Experimental Study Of The Dorsal Root Ganglion (DRG) Neurons Transfected By LV-shCaV2.2e37a

N-type calcium channel of the dorsal root ganglion (DRG) neuron is an essential mediator in transmission of chronic pain. N-type calcium channel inhibitors routinely used can produce powerful analgesic effects, also cause side effects. CaV2.2e37a is expressed particularly in DRG. In our previous study,specific siRNA against CaV2.2e37a was designed, and the restriction of the whole CaV2.2e37a gene expressing by the siRNA was confirmed in 293FT cell. This project proposed to study on the transfection of siRNA carried by lentivirus vector in the dorsal root ganglion neurons and electrophysiology on the transfected DRG to provide evidence to the treatment of neuropathic pain based on the target of CaV2.2e37a.Part I The observation of the dorsal root ganglion (DRG) neurons transfected by LV-shCaV2.2e37aObjective:To establish a kind of cultural method of dorsal root ganglion (DRG) neurons for patch clamp study. To observe the dorsal root ganglion (DRG) neurons transfected by the lentiviral vector of RNA interference (RNAi) of CaV2.2e37a.Methods:DRG neurons were dissociated from newborn Sprague-Dawley rats. Animals were decapitated.The ganglia was removed from spinal with the help of dissecting microscope, then DRG was platted on glass coverslips in neurobasal supplemented with 5%fetal calf serum,100 U/mL penicillin and 0.1 mg/mL streptomycin. Cells were stored in CO2 incubator at 37℃and then for immunofluorescence test and electrophysiology experiments. Dishes were transfected with siRNA which contained green fluorescent protein (GFP). Results: Fluorescence was visualized in the cell cultured and Na+channel currents could be detected.Fluorescence of siRNA was also visualized in DRG neurons,the transfection efficiency is(40.09±2.81)%.Conclusions:The cultured DRG neurons of suckling rat are ideal for experiments. The siRNA can successfully transfected the DRG neurons.PartⅡThe observation of pain threshold alteration and transfection efficiency in CCI rats and electrophysiology research into the DRG neurons after intrathecal injection LV-shCaV2.2e37aObjective:The effects of intrathecal injection of LV-shCaV2.2e37a and transfection efficiency in CCI rats and the voltage-gated Ca2+channels of DRG after intrathecal injection of LV-shCaV2.2e37a were observed. Methods:Twelve healthy male SD rats were randomly divided into two groups (n=6). Normal group, and CCI group, In CCI group, rats were anesthetized,and then we exposed the right sciatic nerve and ligated the sciatic nerve with four gut sutures 1mm apart. In control group, the right sciatic nerve was exposed only,then closed the incision with no treatment. The variation of allodynia and thermal hyperalgesia were observed at stages of 1,3,5,7,9,14,19 days after operation. As we induced allodynia and thermal hyperalgesia,the construction of CCI model was confirmed. CCI Rats were anesthetized with chloral hydrate(10%) and received percutaneous intrathecal injections of 10μl LV-shCaV2.2e37a (1×109TU/mL),19 days after the operation. At various time points of 3d, lw,after intrathecal injection, pain threshold was tested. The rats were killed,26 days after operation.We removed the DRG neurons and dissociated them enzymatically and mechanically for electrophysiology research. Results:Both MWT and TWT of the CCI group were lower than the normal group rats(P<0.01),9 days after the operation. After 1w injection, pain threshold of rats in siRNA therapy group was higher than that of negative control group (P<0.05). Fluorescence of siRNA was visualized in DRG. Outward current could be detected from the transfected DRG. Conclusions:LV-shCaV2.2e37a can successfully transfect the DRG neurons through percutaneous intrathecal injection.Pain of CCI rats could be alleviated a week later after intrathecal injection of LV-shCaV2.2e37a. The current detected may be chloride channel current inhibited byCa2+。...