本文選題：Gadd45b + RNA干擾��； 參考：《重慶醫(yī)科大學(xué)》2014年博士論文

【摘要】：背景與目的：缺血性腦卒中是世界范圍內(nèi)引起死亡和癱瘓的主要病因，并給家庭和社會造成沉重的經(jīng)濟(jì)和社會負(fù)擔(dān)。目前仍缺乏有效的治療藥物幫助腦卒中患者恢復(fù)受損的神經(jīng)功能。成年動物大腦損傷后腦組織神經(jīng)可塑性有限，腦卒中患者常遺留有明顯的神經(jīng)功能障礙。促進(jìn)大腦神經(jīng)可塑性是促進(jìn)缺血性腦損傷后神經(jīng)功能恢復(fù)的重要途徑。近年來多項研究發(fā)現(xiàn)生長抑制與DNA損傷修復(fù)基因b (growtharrest and DNA-damage inducible gene b, Gadd45b)可能為影響神經(jīng)可塑性相關(guān)基因，Gadd45b極有可能成為具有治療潛力的分子指標(biāo)。本研究探討調(diào)控內(nèi)源性Gadd45b表達(dá)對實驗性缺血性卒中后軸突可塑性和運動功能恢復(fù)的影響及其信號調(diào)控通路，同時研究小腦頂核電刺激對腦梗死后內(nèi)源性Gadd45b表達(dá)及其神經(jīng)功能恢復(fù)的影響，為腦梗死后神經(jīng)功能康復(fù)病理生理機(jī)制的了解和臨床干預(yù)提供理論基礎(chǔ)。 方法： 第一部分：構(gòu)建針對大鼠Gadd45b基因的慢病毒介導(dǎo)的RNA干擾載體，，并檢測該RNA干擾載體的轉(zhuǎn)染效率和干擾效率。將高、中、低三種不同滴度(劑量)的慢病毒載體和陰性對照病毒載體立體定向至大鼠腦組織。采用激光共聚焦顯微鏡觀察綠色熒光蛋白(green fluorescentprotein, GFP)的表達(dá)；采用熒光定量PCR (Q-PCR)檢測三種不同滴度慢病毒載體對大鼠腦組織Gadd45b mRNA的抑制效率。通過以上檢測篩選出慢病毒載體的最佳轉(zhuǎn)染滴度。 第二部分：采用大腦中動脈栓塞(middle cerebral artery occlusion,MCAO)建立成年雄性Sprague-Dawley (SD)大鼠實驗性缺血性卒中模型。通過立體定向腦內(nèi)注射生物素葡聚糖胺(biotinylated dextranamine, BDA)追蹤MCAO大鼠皮質(zhì)紅核束(corticorubral tract, CRT)的新生神經(jīng)軸突出芽。通過檢測生長相關(guān)蛋白43(Growth associatedprotein-43, GAP43)表達(dá)變化反映腦缺血后軸突再生。采用“樓梯測試”和“平衡木實驗”評價大鼠腦缺血后癱瘓前肢運動功能。我們采用RNA干擾技術(shù)抑制內(nèi)源性Gadd45b表達(dá)研究其對MCAO模型大鼠軸突可塑性和運動功能恢復(fù)的影響。 第三部分：采用MCAO建立成年大鼠實驗性缺血性卒中模型。采用RNA干擾抑制大鼠腦內(nèi)Gadd45b內(nèi)源性表達(dá)。通過免疫組化、Q-PCR和Western Blot分別檢測腦缺血后2天和14天腦源性神經(jīng)營養(yǎng)因子(brain-derived neurotrophic factor, BDNF)的免疫反應(yīng)、mRNA和蛋白表達(dá)水平。通過酶聯(lián)免疫吸附測定(enzyme-linked immunosorbent assay,ELISA)、免疫組化和Western Blot檢測大鼠腦缺血后2天和14天缺血側(cè)腦組織cAMP/PKA/pCREB信號通路表達(dá)水平。采用Q-PCR和Western Blot分別檢測腦缺血后2天和14天ROCK mRNA和蛋白表達(dá)水平。藉此探討Gadd45b調(diào)控軸突可塑性的信號調(diào)控通路。 第四部分：采用MCAO建立成年SD大鼠實驗性缺血性卒中模型。腦缺血再灌注(ischemia-reperfusion, I/R)后立即給予1小時小腦頂核電刺激(fastigial nucleus electrostimulation, FNS)研究其對大鼠腦缺血后Gadd45b表達(dá)和神經(jīng)功能恢復(fù)的影響。采用免疫組化、Q-PCR和Western blot檢測Gadd45b表達(dá)水平。藉此研究小腦頂核電刺激對腦梗死后Gadd45b表達(dá)變化的影響，探討小腦頂核電刺激應(yīng)用于臨床促進(jìn)腦梗死后功能康復(fù)的分子機(jī)制。 結(jié)果： 第一部分：成功構(gòu)建出針對大鼠Gadd45b基因的慢病毒介導(dǎo)的RNA干擾載體。激光共聚焦結(jié)果顯示，高、中、低三種不同劑量組大鼠皮層和海馬組織注射點周圍均出現(xiàn)GFP陽性表達(dá)。高滴度和中滴度組GFP陽性表達(dá)高而低滴度組GFP陽性表達(dá)較低。Q-PCR結(jié)果顯示，與正常對照組相比，高滴度和中滴度組Gadd45b mRNA表達(dá)下降超過70%(P0.05),高滴度和中滴度組之無明顯差異(P0.05)，低滴度組與正常對照組相比無明顯差異(P0.05)。 第二部分：免疫熒光結(jié)果顯示，與假手術(shù)組(sham)相比，腦缺血后2d和14d缺血側(cè)皮質(zhì)GAP43陽性細(xì)胞數(shù)明顯增加(P0.05)，陰性病毒對照組(LV-Control)與缺血組(MCAO)相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組(LV-shGadd45b)大鼠GAP43陽性細(xì)胞數(shù)則明顯下降(P0.05)。Q-PCR和Western blot結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血后2d和14d缺血側(cè)皮質(zhì)GAP43mRNA和蛋白水平明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組大鼠GAP43mRNA和蛋白水平明顯下降(P0.05)。BDA神經(jīng)示蹤結(jié)果顯示，與缺血組相比，Gadd45bRNA干擾組大鼠腦缺血后皮質(zhì)紅核束代償性的新生軸突明顯下降(P0.05)。與單純?nèi)毖M相比，Gadd45b-RNAi干預(yù)處理明顯抑制大鼠腦缺血后的運動功能恢復(fù)(P0.05)。 第三部分：免疫組化結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血后2d及14d缺血側(cè)皮質(zhì)BDNF陽性細(xì)胞數(shù)明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45bRNA干擾組大鼠BDNF陽性細(xì)胞數(shù)明顯下降(P0.05)。Q-PCR和Western blot結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血后2d和14d缺血側(cè)皮質(zhì)BDNF mRNA和蛋白水平明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組大鼠BDNF mRNA和蛋白水平明顯下降(P0.05)。免疫組化結(jié)果顯示，與假手術(shù)組相比，腦缺血后2d及14d缺血側(cè)皮質(zhì)PKA和pCREB陽性細(xì)胞數(shù)明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組大鼠PKA和pCREB陽性細(xì)胞數(shù)均明顯下降(P0.05)。 ELISA結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血再灌注損傷后2d和14d缺血側(cè)皮質(zhì)cAMP和PKA蛋白水平明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組大鼠cAMP和PKA蛋白水平明顯下降(P0.05)。Western blot結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血再灌注損傷后2d和14d缺血側(cè)皮質(zhì)pCREB蛋白水平明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組大鼠pCREB蛋白水平明顯下降(P0.05)。Q-PCR和Western blot結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血再灌注損傷后2d和14d缺血側(cè)皮質(zhì)ROCK mRNA和蛋白水平明顯增加(P0.05)，陰性病毒對照組與缺血組相比無明顯差異(P0.05)。與缺血組相比，Gadd45b RNA干擾組大鼠ROCK mRNA和蛋白水平進(jìn)一步明顯增加(P0.05)。 第四部分：免疫組化結(jié)果顯示，與假手術(shù)組相比，大鼠腦缺血后缺血側(cè)皮質(zhì)Gadd45b陽性細(xì)胞數(shù)在缺血后6h至3d均表達(dá)升高(P0.05)，缺血組和假刺激組相比無明顯差異(P0.05)。與缺血組相比，小腦頂核電刺激組大鼠缺血側(cè)皮質(zhì)Gadd45b陽性細(xì)胞數(shù)進(jìn)一步增加(P0.05)。Q-PCR和Western blot結(jié)果顯示，與假手術(shù)組相比，大鼠Gadd45b mRNA和蛋白水平在腦缺血再灌注后6h開始增加，24h達(dá)高峰，3d下降到比較低的水平但仍高于假手術(shù)組(P0.05)。與缺血組相比，小腦頂核電刺激組大鼠Gadd45bmRNA和蛋白水平在腦缺血后各時間點進(jìn)一步明顯增加(P0.05)。此外，與缺血組相比，小腦頂核電刺激組大鼠腦缺血損傷后神經(jīng)功能評分顯著下降(P0.05)。 結(jié)論：慢病毒載體能高效地轉(zhuǎn)染大鼠腦組織，有效地進(jìn)行大鼠Gadd45b特異性shRNA基因轉(zhuǎn)導(dǎo)。Gadd45b-RNAi有效抑制了大鼠腦組織內(nèi)源性Gadd45b表達(dá)。Gadd45b-RNAi干預(yù)處理同時還抑制cAMP/PKA/pCREB信號通路激活并促進(jìn)ROCK表達(dá)。因此，Gadd45b可通過調(diào)控BDNF-cAMP/PKA-CREB信號通路激活抑制ROCK通路，從而對腦缺血后的軸突可塑性發(fā)揮作用。腦缺血后電刺激小腦頂核可能是通過激活Gadd45b促進(jìn)功能恢復(fù)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R743.3

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