Experimental Study Of Specific SiRNA In Treatment Of Neuropathic Pain Of CCI Rats Model

N-type calcium channel is a pharmacological key target in treatment of chronic pain. N-type calcium channel inhibitors of common practice always cause side effects, such as the function of nonceciptice comductine pathway and centrol or auto neuro system. N-type calcium channels (CaV2.2) have two isoforms, CaV2.2e37a and CaV2.2e37b. CaV2.2e37a expressed particularly in DRG. Specific siRNA against CaV2.2e37a was searched and designed in our previous study, and the siRNA could specifically restrain the whole CaV2.2e37a gene expressing in 293FT cell. This project proposed to study on the analgesia effects of siRNA mediated by lentiviral vector in neuropathic pain model of CCI rats. Side effects were also observed. This study could provide new evidence to relieve neuropathic pain based on the target of CaV2.2e37a.PartⅠConstruction and identification of lentiviral vector of siRNA of CaV2.2e37a geneObjective:The lentiviral vector of RNA interference (RNAi) of CaV2.2e37a gene was constructed to study the neuropathic pain by restrainning the CaV2.2e37a gene expressing. Methods:The effective sequence of siRNA targeting CaV2.2e37a gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLL3.7 vector by Hpa I and Xho I, which contained green fluorescent protein (GFP). The resulting vector containing CaV2.2e37a was confirmed by sequencing.293T cells were cotransfected with pLL3.7-CaV2.2e37a, pRsv-REV, pMDlg-pRRE and pMD2G. The titer of concentrated virus was tested by Real-time quantity PCR.Results:DNA sequence results showed that the RNAi vector of CaV2.2e37a (pLL3.7-CaV2.2e37a) was constructed successfully. The titer of concentrated virus was 1×109 Tu/ml. Conclusions:The lentivirus RNAi vector against CaV2.2 e37a was constructed successfully.PartⅡConstruction of CCI rats model and variation of CaV2.2 expression in CCI ratsObjective:Chronic constriction injury (CCI) rats pain model was constructed to investigate the variation of the expression of CaV2.2 in CCI rats" DRG. Methods:Twenty healthy male SD rats were randomly divided into two groups(n=10). In CCI group, rats were anesthetized with chloral hydrate (10%), the right sciatic nerve was exposed at mid-thigh level and four 4-0 chromic gut sutures were loosely ligated around the sciatic nerve approximately 1mm apart. The incision was closed with 4-0 silk suture. In control group, the right sciatic nerve was exposed as CCI group,then the incision was closed without any treatment. The variation of allodynia and thermal hyperalgesia were observed at stages of 3,7,10,15,20,30 and 40 days after ligation of the right sciatic nerve by Von-Frey filaments and CO2 laser. Successful rat model was set up when allodynia and thermal hyperalgesia was induced.40 days after the ligation of the sciatic nerve, rats were sacrificed by decapitation and the L4-6 dorsal root ganglias were dissected out on the ice. The variation of CaV2.2 mRNA and protein were tested by Real-time PCR and Western blotting. Results:The threshold of mechanical allodynia in right sides of rats in CCI group began to decrease 3 days after the ligation(P<0.01). Pain threshold came to peak 7 days after the ligation. The MWT and TWT of the CCI group rats were lower than the normal group rats(P<0.01). The mRNA level and protein level of CaV2.2 were significantly higher in CCI group than that of the control group (P<0.01). Conclusions:CCI pain model could be successfully constructed using chromic gut and it could be used for the research of neuropathic pain. Both of mRNA and protein expression level of CaV2.2 in CCI rats'DRG were increased, indicate that CaV2.2 is potentially involved in the neuropathic pain.Part III The observation of pain threshold alteration and side effects after intrathecal injection LV-shCaV2.2e37a in CCI ratsObjective:The effects and side effects of intrathecal injection of LV-shCaV2.2e37a were observed in CCI rats. Methods:Sixty-four healthy male rats were randomly divided into five groups, the siRNA therapy group and the active control group contained fourteen rats. Normal group, CCI group and the negative control group contained twelve seperately. In siRNA therapy group,CCI rats were intrathecally injected with 10μl LV-shCaV2.2e37a (1×109TU/mL) 7 days after the ligation of the sciatic nerve. In active control group, CCI rats were intrathecally injected with Ziconotide (600ng/d) 7 days after the ligation.In normal group was normal rats without any treatment. In CCI group were CCI rats without any other treatment. In negative control group,CCI rats were intrathecally injected with letiviral vector (1×109 TU/mL) without interfering sequence 7 days after the ligation. At various time points of 3d, 1w, 2w,3w,4w and 6w after intrathecal injection, pain threshold was tested. Two rats of the siRNA therapy group were sacrificed 1 week after intrathecal injection, L4-6 dorsal root ganglias were dissected which frozen sections observed by confocal laser scanning microscope. Rats were killed six weeks after injection. Heart, liver, lung, kidney, spine and spleen were taken out and HE staining slices of those organs were made to observe side effects of LV-shCaV2.2e37a intrathecal injection. Results:Pain threshold of rats in active control group was significantly higher than that of CCI group, siRNA therapy group and negative control group 3d after injection (p<0.01). After 1w injection, pain threshold of rats in siRNA therapy group was higher than that of CCI group and negative control group (P<0.01) while lower than that of normal group and active control group (P<0.01). Two weeks after injection, pain threshold of rats in siRNA therapy group was higher than that of CCI group and negative control group (P<0.01), had no significant difference with that of normal group and active control group (P>0.05). GFP was significantly expressed in DRG cells seven days after injection. No side effects of rats were observed after intrathecal injection of LV-shCaV2.2e37a. Conclusions:Pain of CCI rats could be alleviated for 6 weeks after intrathecal injection of LV-shCaV2.2e37a. Meanwhile, no side effects were observed throughout the whole process.