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Differential gene expression in primary cultured sensory and motor fibroblasts

Meng Cong  Qianru He  Shu Yu  Fei Ding  
【摘要】:Objective The peripheral nerve is composed by sensory and motor nerves. The proteomics technique has been used to determine the protein profile of them, and 100 proteins showed differential expressions between sensory and motor nerves. The differential expression protein has difference expression of neuron, also of SCs and fibroblasts. This study aimed to compare the gene expression of sensory and motor fibroblasts by using Whole rat genome microarray analysis, thus contributing to our understanding of different functions of both fibroblasts in sensory and motor nerves. Methods Cell culture and immunocytochemistry. All animal experiments were performed in accordance with institutional guidelines. Sprague-Dawley(SD) rats(7-8 day old). The purity of motor and sensory fibroblasts was further evaluated by flow cytometric analysis(FCA). The motor and sensory fibroblasts were harvested after purification and the samples were subjected to whole rat genome microarray analysis.Total RNA was extracted from sensory and motor fibroblasts using Trizol method, and c DNA was synthesized in accordance with the instructions of Omniscript? RT kit. Quantitative real time RT-PCR(q RT-PCR) was conducted in CFX96? Real-Time PCR System using Fast Start? SYBR Green q PCR Master Mix according to the manufactur's specification. The real time cell analyzer and E-plate 16 system allows for label-free, noninvasive monitoring, real time of the viability of cultured cells using impedance. The proliferation capacity of motor and sensory fibroblasts was further evaluated by 5-ethynyl-2'-deoxyuridine(Ed U) labeling kit. The cell migration of sensory and motor fibroblasts was examined in 6.5 mm transwell chambers with 8 μm pores as described previously. For fibroblasts transfection, si RNAs(1, 2 and 3) for Cxcl 3, si RNAs(1, 2 and 3) for Cxcl 10, and nontargeting negative control. Results(1) Immunohistochemistry and FCA with the antibody against Thy-1, a specific marker for fibroblasts, provided further evidence for the purity of primary sensory and motor fibroblasts.(2) We noted that 95 genes were significantly higher expressed in sensory fibroblasts than in motor fibroblasts, and 26 genes were significantly higher expressed in motor fibroblasts than in sensory fibroblasts.(3) The q PCR results showed that 7 genes, including Cft, Cxcl10, Cxcl3, Syk, C5 ar1, Pf4, Ptgs1 were higher expression in sensory fibroblasts versus motor fibroblasts.(4) Edu(red)/Hoechst(blue) immunostaining showed that the number of Ed U positive cells was significantly higher in sensory fibroblasts than that of motor fibroblasts, and the results of RTCA showed that the proliferation rate of sensory fibroblasts(5 K, 10 K, 20 K) was significantly faster than that of motor fibroblasts.(5) The transwell migration assay showed that after culture for 4 h or 24 h, the migration rate of sensory fibroblasts was higher than that of motor fibroblasts, and the results of RTCA showed that the migration rate of sensory fibroblasts(20 K, 40 K) was significantly faster than that of motor fibroblasts. Conclusion There are significant differences in fibroblasts between sensory and motor nerve in rats. The rate of proliferation and migration in sensory fibroblasts were higher than in motor fibroblasts.

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