Gadd45b对大鼠脑缺血后轴突可塑性和运动功能恢复的调控机制研究

发布时间：2018-07-12 20:51

本文选题：Gadd45b + RNA干扰　；参考：《重庆医科大学》2014年博士论文

【摘要】：背景与目的：缺血性脑卒中是世界范围内引起死亡和瘫痪的主要病因，并给家庭和社会造成沉重的经济和社会负担。目前仍缺乏有效的治疗药物帮助脑卒中患者恢复受损的神经功能。成年动物大脑损伤后脑组织神经可塑性有限，脑卒中患者常遗留有明显的神经功能障碍。促进大脑神经可塑性是促进缺血性脑损伤后神经功能恢复的重要途径。近年来多项研究发现生长抑制与DNA损伤修复基因b (growtharrest and DNA-damage inducible gene b, Gadd45b)可能为影响神经可塑性相关基因，Gadd45b极有可能成为具有治疗潜力的分子指标。本研究探讨调控内源性Gadd45b表达对实验性缺血性卒中后轴突可塑性和运动功能恢复的影响及其信号调控通路，同时研究小脑顶核电刺激对脑梗死后内源性Gadd45b表达及其神经功能恢复的影响，为脑梗死后神经功能康复病理生理机制的了解和临床干预提供理论基础。方法：第一部分：构建针对大鼠Gadd45b基因的慢病毒介导的RNA干扰载体，，并检测该RNA干扰载体的转染效率和干扰效率。将高、中、低三种不同滴度(剂量)的慢病毒载体和阴性对照病毒载体立体定向至大鼠脑组织。采用激光共聚焦显微镜观察绿色荧光蛋白(green fluorescentprotein, GFP)的表达；采用荧光定量PCR (Q-PCR)检测三种不同滴度慢病毒载体对大鼠脑组织Gadd45b mRNA的抑制效率。通过以上检测筛选出慢病毒载体的最佳转染滴度。第二部分：采用大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)建立成年雄性Sprague-Dawley (SD)大鼠实验性缺血性卒中模型。通过立体定向脑内注射生物素葡聚糖胺(biotinylated dextranamine, BDA)追踪MCAO大鼠皮质红核束(corticorubral tract, CRT)的新生神经轴突出芽。通过检测生长相关蛋白43(Growth associatedprotein-43, GAP43)表达变化反映脑缺血后轴突再生。采用“楼梯测试”和“平衡木实验”评价大鼠脑缺血后瘫痪前肢运动功能。我们采用RNA干扰技术抑制内源性Gadd45b表达研究其对MCAO模型大鼠轴突可塑性和运动功能恢复的影响。第三部分：采用MCAO建立成年大鼠实验性缺血性卒中模型。采用RNA干扰抑制大鼠脑内Gadd45b内源性表达。通过免疫组化、Q-PCR和Western Blot分别检测脑缺血后2天和14天脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)的免疫反应、mRNA和蛋白表达水平。通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)、免疫组化和Western Blot检测大鼠脑缺血后2天和14天缺血侧脑组织cAMP/PKA/pCREB信号通路表达水平。采用Q-PCR和Western Blot分别检测脑缺血后2天和14天ROCK mRNA和蛋白表达水平。藉此探讨Gadd45b调控轴突可塑性的信号调控通路。第四部分：采用MCAO建立成年SD大鼠实验性缺血性卒中模型。脑缺血再灌注(ischemia-reperfusion, I/R)后立即给予1小时小脑顶核电刺激(fastigial nucleus electrostimulation, FNS)研究其对大鼠脑缺血后Gadd45b表达和神经功能恢复的影响。采用免疫组化、Q-PCR和Western blot检测Gadd45b表达水平。藉此研究小脑顶核电刺激对脑梗死后Gadd45b表达变化的影响，探讨小脑顶核电刺激应用于临床促进脑梗死后功能康复的分子机制。结果：第一部分：成功构建出针对大鼠Gadd45b基因的慢病毒介导的RNA干扰载体。激光共聚焦结果显示，高、中、低三种不同剂量组大鼠皮层和海马组织注射点周围均出现GFP阳性表达。高滴度和中滴度组GFP阳性表达高而低滴度组GFP阳性表达较低。Q-PCR结果显示，与正常对照组相比，高滴度和中滴度组Gadd45b mRNA表达下降超过70%(P0.05),高滴度和中滴度组之无明显差异(P0.05)，低滴度组与正常对照组相比无明显差异(P0.05)。第二部分：免疫荧光结果显示，与假手术组(sham)相比，脑缺血后2d和14d缺血侧皮质GAP43阳性细胞数明显增加(P0.05)，阴性病毒对照组(LV-Control)与缺血组(MCAO)相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组(LV-shGadd45b)大鼠GAP43阳性细胞数则明显下降(P0.05)。Q-PCR和Western blot结果显示，与假手术组相比，大鼠脑缺血后2d和14d缺血侧皮质GAP43mRNA和蛋白水平明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组大鼠GAP43mRNA和蛋白水平明显下降(P0.05)。BDA神经示踪结果显示，与缺血组相比，Gadd45bRNA干扰组大鼠脑缺血后皮质红核束代偿性的新生轴突明显下降(P0.05)。与单纯缺血组相比，Gadd45b-RNAi干预处理明显抑制大鼠脑缺血后的运动功能恢复(P0.05)。第三部分：免疫组化结果显示，与假手术组相比，大鼠脑缺血后2d及14d缺血侧皮质BDNF阳性细胞数明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45bRNA干扰组大鼠BDNF阳性细胞数明显下降(P0.05)。Q-PCR和Western blot结果显示，与假手术组相比，大鼠脑缺血后2d和14d缺血侧皮质BDNF mRNA和蛋白水平明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组大鼠BDNF mRNA和蛋白水平明显下降(P0.05)。免疫组化结果显示，与假手术组相比，脑缺血后2d及14d缺血侧皮质PKA和pCREB阳性细胞数明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组大鼠PKA和pCREB阳性细胞数均明显下降(P0.05)。 ELISA结果显示，与假手术组相比，大鼠脑缺血再灌注损伤后2d和14d缺血侧皮质cAMP和PKA蛋白水平明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组大鼠cAMP和PKA蛋白水平明显下降(P0.05)。Western blot结果显示，与假手术组相比，大鼠脑缺血再灌注损伤后2d和14d缺血侧皮质pCREB蛋白水平明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组大鼠pCREB蛋白水平明显下降(P0.05)。Q-PCR和Western blot结果显示，与假手术组相比，大鼠脑缺血再灌注损伤后2d和14d缺血侧皮质ROCK mRNA和蛋白水平明显增加(P0.05)，阴性病毒对照组与缺血组相比无明显差异(P0.05)。与缺血组相比，Gadd45b RNA干扰组大鼠ROCK mRNA和蛋白水平进一步明显增加(P0.05)。第四部分：免疫组化结果显示，与假手术组相比，大鼠脑缺血后缺血侧皮质Gadd45b阳性细胞数在缺血后6h至3d均表达升高(P0.05)，缺血组和假刺激组相比无明显差异(P0.05)。与缺血组相比，小脑顶核电刺激组大鼠缺血侧皮质Gadd45b阳性细胞数进一步增加(P0.05)。Q-PCR和Western blot结果显示，与假手术组相比，大鼠Gadd45b mRNA和蛋白水平在脑缺血再灌注后6h开始增加，24h达高峰，3d下降到比较低的水平但仍高于假手术组(P0.05)。与缺血组相比，小脑顶核电刺激组大鼠Gadd45bmRNA和蛋白水平在脑缺血后各时间点进一步明显增加(P0.05)。此外，与缺血组相比，小脑顶核电刺激组大鼠脑缺血损伤后神经功能评分显著下降(P0.05)。结论：慢病毒载体能高效地转染大鼠脑组织，有效地进行大鼠Gadd45b特异性shRNA基因转导。Gadd45b-RNAi有效抑制了大鼠脑组织内源性Gadd45b表达。Gadd45b-RNAi干预处理同时还抑制cAMP/PKA/pCREB信号通路激活并促进ROCK表达。因此，Gadd45b可通过调控BDNF-cAMP/PKA-CREB信号通路激活抑制ROCK通路，从而对脑缺血后的轴突可塑性发挥作用。脑缺血后电刺激小脑顶核可能是通过激活Gadd45b促进功能恢复。
[Abstract]:Background and purpose: ischemic stroke is the main cause of death and paralysis in the world and causes a heavy economic and social burden on family and society. There is still a lack of effective treatment drugs to help the stroke patients recover the damaged nerve function. The brain tissue of adult animal brain injury is limited and the cerebral apoplexy is limited. In recent years, a number of studies have found that growth inhibition and DNA damage repair gene B (growtharrest and DNA-damage inducible gene B, Gadd45b) may affect the neural plasticity related groups. In this study, the effects of endogenous Gadd45b expression on the axonal plasticity and motor function recovery after experimental ischemic stroke and the signal regulation pathway were investigated. The endogenous Gadd45b expression and neural function recovery after cerebral infarction were studied in this study. It provides a theoretical basis for understanding the pathophysiological mechanism and clinical intervention of neurological rehabilitation after cerebral infarction.
Method:
The first part: to construct the RNA interfering carrier mediated by lentivirus for rat Gadd45b gene, and to detect the transfection efficiency and interference efficiency of the RNA interference carrier. The high, middle and low three different titer (dose) lentivirus vectors and negative control virus vectors were stereospecific to the rat brain tissue. The green laser confocal microscope was used to observe the green color. The expression of green fluorescentprotein (GFP) and fluorescence quantitative PCR (Q-PCR) were used to detect the inhibition efficiency of three different titer lentivirus vectors to Gadd45b mRNA in rat brain tissue. The best transfection titer of lentivirus vector was screened by the above detection.
The second part: using middle cerebral artery occlusion (MCAO) to establish an experimental ischemic stroke model in adult male Sprague-Dawley (SD) rats. Through stereotactic intracerebral injection of biotin dextran amine (biotinylated dextranamine, BDA), the cortical red nucleus (Corticorubral) of MCAO rats was traced. Newborn neurite buds. The expression of growth related protein 43 (Growth associatedprotein-43, GAP43) was detected to reflect the axonal regeneration after cerebral ischemia. "Staircase test" and "balance wood experiment" were used to evaluate the motor function of the paralyzed forelimb after cerebral ischemia in rats. We used RNA interference to inhibit endogenous Gadd45b expression. Effects of MCAO on axonal plasticity and motor function recovery in rats.
The third part: the experimental ischemic stroke model of adult rats was established by MCAO. The endogenous expression of Gadd45b in the brain of rats was suppressed by RNA interference. The immunoreaction, mRNA and egg of the brain derived neurotrophic factor (brain-derived neurotrophic factor, BDNF) were detected by immunohistochemistry and Q-PCR and Western Blot respectively on the 2 and 14 days after cerebral ischemia. The level of white expression. Enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western Blot were used to detect the expression level of cAMP/PKA/pCREB signaling pathway in ischemic side brain tissue on 2 and 14 days after cerebral ischemia. Q-PCR and Western Blot were used to detect ROCK mRNA and protein expression water at 2 and 14 days after cerebral ischemia, respectively. To explore the signal regulatory pathway of Gadd45b regulating axonal plasticity.
The fourth part: the experimental ischemic stroke model of adult SD rats was established by MCAO. After cerebral ischemia reperfusion (ischemia-reperfusion, I/R), the effect of the 1 hour cerebellar nucleus stimulation (fastigial nucleus electrostimulation, FNS) on the expression of Gadd45b and the recovery of nerve function after cerebral ischemia in rats was studied. The expression level of Gadd45b was detected by Q-PCR and Western blot. The effect of cerebellar apex nuclear stimulation on the changes of Gadd45b expression after cerebral infarction was investigated, and the molecular mechanism of the application of the cerebellar nucleus stimulation to the function recovery after cerebral infarction was investigated.
Result:
The first part: successfully constructed the RNA interfering vector mediated by the rat Gadd45b gene. The laser confocal results showed that the GFP positive expression was found around the injection points of the cortex and hippocampus in the high, middle and low three different doses groups. The high titer and the middle titer group GFP positive expression was high and the low titer group GFP positive expression was lower.Q -PCR results showed that compared with the normal control group, the Gadd45b mRNA expression in the high titer and the middle titer group decreased by more than 70% (P0.05), and there was no significant difference between the high titer and the middle titer group (P0.05), and there was no significant difference between the low titer group and the normal control group (P0.05).
The second part: the results of immunofluorescence showed that compared with the sham group (sham), the number of GAP43 positive cells in the ischemic side cortex of 2D and 14d increased significantly after cerebral ischemia (P0.05), and there was no significant difference between the negative virus control group (LV-Control) and the ischemic group (MCAO). Compared with the blood deficiency group, the Gadd45b RNA interference group (LV-shGadd45b) rat GAP43 positive cells were compared. The P0.05.Q-PCR and Western blot results showed that compared with the sham group, the level of GAP43mRNA and protein in the ischemic side cortex of 2D and 14d increased significantly (P0.05), and the negative virus control group had no significant difference compared with the ischemic group (P0.05). Compared with the ischemic group, the GAP43mRNA and protein level of the Gadd45b RNA interference group was compared with the ischemic group. The significantly decreased (P0.05).BDA nerve tracer results showed that the compensatory axons of the cortical red nucleus in the Gadd45bRNA interference group decreased significantly compared with the ischemic group (P0.05). Compared with the ischemic group, the Gadd45b-RNAi intervention significantly inhibited the recovery of motor function after cerebral ischemia in rats (P0.05).
The third part: the results of immunohistochemistry showed that compared with the sham group, the number of BDNF positive cells in the ischemic side cortex of 2D and 14d increased significantly (P0.05), and the negative virus control group had no significant difference compared with the ischemic group (P0.05). The number of BDNF positive cells in the Gadd45bRNA group was significantly decreased (P0.05).Q-PCR and West compared with the ischemic group (P0.05). Ern blot results showed that compared with the sham group, the level of BDNF mRNA and protein in the ischemic side cortex of 2D and 14d increased significantly after cerebral ischemia in rats (P0.05). The negative virus control group had no significant difference compared with the ischemic group (P0.05). Compared with the ischemic group, the BDNF mRNA and protein levels in the Gadd45b RNA interference group were significantly decreased. Compared with the sham operation group, the number of PKA and pCREB positive cells in the ischemic side cortex of 2D and 14d increased significantly after cerebral ischemia (P0.05), and there was no significant difference between the negative virus control group and the ischemic group (P0.05). Compared with the ischemic group, the PKA and pCREB positive fine cell numbers of the Gadd45b RNA interference group were significantly decreased (P0.05). The ELISA results showed that the false operation was with the sham operation. The level of cAMP and PKA protein in the ischemic side cortex of 2D and 14d increased significantly after cerebral ischemia reperfusion injury in rats (P0.05), and there was no significant difference between the negative virus control group and the ischemic group (P0.05). Compared with the ischemic group, the cAMP and PKA protein levels of the Gadd45b RNA interference group decreased significantly (P0.05). Compared with the ischemic reperfusion injury, the level of pCREB protein in the ischemic side cortex of 2D and 14d increased significantly (P0.05), and the negative virus control group had no significant difference compared with the ischemic group (P0.05). Compared with the ischemic group, the level of pCREB protein in the Gadd45b RNA interference group was significantly decreased (P0.05).Q-PCR and Western blot results, compared with the sham operation group. The level of ROCK mRNA and protein in the ischemic side cortex of 2D and 14d increased significantly after cerebral ischemia reperfusion injury in rats (P0.05), and there was no significant difference between the negative virus control group and the ischemic group (P0.05). The ROCK mRNA and protein levels in the Gadd45b RNA interference group were significantly increased (P0.05) compared with the ischemic group.
The fourth part: the results of immunohistochemistry showed that compared with the sham group, the number of Gadd45b positive cells in the ischemic lateral cortex increased in 6h to 3D after ischemia in rats (P0.05), and there was no significant difference between the ischemic group and the pseudo stimulus group (P0.05). Compared with the ischemic group, the number of Gadd45b positive cells in the ischemic cortex of the cerebellar nucleus stimulation group was the number of Gadd45b positive cells. The further increase of (P0.05).Q-PCR and Western blot results showed that the Gadd45b mRNA and protein levels in the rats began to increase in 6h after cerebral ischemia and reperfusion, and the 24h reached a peak, and the 3D decreased to a lower level but still higher than that of the sham group (P0.05). The Gadd45bmRNA and protein levels of the rats in the cerebellar nucleus stimulation group were compared with the ischemic group. At each time point after cerebral ischemia, it was further increased (P0.05). In addition, compared with the ischemic group, the nerve function score of the rats after cerebral ischemia was significantly decreased (P0.05).
Conclusion: lentivirus vector can efficiently transfect rat brain tissue, effectively carry out Gadd45b specific shRNA gene transduction in rat.Gadd45b-RNAi effectively inhibit the endogenous Gadd45b expression.Gadd45b-RNAi intervention in rat brain tissue and inhibit the activation of cAMP/PKA/pCREB signaling pathway and promote ROCK expression. Therefore, Gadd45b can regulate B by regulating B. The activation of DNF-cAMP/PKA-CREB signaling pathway inhibits the ROCK pathway and plays a role in the plasticity of axon after cerebral ischemia. After cerebral ischemia, the electrical stimulation of the fastigial nucleus may promote functional recovery by activating Gadd45b.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

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