Rna Interference Suppression Of Trpv4 Expression And Function Of Rat Dorsal Root Ganglion Cells

Part one Effective transfection DRG cells TRPV4SiRNA concentration screeningObjective Detection and transmission of nociceptive stimuli in dorsal root ganglion(DRG) neurons is mediated by several types of ion channels,which regulate either Cellular excitability or synaptic transmission.TRPV4 in DRG was reported as mechanosensor or osmosensoro However,limited research was carried out on the function change of TRPV4 in DRG in response to RNA interference(RNAi).To establish a RNAi model of rat dorsal root ganglion neurons in vitro and observe the changes in appearance and activity in cultured rat dorsal root ganglion(DRG)neurons.Methods The spinal cord was isolated,and the ganglia were dissected. According to the pressure exerting time,the cultured DRG cells was assigned to three groups:group,group and normal control group,the changes of appearance and activity were demonstrated by fluorescent microscope,and MTT assay.In this study, the effect of tetrandrine on free calcium in dorsal root ganglion(DRG) cells was investigated.(and related SP and PKC production was also studied).The effect of Tet on substance P(SP) release and protein kinase C(PKC) production with differentiation density in DRG was also studied.Results Our data clearly demonstrated that TRPV4 siRNA were uptaken into DRG cells.There was no significant difference among groups under fluorescent microscope.In the siRNA group and mismatch group,compared with control group,the microscopy results of ultra-structure suggested that being cultured with SiRNA environment would influence the.No significant activity changes was detected by MTT method among these groups(P＞0.05).We demonstrated that SiRNA dose-dependently decreased enzyme-linked immunosorbent assay(ELISA)of SP activities of DRG cells.These results indicate that SiRNA effectively inhibits free calcium on DRG cells,suggesting that SiRNA could be used for abirritation. Conclusion DRG neuron could be used in SiRNA methods in vitro,we have the convenience to observe the function.The model would provide an effective tool for the studies of tTRPV4 funetion DRG neurons. Part two Inhibition of the TRPV4 expression by RNA interference in dorsal root ganglionBackground Transient receptor potential vanilloid 4(TRPV4) calcium channel is an essentialmediator of spinal noeiceptive transmission in dorsal root ganglion(DRG), but its mechanism in pain transmission is poorly understood.The present study intended to evaluate the effect of small interfering RNA(siRNA) on the expression and function of TRPV4 in DRG neurons and its possible usage in pain relief treatment. Methods Two pairs of siRNA targeting TRPV4(siRNAa and siRNAb) were designed and synthesized.A mismatched double-strand RNA was chosen as negative control siRNAs were transfected into primary culture DRG neurons with cationic lipid-based CodeBreaker reagent.Real-time reverse transcription polymerase chain reaction and Western blot analysis were performed to assess the change of TRPV4 messenger RNA (mRNA) and protein expression in DRG.Results TRPV4 mRNA levels in DRG of mismatch and control groups were significantly higher(3.88- and 3.75-fold) than normal However,siRNAa and siRNAb increased only 1.26- and 1.87-fold,respectively.TRPV4 protein expression was also decreased significantly,as indicated by Western blot.Conclusion designed siRNA inhibits mRNA level,protein expression and function of TRPV4 in DRG neurons.Therefore,synthesized siRNA is suggested to be a potential treatment for TRPV4-related pathologieal Part three Inhibition of the TRPV4 function by RNA interference in dorsal root ganglionBackground Although we have clarified that the TRPV4 siRNA can effectively inhibit TRPV4 mRNA and TRPV4 protein expression,we are interested in whether TRPV4 siRNA affects the function of TRPV4.Therefore,TRPV4 could be a potential therapeutic target for pharmaceutical interventions especially pain related with chronic compression11,12.However,limited research was carried out on the function change of TRPV4 in DRG in response to RNA interference(RNAi)Methods o we used a specific siRNA silencing approach to mediate a selective and localized knockdown of TRPV4.The aim of this study was to determine the influence of siRNA on TRPV4 function in DRG Calcium imaging and We also use ELISA of substance P were carried out to evaluate the function change of TRPV4.The relationship between TRPV4 and PKC was also observed by immunocytochemistry.Results Inhibition of TRPV4 activity was observed after transfection with both siRNA sequences.Protein kinase C expression,which was thought to be related with TRPV4 function,was inhibited as well.ConclusionTRPV4 is a key component in hypotonic signal transduction.