RESUMO
For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained.
Assuntos
Ambystoma mexicanum/genética , Perfilação da Expressão Gênica/normas , Genes Essenciais , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Ambystoma mexicanum/fisiologia , Animais , Extremidades/fisiologia , Perfilação da Expressão Gênica/métodos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Ornitina Descarboxilase/genética , Estabilidade de RNA , Padrões de Referência , Regeneração/genética , Proteínas Ribossômicas/genética , Estudos de Validação como AssuntoRESUMO
INTRODUCTION: Adipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs. METHODS: ASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni's post hoc test. RESULTS: High initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration. CONCLUSIONS: Our study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.
Assuntos
Tecido Adiposo/fisiologia , Lipoxigenase/genética , Transfecção/métodos , Tecido Adiposo/citologia , Tecido Adiposo/enzimologia , Ambystoma mexicanum/genética , Ambystoma mexicanum/metabolismo , Animais , Expressão Gênica , Lipoxigenase/biossíntese , Masculino , Ratos , Ratos Endogâmicos LewRESUMO
OBJECTIVE: The Mexican axolotl (Ambystoma mexicanum) is a well-characterized example for intrinsic regeneration. As lipoxygenase signaling is of crucial importance to scarless mammalian wound healing, we postulated that lipoxygenases might be expressed during amphibian regeneration and they might also influence human cells under appropriate conditions. In this study we identified an amphibian lipoxygenase and evaluated its impact on human cells in an in vitro wound model. METHODS: cDNA encoding for amphibian epidermal lipoxygenase (AmbLOXe) was polymerase chain reaction amplified and sequenced followed by phylogenic classification based on T-coffee alignment. Distribution of AmbLOXe was examined in various Ambystoma tissues, using polymerase chain reaction and in situ hybridization. Lipoxgenase influence was investigated using an outgrowth model of amphibian epidermal cells. Human osteosarcoma, as well as keratinocyte cell lines expressing AmbLOXe, were tested concerning in vitro wound closure in a monolayer scratch model. RESULTS: We isolated AmbLOXe from Ambystoma limb bud blastema identified as a homologue of human epidermal lipoxygenase. Amphibian epidermal lipoxygenase is expressed in Axolotl limb blastema and in epidermal cells which show decreased cell migration and proliferation rates when treated with LOX inhibitors. Furthermore, human osteosarcoma and keratinocyte cells showed increased rates of cell migration if transfected with AmbLOXe. CONCLUSION: In this study, AmbLOXe, a new effector of amphibian regeneration is described. In consideration of the presented data, AmbLOXe is important for amphibian epidermal cell proliferation and migration. As AmbLOXe expressing human osteosarcoma and keratinocyte cell lines showed increased rates of in vitro wound closure, an influence of amphibian mediators on human cells could be described for the first time.