Establishment,Characterization and Expression Pattern of a Spleen Cell Line,SMSP,Derived from Turbot(Scophthalmus maximus)

A turbot(Scophthalmus maximus)cell line named SMSP was obtained from the spleen.The origin of the cells was identified by morphology,chromosome number and COI gene.The optimal basic medium,serum concentration and growth temperature of the cells were detected.SMSP cell line is mainly composed of fibroblast-like cells.Most of the SMSP cells contained 44 chromosomes,and the sequence of COI gene confirmed that the cells were originated from turbot.The optimal culture conditions were 24℃,DMEM+10%FBS.The cell line had high transfection efficiency for siRNA and plasmid.After stimulation with lipopolysaccharide(LPS)or poly(I:C),the expressions of immune-related genes such as TNF-β,IL-12s,IL-10 and IL-1βwere up-regulated significantly in the early stage(P<0.05).This study will provide a model for exploring immune mechanism of turbot against pathogen in vitro.

First report on establishment and characterization of the extrahepatic cholangiocarcinoma sarcoma cell line CBC2T-2

BACKGROUND Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice.These cells consist of both epithelial and mesenchymal cells.Patient-derived cell lines that maintain tumor characteristics are valuable tools for studying the molecular mechanisms associated with carcinosarcoma.However,cholangiocarcinoma sarcoma cell lines are not available in cell banks.AIM To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line,namely CBC2T-2.METHODS We conducted a short tandem repeat(STR)test to confirm the identity of the CBC2T-2 cell line.Furthermore,we assessed the migratory and invasive properties of the cells and performed clonogenicity assay to evaluate the ability of individual cells to form colonies.The tumorigenic potential of CBC2T-2 cells was tested in vivo using nonobese diabetic/severe combined immunodeficient(NOD/SCID)mice.The cells were injected subcutaneously and tumor formation was observed.In addition,immunohistochemical analysis was carried out to examine the expression of epithelial marker CK19 and mesenchymal marker vimentin in both CBC2T-2 cells and xenografts.The CBC2T-2 cell line was used to screen the potential therapeutic effects of various clinical agents in patients with cholangiocarcinoma sarcoma.Lastly,whole-exome sequencing was performed to identify genetic alterations and screen for somatic mutations in the CBC2T-2 cell line.RESULTS The STR test showed that there was no cross-contamination and the results were identical to those of the original tissue.The cells showed round or oval-shaped epithelioid cells and mesenchymal cells with spindle-shaped or elongated morphology.The cells exhibited a high proliferation ratio with a doubling time of 47.11 h.This cell line has migratory,invasive,and clonogenic abilities.The chromosomes in the CBC2T-2 cells were polyploidy,with numbers ranging from 69 to 79.The subcutaneous tumorigenic assay confirmed the in vivo tumorigenic ability of CBC2T-2 cells in NOD/SCID mice.CBC2T-2 cells and xenografts were positive for both the epithelial marker,CK19,and the mesenchymal marker,vimentin.These results suggest that CBC2T-2 cells may have both epithelial and mesenchymal characteristics.The cells were also used to screen clinical agents in patients with cholangiocarcinoma sarcoma,and a combination of paclitaxel and gemcitabine was found to be the most effective treatment option.CONCLUSION We established the first human cholangiocarcinoma sarcoma cell line,CBC2T-2,with stable biogenetic traits.This cell line,as a research model,has a high clinical value and would facilitate the understanding of the pathogenesis of cholangiocarcinoma sarcoma.

Establishment and characterization of a new human ampullary carcinoma cell line,DPC-X1

BACKGROUND An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have been reported, and a mixed-type ampullary carcinoma cell line has not yet been reported.AIM To establish a stable mixed-type ampullary carcinoma cell line originating from Chinese.METHODS Fresh ampullary cancer tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, short tandem repeat(STR) analysis and transmission electron microscopy. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-FU were evaluated by cell counting kit-8 assay. Subcutaneous injection 1 × 106 cells to three BALB/c nude mice for xenograft studies. The hematoxylin-eosin staining was used to detect the pathological status of the cell line. The expression of biomarkers cytokeratin 7(CK7), cytokeratin 20(CK20), cytokeratin low molecular weight(CKL), Ki67 and carcinoembryonic antigen(CEA) were determined by immunocytochemistry assay.RESULTS DPC-X1 was continuously cultivated for over a year and stably passaged for more than 80 generations;its population doubling time was 48 h. STR analysis demonstrated that the characteristics of DPC-X1 were highly consistent with those of the patient’s primary tumor. Furthermore, karyotype analysis revealed its abnormal sub-tetraploid karyotype. DPC-X1 could efficiently form organoids in suspension culture. Under the transmission electron microscope, microvilli and pseudopods were observed on the cell surface, and desmosomes were visible between the cells. DPC-X1 cells inoculated into BALB/C nude mice quickly formed transplanted tumors, with a tumor formation rate of 100%. Their pathological characteristics were similar to those of the primary tumor. Moreover, DPC-X1 was sensitive to oxaliplatin and paclitaxel and resistant to gemcitabine and 5-FU. Immunohistochemistry showed that the DPC-X1 cells were strongly positive for CK7, CK20, and CKL;the Ki67 was 50%, and CEA was focally expressed.CONCLUSION Here, we have constructed a mixed-type ampullary carcinoma cell line that can be used as an effective model for studying the pathogenesis of ampullary carcinoma and drug development.

Establishment of a human hepatoma multidrug resistant cell line in vitro

AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malignant hepatoma was incubated with a high concentration of cisplatin(CDDP) to establish a CDDP-resistant cell subline(SK-Hep-1/CDDP).The 50% inhibitory dose(IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cells were all evaluated using cell counting kit-8 assays.The distribution of the cell cycles were detected by flow cytometry.Expression of acquired multidrug resistance P-glycoprotein(MDR1,ABCB1) and multidrug resistance-associated protein 1(MRP1,ABCC1) was compared with that in parent cells by Western blotting and immunofluorescence combined with laser scanning confocal microscopy.RESULTS:The SK-Hep-1/CDDP cells(IC50 = 70.61 ± 1.06 μg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells(IC50 = 5.13 ± 0.09 μg/mL),and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin,5-fluorouracil and vincristine.Similar morphologies were determined in both SK-Hep-1 and SK-Hep-1/CDDP groups.The cell cycle distribution of the SK-Hep-1/CDDP cell line exhibited a significantly increased percentage of cells in S(42.2% ± 2.65% vs 27.91% ± 2.16%,P < 0.01) and G2/M(20.67% ± 5.69% vs 12.14% ± 3.36%,P < 0.01) phases in comparison with SK-Hep-1 cells,while the percentage of cells in the G0/G1 phase decreased(37.5% ± 5.05% vs 59.83% ± 3.28%,P < 0.01).The levels of MDR1 and MRP1 were overexpressed in the SK-Hep-1/CDDP cells exhibiting the MDR phenotype.CONCLUSION:Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of MDR1 and MRP1.

Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.

Reversing effect of Tanshinone on malignant phenotypes of human hepatocarcinoma cell line

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Apoptotic activity of caged xanthones from Garcinia hanburyi in cholangiocarcinoma cell lines

AIM:To investigate the growth inhibitory mechanism of four caged xanthones from Garcinia hanburyi in cholangiocarcinoma(CCA) KKU-100 and KKU-M156 cells.METHODS:Four caged xanthones,selected on the basis of their anticancer potency and chemical structure diversities(i.e.isomorellin,isomorellinol,forbesione and gambogic acid) were used in this study.Growth inhibition of these caged xanthones was determined using the sulforhodamine B assay.Induction of apoptosis was assessed by observing cell morphology,ethidium bromide and acridine orange staining and DNA fragmentation assay.Levels of apoptotic-related gene and protein expressions were determined by a real-time reverse transcriptase polymerase chain reaction and Western blotting analysis,respectively.RESULTS:The compounds were found to inhibit growth of both cell lines in a dose-dependent manner and also showed selective cytotoxicity against the cancer cells when compared with normal peripheral blood mononuclear cells.Growth suppression by these compounds was due to apoptosis,as evidenced by the cell morphological changes,chromatin condensation,nuclear fragmentation,and DNA ladder formation.At the molecular level,these compounds induced down-regulation of Bcl-2 and survivin proteins with up-regulation of Bax and apoptosisinducing factor proteins,leading to the activation of caspase-9 and-3 and DNA fragmentation.The functional group variations did not appear to affect the anticancer activity with regard to the two CCA cell lines;however,at a mechanistic level,isomorellinol exhibited the highest potency in increasing the Bax/Bcl-2 protein expression ratio(120 and 41.4 for KKU-100 and KKU-M156,respectively) and in decreasing survivin protein expression(0.01 fold as compared to control cells in both cell lines).Other activities at the molecular level indicate that functional groups on the prenyl side chain may be important.CONCLUSION:Our findings for the first time demonstrate that four caged xanthones induce apoptosis in CCA cells which is mediated through a mitochondriadependent signaling pathway.

Establishment of a Spleen Cell Line from Large Yellow Croaker Pseudosciaena crocea and its Primitive Application in Foreign Gene Transfection

A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco's modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.

TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-x_L mRNA in Extended Drug Resistance Leukemia Cell Line HL- 60/E6

To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.

Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami(Cypriniformes:Cyprinidae)

Though Yunnan province contains some 562 known species of fish,no cell lines from any of these have been made available to date.To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami,a fish endemic to Fuxian Lake,Yunnan,China,we established and characterized the major features of a continuous cell line(AGF II)from the caudal fin tissue of A.grahami.This AGF II cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year.The cell line was maintained in DMEM/F12 supplemented with 10%FBS,with a cellular doubling time of 51.1 h.We continued with more experiments to optimize the culture and storage conditions,and found a variety of interesting results:cells could grow at temperature between 24℃and 28℃,with the optimal temperature of 28℃.Likewise,the growth rate of A.grahami fin cells increased when the FBS proportion increased from 5%to 20%,with the optimal growth at the concentrations of 20%FBS;cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15;DMSO is a better cryoprotectant than Glycerol,EG and MeOH for AGFII cells with optimal concentration of 5%DMSO.Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52,with a modal peak at 48 chromosomes,accounting for 39.8%of all cells.Using the same primer pairs specific to mtDNA,the AGF II cell sequences obtained by PCR were identical to those from muscle tissues of A.grahami.Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A.grahami,also indicating that that current long-term artificial propagation of A.grahami has been successful.Finally,we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid,bright fluorescent signals were observed,suggesting that this cell line may be suitable for use in transfection and future gene expression studies。

Dynamic Investigation on Chromosome Aberration of a Human Retinoblastoma Cell Line SO-Rb_(50)

G-banding and karyotype analyses of cells in seventeen passages of SO-Rb_(50) during a long period of culture for about four years were performed. Three chromosome markers 13q14^-, 1p36^+ and 12p13^+ were found. Cells possessed 13q14^- reduced to zero after the 200th passage while 1p^+ and 12p^+ cells increased to 100% after 30 and 200 passages respectively. Abnormal chromosomes, ring chromosomes, chromosome radiuses and double minutes were also observed. These chromosomal changes were more often seen b...

Enhanced cytotoxic effect on human lung carcinoma cell line(A549) by gold nanoparticles synthesized from Justicia adhatoda leaf extract

Objective: To synthesize bio-inspired gold nanoparticles(AuNPs) using the leaf extract of Justicia adhatoda and evaluate the anti-cancer activity on human lung cancer cell line(A549).Methods: Synthesis of AuNPs was done using an aqueous leaf extract of Justicia adhatoda as a green route. The bio-synthesized AuNPs were confirmed and characterized by using various spectral studies such as UV-Vis spectrum, Scanning Electron Microscope with EDAX, Transmission Electron Microscope, Fourier Transmission Infrared Spectroscope analysis and Surface Enhanced Raman Spectroscopy. The cell viability was determined by MTT reduction assay. In addition, cytomorphology and the nuclear morphological study of A549 cell line was observed under fluorescence microscope. Results: UV-Vis spectrum showed surface plasmon resonance peak at 547 nm, scanning electron microscope and transmission electron microscope studies showed the monodispersed spherical shape and its average size in the range of 40.1 nm was noticed. Fourier Transmission Infrared Spectroscope analysis confirmed that the C=O group of amino acids of proteins had strong ability to bind with the surface of nanoparticle. Interestingly, our results also demonstrated inhibited proliferation of A549 cell line by MTT(IC50 value: 80 μg/mL). Cell morphology was observed and cell death was caused by apoptosis as revealed by propidium iodide staining. Conclusions: The current study proves the anticancer potential of bio-synthesized AuNPs. Thus, synthesized AuNPs can be used for the treatment of human lung cancer cell(A549) and it can be exploited for drug delivery in future.

Identification of differentially expressed genes in two new human bladder carcinoma cell lines

Objective To screen and identify differentially expressed genes in two new human urothelial carcinoma cell lines, BLS-211 and BLX. Methods Suppression subtractive hybridization (SSH) was used to createa subtracted library, and clones were sequenced. Results Totally 13 over-expressed genes in BLX and 9 in BLS-211 cells were obtained, respectively. Among them, 18 were known genes and 4 were new ESTs (Expressed Sequence Tag), and were collected by GenBank dbEST database (The access number was EB390424-7). Conclusion SSH is a powerful method for the identification of differentially expressed genes. The differential expression of some BCG-associated genes in different cells may be related to the different responses to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.

Establishment and Characterization of Four Long-Term Cultures of Neural Stem/Progenitor Cell Lines from the Japanese Flounder Paralichthys olivaceus

Neurogenesis is an important progress wherein the neural stem cells(NSCs) differentiate into functional neurons under conductive conditions. Neurogenesis occurs continuously in different areas of the central neural system in adult teleosts compared with adult mammals. Therefore, NSC cell lines must be established to offer a valuable in vitro system for studies on neurogenesis and other related functions. In this study, four cell lines designated as PoB1, PoB2, PoBf and PoBh were established from the brain of the Japanese flounder Paralichthys olivaceus. The cell lines were sub-cultured over 150 times and still grew well in DMEM/F12 medium at 24℃. PoB1, PoB2, PoBf and PoBh were identified as neural stem/progenitor cell lines on the basis of the mRNA expression of nestin and/or aldh111 or slc6 a4 and the formation of neurospheres. The cells transfected with the pEGFP-C1 plasmid showed fluorescent signals with distinct reagent dependencies. The established cell lines from the brain of P. olivaceus offer a valuable system in vitro for the study of neurogenesis, fish neural regulation and endocrinology-related functions.

Establishment and characterization of a new cell line derived from half-smooth tongue sole Cynoglossus semilaevis kidney

In this study,we established a new cell line(Cynoglossus semilaevis kidney cells,CSK)from kidney of the half-smooth tongue sole(Cynoglossus semilaevis).The cells were subcultured over 1000 days and passaged for more than 100 times.Additionally,CSK cells were optimally maintained in Dulbecco's modified Eagle medium nutrient mixture F-12 supplemented with HEPES,antibiotics,fetal bovine serum,2-mercaptoethanol,and basic fibroblast growth factor.The optimum growth temperature for CSK cells was 25℃,and the cells showed a fibroblast-like phenotype.Chromosome analysis revealed that CSK cells had a normal diploid karyotype with 2 n=42.CSK cells were susceptible to Grouper nervous necrosis virus(NNV),and cytopathic ef fects were observed at 3–5 days postinfection.The NNV sensitivity of CSK cells was related to the high abundance of virions in the cytoplasm,as observed by electron microscopy.Additionally,CSK cells could be successfully transfected with a green fluorescent protein reporter plasmid,and fluorescent signals were easily observed.Finally,immunocytochemistry analysis showed that CSK cells were supporting cells.Overall,we established this new cell line,which may have potential applications in the identification of viral pathogens af fecting the half-smooth tongue sole.

Qu-2,a robust poplar suspension cell line for molecular biology

Populus spp.have long been used as model woody plant species for molecular biology research.However,tissues of poplar are often recalcitrant to experimental procedures for molecular studies.We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba×P.berolinensis‘Yinzhong’,named Qu-2.Qu-2 cells are suitable as a model biological system for studying woody plants.Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants.Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone.They show exceptionally high growth rates,reaching an approximately 150-fold increase in biomass after one week of culturing.Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated.Qu-2 cells are suitable for molecular manipulations such as protoplast production,transient transformation,and RNA-seq analysis.Therefore,Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research,ranging from physiology to gene function.The Qu-2 cells will be made available to the plant community for research.

Establishment and Characterization of a Fin Cell Line Derived from the Atlantic Salmon Salmo salar and Its Application to Fish Virology Study

Atlantic salmon(Salmo salar)is an important economic fish that is seriously threatened by various viruses.A cell line designated as ASF derived from the caudal fin tissue of Atlantic salmon was established and characterized in this study.ASF cells grew well in Dulbecco’s modified Eagle’s medium(DMEM)containing 20%fetal bovine serum at 20℃.DNA sequencing and comparative analysis of the cytochrome B gene verified that the ASF cell line originated from Atlantic salmon.Chromosome analysis indicated that the modal chromosome number of ASF cells was 58.Viral susceptibility test showed that ASF cells were susceptive to two important fish viruses,viral hemorrhagic septicemia virus(VHSV)and red-spotted grouper nervous necrosis virus(RGNNV).Viral replication in ASF cells was further confirmed by qRT-PCR and transmission electron microscopy.Moreover,VHSV and RGNNV infections could induce the cellular responses in ASF cells,as indicated by the differential expression of cellular antiviral response-related genes,interferon-1 and Mx-1.In conclusion,the newly established ASF cell line can be applied as an in vitro tool in fish virology and immunity studies.

Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines

The effects of Sonic hedgehog(Shh) signaling pathway activation on S-type neuroblastoma(NB) cell lines and its role in NB tumorigenesis were investigated.Immunohistochemistry was used to detect the expression of Shh pathway components- Patched1(PTCH1) and Gli1 in 40 human primary NB samples.Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines(SK-N-AS,SK-N-SH and SHEP1),respectively.Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway.S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations.Cell viability was measured by using MTT method.Apoptosis rate and cell cycle were assayed by flow cytometry.The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice.High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines.Cyclopamine decreased the survival rate of the three cell lines while Shh increased it,and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment.Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase,while Shh induced the reverse effects and could partially prevent effects of cyclopamine.Cyclopamine could also inhibit the growth of NB in vivo.Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle,suggesting a new therapeutic approach to NB.