Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

Magnetic resonance image （MRI） systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields （SMF）. With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid （AL） cells, Chinese hamster ovary （CHO） cells, DNA double-strand break repair deficient mutant （XRS-5） cells, and human primary skin fibroblasts （AG1522） cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster ceils after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

RNA sequencing of exosomes secreted by fibroblast and Schwann cells elucidates mechanisms underlying peripheral nerve regeneration

Exosomes exhibit complex biological functions and mediate a variety of biological processes,such as promoting axonal regeneration and functional recove ry after injury.Long non-coding RNAs(IncRNAs)have been reported to play a crucial role in axonal regeneration.Howeve r,the role of the IncRNA-microRNAmessenger RNA(mRNA)-competitive endogenous RNA(ceRNA)network in exosome-mediated axonal regeneration remains unclear.In this study,we performed RNA transcriptome sequencing analysis to assess mRNA expression patterns in exosomes produced by cultured fibroblasts(FC-EXOs)and Schwann cells(SCEXOs).Diffe rential gene expression analysis,Gene Ontology analysis,Kyoto Encyclopedia of Genes and Genomes analysis,and protein-protein intera ction network analysis were used to explo re the functions and related pathways of RNAs isolated from FC-EXOs and SC-EXOs.We found that the ribosome-related central gene Rps5 was enriched in FC-EXOs and SC-EXOs,which suggests that it may promote axonal regeneration.In addition,using the miRWalk and Starbase prediction databases,we constructed a regulatory network of ceRNAs targeting Rps5,including 27 microRNAs and five IncRNAs.The ceRNA regulatory network,which included Ftx and Miat,revealed that exsosome-derived Rps5 inhibits scar formation and promotes axonal regeneration and functional recovery after nerve injury.Our findings suggest that exosomes derived from fibro blast and Schwann cells could be used to treat injuries of peripheral nervous system.

Biocompatibility Evaluation of Polyethylene Terephthalate Artificial Ligament Coating Hydroxyapatite by Fibroblasts Cells in Vitro

Hydroxyapatite(HA) based materials have been widely used in the field of ligament tissue engineering in the past decades.It has been previously reported that HA can increase the penetration of marrow-derived mesenchymal stem cells(MSCs) and MSCs cells into scaffolds due to increased cell differentiation in biological media.Additionally,it was found that there are much difference between MSCs and anterior cruciate ligament (ACL) cells.For that reason,we mainly evaluate the biocompatibility of polyethylene terephthalate(PET) silk scaffold with fibroblasts cells in vitro.We cultured mouse fibroblasts cells on the substrate of PET fiber and PET-HA scaffold,respectively,and then observed the morphology by using scanning electron microscopy.Our data indicate that PET-HA scaffold has good biocompatibility with fibroblasts cells and can potentially be useful in enhancing the fibroblasts cell differentiation and proliferation.

Differential Expression of APOEGene in Porcine Fetal Fibroblast Cells

[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porcine fetal fibroblast cells,which were normally grown and passed,were collected before total RNA was extracted respectively.The expression ofAPOEgene in different generations of porcine fetal fibro-blast cells was detected by RT-PCR technique.[Result]The expression level of porcine APOE mRNA in the first generation of porcine fetal fi-broblast cells was the highest,and then it gradually decreased with the increase of cell generations and was the lowest in the fiftieth generation.[Conclusion]The expression ofAPOEgene had the selective trend in different generations of porcine fetal fibroblast cells.

Skin care efficacy study of recombinant humanized collagen based on in vitro level

Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro.Experimental results showed that,compared with the control cells,the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis,while the treatment of 25 s plasma resulted in a remarkable decrease.Exploration of related mechanisms suggested that cold plasma could up-regulate Cyclin D1 gene expression and down-regulate p27 gene expression at a low dose,while it could down-regulate Cyclin D1 expression and up-regulate p27 expression at a higher dose,thus altering the cell cycle progression,and then affecting cell viability and collagen synthesis of fibroblasts.

Cytotoxicity of Modified Nonequilibrium Plasma with Chlorhexidine Digluconate on Primary Cultured Human Gingival Fibroblasts

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate（CHX） on human gingival fibroblasts（HGFs）, and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min（control group）, 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8（CCK-8） assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group（P〉0.05）. However, there were significant differences between all the other treated groups and the control group（P〈0.05）. When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.

Expressions of TGF-β2, bFGF and ICAM-1 in lens epithelial cells of complicated cataract with silicone oil tamponade

AIM： To investigate the expression differences of transforming growth factor-β2（TGF-β2）, basic fibroblast growth factor（b FGF） and intercellular cell-adhesion molecule-1（ICAM-1） in lens epithelial cells（LECs） of complicated cataract with silicone oil tamponade and agerelated cataract. METHODS： Totally 150 eyes of 150 patients（aged 35 to 77y） were investigated, including 75 patients with complicated cataract after silicone oil tamponade and 75 patients with age-related cataract. The central piece of anterior capsules was collected during cataract surgery. TGF-β2, b FGF and ICAM-1 were detected in the 60 specimens of the two groups by immunohistochemistry. The expression levels of the three kinds of messenger ribonucleic acid（m RNA） were determined by real-time quantitative reverse transcriptionpolymerase chain reaction in the 90 specimens of the two groups.RESULTS： TGF-β2 was detected in the cytomembrane and cytoplasm of the LECs and b FGF was detected in the nucleus. ICAM-1 was positive in the cytomembrane of the LECs and the distribution of positive cells was uneven. The m RNA genes expression of the TGF-β2, b FGF and ICAM-1 was significant differences between the two groups and markedly increased in complicated cataract group（P〈0.05）.CONCLUSION： The up-regulated TGF-β2, b FGF and ICAM-1 maybe associate with the occurrence and development of complicated cataract with silicone oil tamponade.

A spectroscopic study on the effect of ultra-violet solar radiation in Antarctica on the human skin fbroblast cells

A study on the effect of the solar ultra-violet radiation on the human skin fibroblast cells revealed that the production of matrix metalloproteinase-2 was inhibited by the radiation.A CO2 incubator connected by optical fibers to a reflector telescope for collecting the solar light was built at Syowa station by the 49th Japanese Antarctica Research Expedition.The direction of the telescope was continuously controlled by a sun-tracker to follow the movement of the Sun automatically.The intensity of the collected light was monitored by a portable spectrophotometer housed inside.The human skin fibroblast cells were incubated in the CO2 chamber to investigate the effect of the solar radiation at Syowa station and were compared with those reference experiments at a laboratory in Japan.The results showed cell damage by strong UV radiation.The production of matrix metalloproteinase-2 was prompted by the moderate UV-B,but was inhibited by the strong UV-B radiation,as studied under laboratory conditions in Japan.The effect of strong solar radiation at Syowa station involving the radiation of UV-B region was estimated to be of the same extent of the radiation caused by an artificial UV-B light with the intensity more than 50 mJ/cm2.

THE STUDY ON RELATED GENES IN THE NEOPLASTIC TRANSFORMATION OF IMMORTALIZED HUMAN FETAL TRACHEAL FIBROBLAST CELLS INDUCED BY IRRADIATION

In this study, we investigated the genes related to the transformation of immortalized human fetal tracheal fibroblast cell line induced by alpha particles by means of differential display mRNA method. The result revealed that there were 23 DNA fragments that were expressed intensively in alphaSHTF cells (SHTF cells forming clone on agar after irradiated by alpha particles emitted by 238Pu) only and not in SHTF (SV40-immortalized human fetal tracheal fibroblast) cells. Northern dot confirmed two fragments, C17-5, C23-1 which showed intensive mRNA expression in alphaSHTF cells, but not in SHTF cells. The length of the C17-5 fragment was 310bp. Searching in BLAST database revealed that the C17-5 fragment might be an unknown sequence.

Human ovarian neoplasm cell CD147 stimulates production and activation of matrix metalloproteinases in co-cultures with mouse fibroblasts

Objective: To investigate the expression of CD147 on human ovarian neoplasm cell lines and its influence on production and activation of matrix metallproteinases(MMPs). Methods: The expression of CD147 on different human ovarian neoplasm cell lines was studied by western blotting. Co-culture was carried out to investigate the stimulative effect of the positive expression CD147 cell HO-8910 on the production of MMPs of fibroblast cell in vitro. Zymography and immune blotting were used to study the production and activity of positive MMPs, at the time, to explore the relation between CD147 and MMPs. Results: CD147 was positively presented in 2 ovarian neoplasm cell lines(HO-8910,3-AO), but in SKOV3, TC-1,NIN3T3 cell was negative. MMP-2 and MMP-9 were detected by HO-8910 cell line, mouse fibroblast cell and co-culture cells; but the expression in co-culture cell is obviously higher than individual cultures of each type alone.CD147 stimulated MMPs in dose-dependent manner. Conclusion: CD147 causes increased production and activation of MMP-2, MMP-9.CD147 is probably a indirect marker of some ovarian cancer cells with invasion and metastasis.

Effect of the implant composite of poly lactide-co-glycolide and bone mesenchymal stem cells modified by basic fibroblast growth factor on injured spinal cord in rats

Objective To investigate the effect of the implant composite of poly lactide-co-glycolide(PLGA)and bone mesenchymal stem cells (BMSCs) modified by basic fibroblast growth factor (bFGF) on injured spinal cord in rats.Methods Two hundred and

Alterations in genes other than EGFR/ALK/ROS1 in non-small cell lung cancer:trials and treatment options

During the last decade, we have seen tremendous progress in the therapy of lung cancer. Discovery of actionable mutations in EGFR and translocations in ALK and ROS1 have identified subsets of patients with excellent tumor response to oral targeted agents with manageable side effects. In this review, we highlight treatment options including corresponding clinical trials for oncogenic alterations affecting the receptor tyrosine kinases MET, FGFR, NTRK, RET, HER2, HER3, and HER4 as well as components of the RAS-RAF-MEK signaling pathway.

Construction and Screening of Bovine Bax Gene shRNA Interference Expression Vector

Bax is a pro-apoptotic member of the Bcl-2 family genes which regulate programmed cell death. To decrease the apoptosis of bovine fibroblast cells, we construsted specific short hairpin （shRNA） expression vector encoding shRNA targeting Bax gene to screen the most effective vector. Four shRNAs sequences based on the sequence of bovine Bax mRNA in the GenBank were designed, and one scrambled shRNA sequence was regarded as negative control. The designed and synthesised single-stranded primer were annealed to double-stranded oligo sequences and cloned into linear pRI-GFP vector digested by enzymes Xho I and Bgl II. Screening positive cloning after transformed into DH5a competent cells and identified by PCR amplification and DNA sequencing. Named the correct vectors as pRI-GFP-Bax-190, pRI-GFP-Bax-206, pRI-GFP-Bax-215, pRI -GFP-Bax-389, pRI-GFP- Bax-NC （the negative control） and seleced them by quantitative PCR after transfected after 24 h and 48 h. The results showed that pRI-GFP-Bax-190 was the highest efficiency （95.47%）, and significant difference （P〈0.01） after 48 h transfection. RNA interference （RNAi） mediated by shRNA expression vector could significantly down-regulate the expression of Bax gene in bovine fibroblast cells, which laid a foundation for further research.

Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2(hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

Chemical compositions,cytotoxicity and antioxidant activity of the endophytic fungus Fusarium napiforme isolated from Psidium guajava

The bioactive secondary metabolites from the endophytic fungus Fusarium napiforme was evaluated for the cytotoxic effect and antioxidant activity.The total antioxidant capacity(TAC)of the extract was determined by 2,2-diphenyl-1-picrylhydrazyl(DPPH),phosphomolybdate,and reducing power assay methods.The cytotoxicity of the extract was evaluated against lung adenocarcinoma(A549)cells and mouse embryo fibroblast(NIH3T3)cells by methyl thiazolyl tetrazolium(MTT)method.The major composition of the crude extract was identified by the Gas Chromatography-Mass Spectrometry(GC-MS)analysis.Estimation of the endophyte crude extract revealed a high amount of the total flavonoid content(TFC)and total phenolic content(TPC).The extract showed high cytotoxic activity against the A549 cell line with the mean cytotoxicity of 69.74±0.49%.The extract did not show any cytotoxic effect against the NIH3T3 cell line.The extract exhibited high antioxidant activity as a function of the concentrations.At a test concentration of 1 mg ml-1,the extract showed the highest inhibition against DPPH radical at 75.4607%±0.47688,ferric ion reducing power at 0.882±0.0120,and 255.434±21.404 AAE/g of the extract by phosphomolybdenum assay(PMA).There is a significant correlation between TPC and antioxidant activity at p<0.05.The correlation between reducing power and DPPH is significant at p<0.01.The major types of bioactive compounds identified by the GC-MS have shown the presence of nine major compounds.This result strongly exhibits that the endophyte F.napiforme can be a potential source for the formulation of natural anticancer drugs and protecting the body from oxidative damages.

EFFECTS OF EPCs OR b-FGF INTRAMYOCARDIAL INFUSION ON CARDIAC FUNCTION AND NEOVASCULARIZATION FOR DILATED CARDIOMYOPATHY RATS

Objective To compare the different effects of endothelia progenitor cells （ EPCs ） or basic fibroblast growth factor （b-FGF） intromyocardial infusion on cardiac function and neovascularization for dilated cardiomyopathy（ DCM） rats. Methods Fifty adult female rats received inguinal subcutaneous injections of isoproterenol （ISO, 250 mg/kg） for induction of DCM. Four weeks later, the model rats were randomly divided into EPCs group, b-FGF group and control group. The 2×106 EPCs （ resolved in 100 μL PBS） , 100 μL b-FGF （ lO0 μg/mL ） and 100 μL PBS were evenly transplanted into the myocardium of EPCs group, b-FGF group and control group, respectively. Three months later, echocardiographic examination and regional myocardial blood flow （RMBF） measurement were performed. EPCs were traced by fluorescence in situ hybridization （FISH）. The protein and mRNA expression of b-FGF in each group was measured by ELISA assay and reverse transcription-polymerase chain reaction （ RT-PCR ） . Results Three months after transplantation, sry positive cells were detected only in EPCs group. The cardiac function as well as RMBF was significantly improved in EPCs group compared with b-FGF group or control group. There was higher capillary density in EPCs group. The protein and mRNA expression of b-FGF was stronger than b-FGF group and control group. Conclusion Transplantation of EPCs can improve cardiac function, induce neovascularization and increase RMBF for DCM rats. The treatment with EPCs has better effect than administration of b-FGF alone.

Cytokeratin-positive interstitial reticulum cell tumors of lymph nodes： a case report and review of literature

Cytokeratin-positive interstitial reticulum cells （CIRCs） are considered to represent a subset of fibroblastic reticulum cells （FBRCs） belonging to accessory dendritic cells in lymph nodes, the spleen and tonsils.1-3 The tumors arising from CIRCs are so rare that they are easily misdiagnosed as tumors originating from other accessory dendritic cells, myofibroblasts or even metastatic poor-differentiated carcinomas due to their similar histomorphologic features. We report herein one new case of a CIRC tumor （CIRCT） located in the retroperitoneum and analyze the clinicopathologic characteristics, pathogenesis, treatment and prognosis by reviewing the literature.