibility of inducing differentiation of the human amniotic mesenchymal cells (hAMCs) into osteoblasts in vitro, so as to provide the seed cells for bone tissue engineering. METHODS: The hAMCs were isolated from abandoned human amnion and cultured in osteogenic media to induce the osteogenic differentiation in vitro. After hAMCs were induced by osteogenic media for 15 days, morphological observation, immunocytochemistry and western blot were used to study the cellular morphology and expression of alkaline phosphatase (ALP), type I collagen, osteopontin and osteocalcin. RESULTS: The primary cultured hAMCs had long spindle shape or irregular shape, which were distributed evenly. The cells were usually suheultured in 5 or 7 days. After subculture, the cells became larger. After cultured by osteogenic media for 15 days, the hAMCs were detected to express ALP, osteocalcin and osteopontin, and secrete type I collagen. CONCLUSIONS: The hAMCs are isolated, cultured and amplified easily in vitro. The induced differentiated cells by osteogenic media have typical osteoblast morphological and functional characteristics, which can be used as seed cells for bone tissue engineering.

Ferritin heavy chain-mediated iron homeostasis and subsequent increased reactive oxygen species production are essential for epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) plays a critical role in tumor progression. To obtain a broad view of the molecules involved in EMT, we carried out a comparative proteomic analysis of transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes. A total of 36 proteins with significant alterations in abundance were identified. Among these proteins, ferritin heavy chain (FHC), a cellular iron storage protein, was characterized as a novel modulator in TGF-beta1-induced EMT. In response to TGF-beta1, there was a dramatic decrease in the FHC levels, which caused iron release from FHC and, therefore, increased the intracellular labile iron pool (LIP). Abolishing the increase in LIP blocked TGF-beta1-induced EMT. In addition, increased LIP levels promoted the production of reactive oxygen species (ROS), which in turn activated p38 mitogen-activated protein kinase. The elimination of ROS inhibited EMT, whereas H2O2 treatment rescued TGF-beta1-induced EMT in cells in which the LIP increase was abrogated. Overexpression of exogenous FHC attenuated the increases in LIP and ROS production, leading to a suppression of EMT. We also showed that TGF-beta1-mediated down-regulation of FHC occurs via 3' untranslated region-dependent repression of the translation of FHC mRNA. Moreover, we found that FHC down-regulation is an event that occurs between the early and highly invasive advanced stages in esophageal adenocarcinoma and that depletion of LIP or ROS suppresses the migration of tumor cells. Our data show that cellular iron homeostasis regulated by FHC plays a critical role in TGF-beta1-induced EMT.

Comparative proteomic analysis of cell cycle-dependent apoptosis induced by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) can induce G2/M phase-dependent apoptosis and G1/S phase-dependent epithelial-mesenchymal transition (EMT) in hepatocytes, but the underlying mechanism remains poorly understood. In this study, we investigated alterations in the global proteome using two dimensional gel electrophoresis of AML-12 murine hepatocyte cells after treatment with TGF-beta at several time points after synchronization in the G2/M or G1/S phase. Upon TGF-beta treatment, the expression levels of 44 proteins were found to be significantly changed in cells synchronized in the G2/M phase. These proteins were identified by MALDI-TOF/TOF and classified into seven categories according to function. In addition, TGF-beta induced downregulation of glutamine synthetase in cells in G2/M but not G1/S phase, and this was further confirmed by immunoblotting. Moreover, exogenous glutamine completely blocked TGF-beta-induced apoptosis in G2/M and non-synchronized cells, whereas it had no effect on EMT, suggesting that the downregulation of glutamine synthetase is involved in G2/M phase-dependent apoptosis. These results provide new insight into the mechanism of the multifunctional effects of TGF-beta and how apoptosis and EMT are regulated in the same type of cells.