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Effects of Lentiviral Vector Mediated RNA Interference of Protein Kinase C Gamma on Morphine Tolerance in Rats

Author: LiuFeng
Tutor: TianYuKe
School: Huazhong University of Science and Technology
Course: Anesthesiology
Keywords: lentiviral vectors morphine tolerance PKCγ RNA interference
CLC: R96
Type: Master's thesis
Year: 2011
Downloads: 16
Quote: 0
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Abstract


Objective Protein kinase C gamma (PKCγ) is an important intracellular signaling molecule participating morphine tolerance. It can activate NMDA receptor and make mu receptor desensitization. This study observed effects of intrathecal injection of PKCγshort hairpin RNA lentivirus on expressions of PKCγand NMDA receptor and on acute and chronic morphine tolerance in rats, in order to explore new strategies of gene therapy of morphine tolerance. Methods PKCγshort hairpin RNA (shRNA) lentiviruses were packaged and concentrated. The lentiviral titer was detected by the method of 293T cells transfection. Intrathecal catheters were placed in male Sprgue-Dowley rats. Twelve rats were randomly divided into saline control group and experimental group with 6 rats in each, injected intrathecally by normal saline 10ul and PKCγshRNA lentivirus 10ul respectively. After a week, tail flick latency (TFL) was recorded while different doses of morphine were injected subcutaneously in rats. The analgesic effects of morphine of two groups were evaluated by maximum possible effects (%MPE) calculated. Acute and chronic morphine tolerance models were constructed in rats. Effects of PKCγsilencing on morphine tolerance in rats were evaluated by %MPE calculated by measuring the rat TFL. Spinal expressions of PKCγand NMDA receptor were detected by immunohistochemistry in rat chronic morphine tolerance. Results PKCγshRNA lentiviruses were packaged and concentrated successfully. The lentiviral titer was measured to be 1.25×109/ml. The difference of %MPE between experimental group and saline control group in morphine analgesic experiment was significant statistically (p<0.05). The analgesic effect of morphine in experimental group was increased. Acute and chronic morphine tolerance models were stable in rats. In acute morphine tolerance model, the %MPE of PKCγsilencing rats was increased significantly (p<0.05). In chronic morphine tolerance model, the %MPE of PKCγsilencing rats was also increased signifcantly (p<0.05). By immunohistochemical coloring, gray degree values of PKCγand NMDAR1 receptor of spinal cord slices in chronical morphine tolerance rats were increased significantly (p<0.05). And gray degree values of PKCγand NMDAR1 receptor of spinal cord slices in PKCγsilencing rats were decreased (p<0.05). Conclusions It can enhance the analgesic effect of morphine and can reduce acute andchronic morpine tolerance to inject PKCγshRNA lentiviruses intrathecally in rats.

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