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.

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.

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

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.

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.

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.

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.

Effect of Burn Healing Liquid on keratinocyte and fibroblast proliferation and on collagen lattice contraction

Objectives To investigate the effect of Burn Healing Liquid (BHL) on the proliferation of keratinocytes and fibroblasts and to explore the potential effect of BHL on fibroblast contraction Methods Human keratinocytes and dermal fibroblasts were cultured in media containing serial dilutions of BHL followed by cell proliferation determination assessed with MTT (3 [4,5 dimehtylthiazol 2 yl] 2,5 diphenyl tetrazolium bromide) assay at different time intervals The in vitro collagen lattice contraction model was utilized for determining the contractility of fibroblasts cultured in BHL containing medium Results The 1∶10 7 dilution of BHL enhanced the growth of both keratinocytes and fibroblasts whereas the 1∶10 dilution increased the growth of keratinocytes only Collagen lattice contraction was inhibited dose dependently by BHL and such an inhibition could be reversed by switching BHL containing medium to normal medium containing 10% fetal calf serum Conclusion BHL enhances the growth of both keratinocytes and fibroblasts and reversibly inhibits the fibroblast contraction in collagen lattice

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.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis(TED).To solve these problems,we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet(PBMCS)and pre-vascularized fibroblasts cell sheet(PFCS)by cell sheet technology,and then superimposed or folded them together to construct a pre-vascularized TED(PTED),aiming to mimic the real dermis structure.The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1,7 and 14 via the methods of histochemistry and immunohistochemistry.The results showed that PTED could rapidly promote the wound closure,especially at Day 14,and the wound-healing rate of three-layer PTED could reach 97.2%(P<0.01),which was faster than the blank control group(89.1%),PBMCS(92.4%),PFCS(93.8%)and six-layer PTED(92.3%).In addition,the vessel density in the PTED group was higher than the other groups on the 14th day.Taken together,it is proved that the PTED,especially three-layer PTED,is more conducive to the fullthickness dermis-defect repair and the construction of the three-dimensional vascular networks,indicating its potential application in dermis-defect repair.