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Experimental Study of the Dorsal Root Ganglion (DRG) Neurons Transfected by LV-shCaV2.2e37a

Author: MaTao
Tutor: FengZeGuo
School: PLA Postgraduate Medical School
Course: Anesthesiology
Keywords: Dorsal root ganglion Neurons Cell culture Intrathecal injection Dorsal root ganglion neurons Small interfering RNA
CLC: R614
Type: Master's thesis
Year: 2011
Downloads: 23
Quote: 0
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Abstract


Dorsal root ganglion neurons N-type calcium channel peripheral neuralgia signaling has an important role in the regulation of N-type calcium channel blockers has been used in clinical trials either role in pain pathways, as well as on the central and the impact on the autonomic nervous system. The study found that the N-type the calcium channel CaV2.2e37a subtypes specifically expressed in DRG neurons. The research group in the previous study has been designed to filter out have a specific inhibitory effect of siRNA against CaV2.2e37a verified in 293T cells with the siRNA can specifically inhibit their cotransfected CaV2.2e37a length gene expressed. This study lentiviral vector carrying siRNA transfected primary dorsal root ganglion neurons, and observed the neuron cell transfection efficiency and cell transfection electrophysiological changes for further research and development in order to CaV2.2e37a target analgesic drugs provide experimental basis. Observed purpose: the first part the LV-shCaV2.2e37a transfected primary dorsal root ganglion neurons cultured in good condition, suitable for patch clamp studies in primary dorsal root ganglion neurons detect LV-shCaV2.2e37a The primary effect of transfection of dorsal root ganglion neurons. Methods: The primary dorsal root ganglion neurons in cell culture: SD rats with scissors decapitated newborn 24 hours after, under the microscope before with microscopic shear along the spinal ventral midline incision spine in spine both sides of the the foraminal visible dorsal root ganglion hand Surgery microscopic tweezers to remove the dorsal root ganglion. Remove excess nerve and the capsule will be organized to cut into a diameter of 0.2-0.3mm tile trumpet joined NB culture medium petri dishes. At 24h, 48h, recorded in the phase contrast microscope DRG changes in cell morphology. Dorsal root ganglion neurons in cell culture success primary cell immunofluorescence staining, clear cell source, the patch clamp detected in primary dorsal root ganglion neurons electrophysiological state. Use LV-shCaV2.2e37a transfection of primary cells of dorsal root ganglion neurons and observe the transfection efficiency. Results: cultured dorsal root ganglion neuron cells, immunostaining developing cell sodium current leads. Observed under a fluorescence microscope: LV-shCaV2.2e37a transfected primary dorsal root ganglion neurons hair green fluorescent transfection efficiency (40.09 ± 2.81)%. Conclusion: do not use the digestive enzymes, simple means of microsurgical instruments and anatomical microscope isolated dorsal root ganglion neurons and cultured, cultivate good condition, DRG neurons for electrophysiological studies and cell transfection studies, LV the-shCaV2.2e37a transfected with DRG neurons. Preliminary observation of the second part Intrathecal injection the LV-shCaV2.2e37a of the CCI rats analgesic effect of dorsal root ganglion neurons electrophysiological effects Objective: To study the intrathecal injection of LV-shCaV2.2e37a CCI rat model analgesic effect of transfection efficiency of DRG neurons transfected DRG neurons after electrophysiological changes. Methods: 12 healthy male SD rats were randomly divided into 2 groups, CCI group and the control group. Production CCI model, by observing the change with the control group of paresthesia and hyperalgesia threshold for statistical analysis. After the success of the model, the CCI group were randomly divided into two groups, the siRNA treatment group, negative control group. siRNA-treated group at 19 days after making the CCI model, intrathecal injection of LV-shCaV2.2e37a 10μl (1 × 109TU/mL); negative control group 19 days after making CCI model intrathecal injection of air lentiviral particles 10μl (1 × 109 TU / mL). intrathecal injection of the first three days, seven days to detect each group CCI rats mechanical the flinching reflex threshold (MWT) and C02 laser thermal withdrawal threshold (TWT). 7 days after the intrathecal injection, acutely isolated rat dorsal root ganglion cells were observed to LV-shCaV2.2e37a in the DRG neurons transfected cells was 3%. Patch-clamp technique was isolated rat dorsal root ganglion neurons current changes. Results: nine days after MWT and TWT peaked (P <0.01). The intrathecal injection 1w siRNA-treated rats compared with MWT and TWT - the time negative improve the control group (P <0.05). siRNA treatment group DRG neurons can be observed outward currents. Conclusion: intrathecal injection of LV-shCaV2.2e37a transfection to dorsal root ganglion neurons and allows CCI rats with mechanical allodynia flinching threshold (MWT) and CO2 laser thermal withdrawal reflex threshold (TWT ) were significantly increased (P lt; 0.05) siRNA treatment group DRG neurons leads to the nature of the outward current needs further study.

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