| Objective To observe the expression of Nav1.7in dorsal root ganglion (DRG) andactivation of microglia and astrocytes in a rat model of bone cancer pain.Methods30female SD rats weighing180～220g were randomly divided into3groups:Normal group(N, n=6), Sham-operated group(Sham, n=8),Model group(Ca, n=18).Shamand Ca groups were respectively injected10μl normal saline or Walker256breast cancercells(1×107/ml) into the left proximal tibial medullary cavity, while the N group receivedno treatment. On the day before inoculation and3d,6d,9d,12d after inoculation (AI), themechanical allodynia (mechanical withdrawal threshold, MWT) was measured. Rats in theN group and Sham group were executed on18d after inoculation. While rats in Ca groupwere executed on8d,12d and18d after inoculation,6rats respectively. The L4-6spinalcord and DRG was removed. The expression of Nav1.7in dorsal root ganglion and OX42(marker for microglia), glial fibrillary acidic protein (GFAP, marker for astrocytes) inspinal cord was analyzed by RT-PCR and Western blot method.Results The MWT of Rats with tibia tumors decreased from the6d AI with progressivesevere. The expression of Nav1.7mRNA and protein of the rats in Ca group significantlyincreased (P<0.05) on the8dAI, and further increased on the12dAI and18dAI (P<0.05).The expression of GFAP were also increased (P<0.05) on12d and18d. The levels ofmRNA and protein of ox42rose on8d and12d AI (P<0.05).Conclusion The expression of Nav1.7in dorsal root ganglion (DRG) increased in the ratmodel of bone cancer pain. The microglia and astrocytes in the correspondingsegments of the spinal cord were activated accordingly. Objective To construct and identify an effective lentiviral vector containing short hairpinRNA (shRNA) inhibiting rat Nav1.7gene by transfecting PC12cells in vitro.Methods Design and synthesis4shRNA expressing sequences for rat Nav1.7gene. Thenclone them into a lentiviral vector pGLV-U6-GFP respectively, thus4shRNA expressingvector (LV-4495,LV-390,LV-830and LV-3291) were generated. The effectiveness of thevectors was assessed by transfecting PC12cells with nerve growth factor (NGF) treatmentin vitro. Ninety-six hours after transfection, the cells were harvested for RT-PCR andwestern blot analysis of the expression of Nav1.7.Results All of the four shRNA expression vector could integrate into PC12cells with highefficiency. The transfection rate was85%detected by fluorescence microscope. Afterninety-six hours of transfection, LV-3291shRNA expressing vector has the bestinterference effect to block Nav1.7expression (P<0.05).Conclusion Lentiviral interference vector for rat Nav1.7shRNA has been constructedsuccessfully. LV-3291shRNA expressing vector could effectively transfect PC12cells andinhibit the expression of Nav1.7. Objective To observe the effects of intrathecal injection of lentivirus expressing shRNAinhibiting Nav1.7gene (LV-3291) on the rat model of bone cancer pain at its early period.Methods54female SD rats weighing180～220g were randomly divided into3groups:cancer pain+PBS (vehicle group, n=18), cancer pain+empty lentiviral vector with GFP(LV-GFP group, n=18),cancer pain+LV-3291vector (LV-Nav1.7group, n=18). Four daysafter inoculation, rats in vehicle group, LV-GFP group and LV-Nav1.7group received intrathecal injection of10μl PBS, empty lentiviral vector and LV-3291vector respectively.On the day before inoculation and3d,6d,9d,12d after inoculation, MWT was measured.Rats were executed on8d,12d and18d after inoculation,6rats respectively. The L4-6spinal cord and DRG was collected. The expression of Nav1.7, OX42and GFAP weredetermined by RT-PCR and Western blot method. Immunofluorescence technique was usedto detect the tansfection of lentiviral vector upon DRG neurons in vivo.Results Intrathecal injection of LV-3291on the4d AI could prevent the development ofbone cancer pain and still take effect on18d AI. The relative levels of Nav1.7, GFAP andOX42showed no difference between vehicle group and LV-GFP group. Compared withLV-GFP group, the expression of Nav1.7,GFAP, OX42mRNA and protein of the rat inLV-Nav1.7group significantly reduced on12d and18d AI (P<0.05)..Conclusion DRG neurons could be transfected by intrathecal injection of LV-3291.Thetreatment effectively attenuated bone cancer pain in the rat model by inhibit the expressionof Nav1.7on the DRG and the activation of microglia and astrocytes in the spinal cord. Objective To establish a morphine tolerance model in rats with bone cancer pain andexplore the effects of intrathecal injection of lentivirus expressing shRNA inhibitingNav1.7gene (LV-3291) on the model.Methods18female SD rats weighing180～220g received inoculation according toprevious method. Then the rats were treated with intrathecal injection of morphine10μg on6d AI, twice a day for consecutive7days. Morphine tolerance was accessed by heattail-flick latency (TFL) test. The rats with morphine tolerance were randomly allocated into3groups: vehicle group, LV-GFP group and LV-Nav1.7group,6rats each group. Thirteendays after inoculation, rats in vehicle group, LV-GFP group and LV-Nav1.7group were accordingly treated with intrathecal injection of10μl PBS, empty lentivirus vector andLV-3291vector. On the day before inoculation and6d,12d,18d and24d after inoculation,MWT was measured. Rats were executed on24d AI. The L4-6spinal cord and DRG washarvested. RT-PCR and Western blot technique were then performed to analyze theexpression of Nav1.7, OX42and GFAP.Results The TFL of the rats increased significantly after the first morphine injection(P<0.05). After continuous intrathecal injection of morphine,the average TFL afterinjection shortened gradually and returned to baseline value after the7th dayinjection．After intrathecal injection of lentivirus expressing shRNA inhibiting Nav1.7gene(LV-3291), the MWT of rats in the vehicle group and LV-GFP group showed no difference.The MWT of rats in the LV-Nav1.7group increased significantly on18d and24d AI(P<0.05). Compared with vehicle group, the expression of Nav1.7, GFAP and OX42mRNA and protein of the rat in LV-GFP group were not significantly different. Theexpression of Nav1.7and GFAP mRNA and protein of the rat in LV-Nav1.7groupdecreased apparently (P<0.05), while the expression of OX42showed no change.Conclusion Morphine tolerance was successfully induced by consecutive intrathecalinjection of morphine on the rats of bone cancer pain. The expression of Nav1.7on theDRG and the activation of microglia and astrocytes in the spinal cord could also besuppressed by intrathecal injection of LV-3291in the model. The treatment still workedwith obviously antinociceptic effect. |
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