摘要
研究背景:脑缺血发生后,尽早开通闭塞的血管,恢复血液灌流,是限制和缩小梗塞面积,改善预后的关键。但再灌注往往引起脑组织损伤的进一步加重,即再灌注损伤,导致神经细胞坏死或凋亡,神经功能障碍。
超早期溶栓是目前治疗脑缺血唯一有效的措施,但太短的治疗时间窗及再灌注损伤使其临床应用受到限制;降低脑组织代谢、各种神经保护剂(自由基清除剂、钙通道阻断剂等)虽然可以在一定程度上降低患者的死亡率,但存在临床疗效不确定,不良反应严重等问题,且对于改善患者的神经功能症状,效果并不理想,究其原因是并没有从根本上解决神经细胞数量减少的问题。
干细胞移植治疗脑缺血受到研究者的关注。移植的神经干细胞可在缺血脑组织局部迁移、增殖,并分化成神经元部分地替代和修复受损的神经元,从而在一定程度上改善神经功能缺失症状;然而伦理学问题、来源困难及移植后的免疫排斥反应等因素限制了其临床应用。MSCs作为目前研究最广泛的成体干细胞进入了人们的视线;但MSCs在体内向神经细胞的转化效率极低及体外扩增后的成瘤风险等,使人们不得不重新审视MSCs移植在脑缺血治疗中的作用及安全性。
因此,我们迫切需要寻找新的治疗脑缺血再灌注损伤的方法,以保护和修复受损神经细胞的功能。细胞因子因其获得简便、无创伤、且有潜在的抗神经细胞凋亡和促进神经再生等作用而进入研究者的视线。
粒细胞集落刺激因子(G-CSF)是粒系造血生长因子,用于各种原因引起的中性粒细胞减少症以及动员外周血干细胞移植治疗免疫性疾病。最近研究显示,脑缺血时,G-CSF能减少梗塞面积,促进神经功能恢复,可能的机制为:1、促进神经再生;2、促进神经营养因子的分泌;3、抑制细胞凋亡;4、抑制炎症反应;5、促进血管发生等。
促红细胞生成素(EPO)是一种刺激骨髓造血的糖蛋白类激素,主要由成人肾脏和胎肝合成,用于各种原因引起的贫血,主要是肾性贫血的治疗。随着研究的深入,发现EPO还通过非造血相关效应在缺血所致脑损伤中发挥神经保护作用,可能通过:1、抑制细胞凋亡;2、减轻炎症反应;3、减少兴奋性氨基酸的毒性作用;4、抗氧化应激;5、促进神经和血管发生等。
目前认为脑缺血的发病机制复杂,是多种机制、多个环节共同作用的结果,单靠某一种药物治疗,并不能达到最好的治疗效果,联合应用作用于脑缺血后不同环节的神经保护剂,进行多靶点、多环节的综合治疗,成为脑缺血神经保护研究的新方向。
由于脑组织的功能极大程度上依赖于神经元的数量和质量,因此,如果采取措施,使缺血早期神经细胞的凋亡减少,最大限度的保存神经细胞的数量;同时缺血后期使神经细胞的再生增加,促进神经细胞的增殖、分化,必将有利于脑缺血再灌注损伤后的组织修复和功能恢复。
因此,我们首次尝试将G-CSF和EPO联用,以细胞凋亡和神经再生为切入点,观察联合治疗对脑缺血再灌注损伤的保护作用。本课题采用乳鼠皮质神经元氧糖剥夺复糖、复氧模型和局灶性脑缺血再灌注大鼠模型,研究G-CSF联合EPO治疗是否具有防治脑缺血再灌注损伤,促进神经功能恢复的效应,并探讨其可能的作用机制。本实验内容可归纳成四部分,概述如下:
第一部分粒细胞集落刺激因子联合促红细胞生成素对乳鼠皮质神经元氧糖剥夺模型的保护作用
目的:利用体外培养的乳鼠皮质神经元制备氧糖剥夺复糖、复氧(OGD/R)模型,观察G-CSF和EPO单独或联合治疗对皮质神经元OGD/R后的保护作用。
方法:体外培养乳鼠皮质神经元,建立氧糖剥夺(4h)复糖、复氧(24h)模型;研究不同浓度的G-CSF和EPO对皮质神经元的保护作用;研究最佳治疗浓度时,G-CSF和EPO单独或联合治疗对皮质神经元的保护作用,通过CCK-8分析测定细胞活力、LDH漏出量检测细胞损伤程度、AnnexinV/PI法测定细胞凋亡率、Western-blot法检测凋亡相关蛋白Bcl-2、Bax、Caspase-3的表达。
结果:
1、成功制备了乳鼠皮质神经元氧糖剥夺复糖、复氧(OGD/R)模型。
2、G-CSF 0.1μg/ml、1μg/ml和10μg/ml处理组均可提高细胞活力、降低LDH漏出量,1μg/ml保护作用最强,10μg/ml保护作用略有下降。
3、EPO 1U/ml、10U/ml和100U/ml处理组均可提高细胞活力、降低LDH漏出量,10U/ml和100U/ml保护作用相似。
4、G-CSF和EPO联合治疗组较各单独治疗组明显提高细胞活力、降低LDH漏出量、减少细胞凋亡率(P <0.05);与G-CSF比较,EPO的干预明显提高细胞活力、降低LDH漏出量、减少细胞凋亡率,且有统计学差异(P <0.05)。
5、G-CSF和EPO联合治疗组较各单独治疗组明显增加皮质神经元Bcl-2表达、降低Caspase-3表达;与G-CSF比较,EPO的干预使Bcl-2表达增加、Caspase-3表达降低更显著(P <0.05);G-CSF和EPO单独或联合治疗均可降低Bax表达,但三个治疗组间无明显差异。
结论:G-CSF可减少皮质神经元OGD/R后的细胞损伤,最佳治疗浓度为1μg/ml。EPO可减少皮质神经元OGD/R后的细胞损伤,最佳治疗浓度为10U/ml。G-CSF联合EPO明显抑制皮质神经元OGD/R后的细胞损伤,减少细胞凋亡率,增加Bcl-2的表达、降低Bax、Caspase-3的表达。EPO干预使皮质神经元凋亡减轻更明显。
第二部分粒细胞集落刺激因子联合促红细胞生成素对脑缺血再灌注大鼠的保护作用
目的:通过建立局灶性脑缺血再灌注大鼠模型,在整体水平上,观察G-CSF和EPO单独或联合治疗对大鼠脑缺血再灌注后的保护作用,对联合治疗的有效性及安全性进行评价。
方法:制备大脑中动脉阻断、局灶性脑缺血再灌注大鼠(I/R)模型,给予G-CSF和EPO单独或联合治疗,共5天;分别于再灌注的相应时间点进行神经功能评分、TTC染色检测梗塞面积、HE染色检测脑组织的病理改变、干湿法测定脑组织含水量、检测外周血象,实验结束时计算各组大鼠的死亡率。
结果:
1、成功制备局灶性脑缺血再灌注大鼠模型,制模成功率为73.9%。
2、再灌注3d、7d、14d时三个治疗组神经功能评分均高于I/R组,差异有显著性( P <0.05);7d、14d时,联合治疗组较各单独治疗组神经功能评分明显增加,均有统计学差异( P <0.05);各时间点G-CSF组和EPO组比较,神经功能评分均无明显差异。
3、脑梗塞灶主要位于大脑皮质及基底节区;再灌注3d、7d时,三个治疗组梗塞面积均明显小于I/R组;联合治疗组较各单独治疗组梗塞面积明显缩小,且有统计学差异( P <0.05);各时间点G-CSF组和EPO组比较,梗塞面积均无明显差异。
4、HE染色可见I/R组脑组织水肿明显,神经细胞稀疏、间隙增大、体积变小,胞核固缩,核仁不明显,缺血中心区可见核碎裂、核溶解等细胞坏死改变、出现软化坏死灶;各治疗组神经细胞损伤明显减轻,细胞坏死有所减少、组织水肿减轻。
5、I/R组大鼠于再灌注后体重明显下降,7d时体重出现回升,以后持续增加;再灌注1d、3d时三个治疗组与I/R组比较体重无明显差异,7d、14d时,三个治疗组与I/R组比较体重均有所增加(P <0.05);各时间点三个治疗组间大鼠体重均无明显差异。
6、I/R组大鼠死亡率为31.91%;三个治疗组大鼠死亡率分别为20.0%、17.94%、17.94%;三个治疗组均可降低大鼠的死亡率(P <0.05),但三个治疗组间大鼠死亡率无明显差异。
7、再灌注1d、3d时三个治疗组与I/R组比较脑组织含水量无明显差异,7d时三个治疗组均明显减少脑组织含水量( P <0.05);各时间点三个治疗组间脑组织含水量均无明显差异。
8、各组大鼠各时间点HB、RBC、PLT均无差异;I/R组再灌注1d时WBC开始增加,7d时达到高峰,14d时有所下降;各时间点I/R组和三个治疗组间、三个治疗组间WBC均无显著性差异(P >0.05),但均显著高于对照组(P <0.05)。
结论:G-CSF联合EPO治疗明显改善大鼠的神经功能症状、减少梗塞面积、减轻脑组织病理改变、降低大鼠死亡率,对大鼠脑缺血再灌注损伤有保护作用。短期内联合使用G-CSF和EPO,对大鼠的血液指标未见显著影响,所用药物剂量安全。
第三部分粒细胞集落刺激因子联合促红细胞生成素对脑缺血再灌注大鼠神经细胞凋亡影响的研究
目的:观察G-CSF和EPO单独或联合治疗对脑缺血再灌注大鼠神经细胞凋亡的影响及其可能的机制。
方法:分别于再灌注1d、3d、7d时,TUNEL染色检测各组大鼠缺血周围脑组织凋亡神经细胞数;免疫组化法检测凋亡相关蛋白Bcl-2、Bax、Caspase-3的表达;Western-blot法检测p-Akt活性。
结果:
1、I/R组1d时出现大量凋亡细胞,3d时凋亡细胞数达高峰,7d时下降;再灌注1d时三个治疗组与I/R组比较凋亡细胞数无明显差异;3d、7d时三个治疗组凋亡细胞数均明显低于I/R组,联合治疗组较各单独治疗组凋亡细胞数明显减少,均有统计学差异( P <0.05);再灌注3d、7d时,与G-CSF组比较,EPO组凋亡细胞数明显减少(均P <0.05)。
2、G-CSF和EPO联合治疗组较各单独治疗组均可明显增加脑组织Bcl-2的表达、降低Caspase-3的表达;与G-CSF组比较,EPO组Bcl-2表达增加、Caspase-3表达降低更明显,且有统计学差异(P <0.05);与I/R组比较,G-CSF和EPO单独或联合治疗均可降低脑组织Bax表达,但三个治疗组间Bax表达无明显差异。
3、I/R组脑组织p-Akt活性明显升高;与I/R组比较,三个治疗组均可增加p-Akt的活性,联合治疗组较各单独治疗组p-Akt的活性明显增加(P <0.05);G-CSF组与EPO组比较, p-Akt活性无明显差异。
结论:G-CSF联合EPO治疗显著减少缺血再灌注引起的神经细胞凋亡,增加缺血周围脑组织Bcl-2的表达、抑制Bax、Caspase-3的表达;联合治疗可能部分通过激活PI-3K/Akt途径发挥协同的抗凋亡作用。EPO在抑制神经细胞凋亡方面作用显著。
第四部分粒细胞集落刺激因子联合促红细胞生成素对脑缺血再灌注大鼠神经再生作用的研究
目的:观察G-CSF和EPO单独或联合治疗对脑缺血再灌注大鼠神经细胞增殖、分化及神经营养因子表达的影响。
方法:通过Brdu标记增殖细胞,分别于再灌注1d、3d、7d、14d时,采用荧光单染法检测各组大鼠缺血周围脑组织Brdu阳性细胞数;免疫荧光双标法检测Brdu/NeuN、Brdu/GFAP双阳性细胞数;RT-PCR法检测神经营养因子BDNFmRNA、NGFmRNA的表达。
结果:
1、I/R组1d时可见散在的BrdU阳性细胞,7d时阳性细胞数达高峰,14d时下降;再灌注1d、3d时三个治疗组间及与I/R组间BrdU阳性细胞数均无明显差异;7d、14d时三个治疗组BrdU阳性细胞数均高于I/R组,联合治疗组较各单独治疗组BrdU阳性细胞数明显增加(P <0.05);再灌注7d、14d时,与EPO组比较,G-CSF组BrdU阳性细胞数明显增加(均P <0.05)。
2、I/R组可见少量BrdU/NeuN和BrdU/GFAP双阳性细胞;三个治疗组BrdU/NeuN和BrdU/GFAP双阳性细胞数均明显高于I/R组;联合治疗组较各单独治疗组BrdU/NeuN和BrdU/GFAP双阳性细胞数均明显增加,且有统计学差异(P <0.05);与EPO组比较,G-CSF组BrdU/NeuN和BrdU/GFAP双阳性细胞数均明显增加( P <0.05)。
3、I/R组脑组织中BDNFmRNA表达明显增加;与I/R组比较,三个治疗组BDNFmRNA表达均明显升高(P <0.05);但三个治疗组间表达无明显差异。
4、I/R组脑组织中NGFmRNA表达明显增加;与I/R组比较,三个治疗组NGFmRNA表达均明显增加(P <0.05);联合治疗组较各单独治疗组NGFmRNA表达增加显著;与EPO组比较,G-CSF组NGFmRNA表达有显著增加(P <0.05)。
结论:G-CSF联合EPO治疗明显促进大鼠缺血周围脑组织神经细胞的增殖,及向神经元和神经胶质细胞发生分化,同时促进神经营养因子BDNFmRNA、NGFmRNA的表达增加。G-CSF在促进神经细胞再生方面作用显著。
Background: When cerebral ischemia occurred, it is important for confining or deflating infarct volume and improving prognosis to remove obstacles from obstructive blood vessel and to recuperate hemoperfusion as early as possible. But reperfusion often aggravates brain tissue damage, that is reperfusion injury, which can induce neurocyte necrosis or apoptosis and nerve functional disturbance.
Although supra-pristine thrombolysis, lowering brain tissue metabolism, various kinds of nerve protectants, etc may cut down the fatality of patients to some degree, the effect of improving the symptom of patients nerve function is not ideal, and problems such as transient time window of therapy and severe adverse reaction are present. The reason is that decreasing neurocyte is not avoided fundamentally.
It is followed with interest by researchers to treat cerebral ischemia with stem cell transplantation. The transplanted nerve stem cells can migrate and proliferate in the ischemia brain tissue area. They also can differentiate to partly substitute and recover impaired neurons, thus to improve the symptom of loss of nerve function. But problems such as ethics issue, difficult source and immunological rejection, etc limit the clinical application of stem cell transplantation. MSCs are the most widely researched stem cells. But the low transformation of MSCs into nerve cells and the risk of becoming tumour after extraorgan amplification made the effect and safety of MSCs transplantation for cerebral ischemia be suspected.
Therefore original therapy means for cerebral ischemia-reperfusion injury are needed badly to preserve and recover the function of impaired nerve cells. For the reason that the function of brain tissue relies on the quantity and quality of neurocyte to great extent, if we adopt a measure which include diminishing apoptosis of early ischemia nerve cells, preserving the quantity of neurocyte maximatily, meanwhile increasing the regeneration of postischemia neurocyte and promoting the proliferation and differentiation of nerve cells, the tissue repair and functional recovery after ischemia-reperfusion injury will profit from it inevitably. Cytokine is followed with interest by researchers because of its convenient availability, non-insult and potential effect of diminishing apoptosis and increasing the regeneration of nerve cells.
Granulocyte-colony stimulating factor (G-CSF), which is granulocyte-colony hemopoietic growth factor, is used for various kinds of Neutropenia and autologous peripheral blood stem cell transplantation for immune diseases. Recent research showed that when cerebral ischemia happened G-CSF can deflate infarct volume and facilitate nerval function recovery. The possible mechanisms are : 1, promoting nerval regeneration; 2, promoting secretion of neurotrophic factor; 3, inhibiting cell apoptosis; 4, inhibiting inflammatory reaction; 5, promoting angiogenesis.
Erythropoietin (EPO), which is a kind of glucoprotein hormone which can stimulate bone marrow hemopoiesis, is mainly produced by adult kidney and fetus liver. EPO is used for various kinds of anaemia, especially renal anaemia. Along with the developing of research, EPO is discovered to have neuroprotective effect in brain injury induced by ischemia through non-haemopoiesis correlated effect. The possible mechanisms are : 1, inhibiting cell apoptosis; 2, inhibiting inflammatory reaction; 3, diminishing the toxic reaction of excitatory amino acids; 4, resisting oxidization; 5, promoting neurogenesis and angiogenesis.
Pathogenesy of cerebral ischemia is now considered complicated. It may be a result induced by the combined effects of multiple mechanisms. The best therapeutic efficacy can not be achieved through certain single drug treatment. It has become a new direction of research of cerebral ischemia nerve protection to combine different neuro-protectants, which have effect in different link after cerebral ischemia, to undertake multi-target and multi-link combined therapy.
Therefore we attempted to combine two kinds of cytokines, which are different in structure and function, to treat cerebral ischemia-reperfusion injury. In this study, we used the model of oxygen glucose deprivation/reoxygenation of neurons of rats and the model of regional cerebral ischemia-reperfusion of rats, studied the protection effect of combining G-CSF and EPO to treat cerebral ischemia-reperfusion injury in two aspects including cell apoptosis and neural regeneration, and investigated the possible mechanism of action. This experiment content can be divided into four parts, which are summarized as follow:
Part One Protection of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin for the Model of Oxygen Glucose Deprivation of Neurons of Rats
Objective: To establish the model of oxygen glucose deprivation/reoxygenation (OGD/R) of cortical neuron of neonate rats in vitro culture, and to observe the protection of therapy of G-CSF or EPO only or G-CSF combined with EPO.
Method: Cortical neuron of neonate rats were cultured in vitro, oxygen glucose deprivation (4h)/reoxygenation (24h) were established; G-CSF and EPO were used in accordance with different concentration to study the protection for cortical neuron; optimized therapeutic strength was determined for the protection for cortical neuron which was gained by using G-CSF only or G-CSF combined with EPO; CCK-8 assay was used to determine cell viability and LDH outleakage was detected to measure the degree of cell injury; apoptosis rate was detected by means of Annexin V/PI method and the expression of apoptosis-associated protein such as Bcl-2, Bax and Caspase-3 was detected by means of Western-blot method.
Results:
1. The model of oxygen glucose deprivation/reoxygenation (OGD/R) of cortical neuron of neonate rats was established successfully.
2. Treatment with G-CSF of 0.1μg/ml、1μg/ml and 10μg/ml can enhance cell viability,reduce LDH outleakage; the protection of G-CSF(1μg/ml subgroup) was strongest and G-CSF(10μg/ml subgroup) was a little lower.
3. Treatment with EPO of 1U/ml、10U/ml and 100U/ml can enhance cell viability,reduce LDH outleakage; the protection of EPO(10U/ml subgroup) was similar to EPO(100U/ml subgroup).
4. Treatment with G-CSF combined with EPO, compared with treatment with G-CSF or EPO only, obviously enhanced cell viability,reduced LDH outleakage and apoptosis rate (P<0.05). In comparison with G-CSF, EPO intervention obviously enhanced cell viability,reduced LDH outleakage and apoptosis rate (P<0.05).
5. Treatment with G-CSF combined with EPO, compared with treatment with G-CSF or EPO only, obviously augmented the expression of Bcl-2, depressed the expression of Caspase-3. In comparison with G-CSF, EPO intervention obviously augmented the expression of Bcl-2, depressed the expression of Caspase-3 (P<0.05). Treatment with G-CSF or EPO only or G-CSF combined with EPO depressed the expression of Bax, but there was no significant difference in the three subgroups.
Conclusions: G-CSF can lessen the injury of cortical neuron after OGD/R, the optimal treatment concentration is 1μg/ml. EPO can lessen the injury of cortical neuron after OGD/R, the optimal treatment concentration is 10U/ml. G-CSF combined with EPO can obviously inhibit cortical neuron apoptosis after OGD/R, augment the expression of Bcl-2, depress the expression of Bax and Caspase-3. EPO intervention can inhibit cortical neuron apoptosis obviously.
Part Two Protection of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin for the cerebral ischemia-reperfusion in Rats
Objective: To establish the model of focal cerebral ischemia-reperfusion in rats, at the whole level to observe the protection of therapy of G-CSF or EPO only or G-CSF combined with EPO against the cerebral ischemia-reperfusion in rats, and to evaluate the validity and safety of therapeutic alliance.
Method: The model of focal cerebral ischemia-reperfusion in rats was established by middle cerebral artery occlusion. Therapy of G-CSF or EPO only or G-CSF combined with EPO was administered for 5 days. After reperfusion for corresponding time, the neurological function score was evaluated, the infarct volume was measured with TTC stain, the brain tissue pathological changes were observed with HE stain, the brain edema was detected with dry-wet weight method, the peripheral hemogram was determined, and when the experiment completed the fatality of each group was calculated.
Results:
1. The model of focal cerebral ischemia-reperfusion in rats was established successfully, and the success rate of mold is 73.9%.
2. The neurological function score of the three treatment groups after reperfusion for 3d, 7d,and 14d obviously exceeded that of the I/R group, and the difference was significant(P<0.05). Compared to G-CSF or EPO group, the neurological function score was significantly higher in G-CSF+EPO group (P<0.05). There was no significant difference, after each corresponding time, in the neurological function score of G-CSF group compared with that of EPO group.
3. The cerebral infarction focus was mainly situated in cerebral cortex and basal ganglia area. After reperfusion for 3d and 7d, the infarct volume of the three treatment groups was significantly reduced compared to the I/R group. The infarct volume of G-CSF+EPO group was obviously reduced compared to that of the G-CSF or EPO group, which has significant difference (P<0.05 repectively). There was no significant difference, after each corresponding time, in the infarct volume of G-CSF group compared with that of EPO group.
4. In I/R group, tissue HE staining showed that the brain tissue edema was obvious, the neurocyte was rarefied, the inter-space between cells became wide, the cell volume became small, the nucleus was pyknotic, the chromatospherite became not clear, cell necrosis changes such as nuclear fragmentation and caryolysis etc appeared in the center of ischemia. In each treatment group, neurocyte damage was obviously lessened, cell necrosis was reduced, and tissue edema was improved.
5. In I/R group, the body weight of rats was reduced obviously, after 7d the weight appeared to increase and afterward continued to increase. After reperfusion for 1d and 3d, there was no significant difference in the weight of the three treatment groups compared with that of the I/R group. After reperfusion for 7d and 14d, the weight of the three treatment groups increased significantly compared to that of the I/R group (P<0.05). There was no significant difference, after each corresponding time, in the body weight of the three treatment groups when compared with each other.
6. The fatality of the I/R group was 31.91%. The fatality of the three treatment group (G-CSF group 20.0%, EPO group 17.94% and G-CSF+EPO group 17.94%) was significantly reduced compared to that of the I/R group (P<0.05), but there was no significant difference in the fatality of the three treatment groups when compared with each other.
7. There was no significant difference in the water content of brain tissue of the three treatment groups after reperfusion for 1d, and 3d compared with that of the I/R group. After reperfusion for 7d, the water content of brain tissue of the three treatment groups was reduced significantly compared to that of the I/R group (P<0.05). There was no significant difference, after each corresponding time, in the water content of brain tissue of the three treatment groups when compared with each other.
8. There was no significant difference, after each corresponding time, in HB, RBC and PLT of each group when compared with each other. The WBC of I/R group began to increase after 1d, peaked after 7d and then began to decline after 14d. There was no significant difference, after each corresponding time, in the WBC of the three treatment groups when compared with each other (P>0.05); but the WBC of each treatment group was obviously more than that of control group (P<0.05).
Conclusions: Therapy of G-CSF combined with EPO can obviously improve the neurological function symptoms of rats, reduce the infarct volume, lessen the pathological changes of brain tissue, reduce the fatality of rats and has protection against cerebral ischemia-reperfusion injury. Temporarily combining G-CSF with EPO does not has obvious effect on the blood parameters, and the dosage of the used drug is safe.
Part Three Research of the effect of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin on the neurocyte apoptosis of the cerebral ischemia-reperfusion in Rats
Objective: To observe the effect of G-CSF or EPO only or combined treatment on the neurocyte apoptosis of the cerebral ischemia-reperfusion in rats and its mechanism.
Method: After each corresponding time of reperfusion, the quantity of neurocyte apoptosis in the ischemia brain tissue of rats was determined by tissue TUNEL staining. The expression of apoptosis-associated protein such as Bcl-2, Bax and Caspase-3 was detected by means of Immunohistochemistry method. The activity of p-Akt was detected by means of Western-blot method.
Results:
1. The cell apoptosis of I/R group occurred considerably after 1d, peaked after 3d and then began to decline after 7d. There was no significant difference in the cell apoptosis quantity of the three treatment groups after reperfusion for 1d compared with that of the I/R group. After reperfusion for 3d and 7d, the cell apoptosis quantity of the three treatment groups was reduced significantly compared to that of the I/R group (P<0.05), and the cell apoptosis quantity of G-CSF+EPO group was obviously reduced compared to that of the G-CSF or EPO group, which has significant difference (P<0.05 respectively). After reperfusion for 3d and 7d, the cell apoptosis quantity of the EPO group was reduced obviously compared to that of the G-CSF group (P<0.05 respectively).
2. The expression of Bcl-2 and Caspase-3 of the G-CSF+EPO group was obviously augmented and obviouly depressed respectively compared to that of the G-CSF group or EPO group. The expression of Bcl-2 and Caspase-3 of the EPO group was obviously augmented and obviouly depressed respectively compared to that of the G-CSF group, which has significant difference (P<0.05). Compared with I/R group, the expression of Bax of G-CSF group, EPO group and G-CSF+EPO group was depressed, but there was no significant differance in the expression of Bax of the three treatment groups when compared with each other.
3. The activity of the brain tissue p-Akt was obviously increased in I/R group. In the three treatment groups, the activity of p-Akt was increased respectively. The activity of p-Akt of the G-CSF+EPO group was obviously increased compared to that of the G-CSF group and the EPO group (P<0.05). There was no significant difference in the activity of p-Akt when compared the G-CSF group with the EPO group.
Conclusions: Therapy of G-CSF combined with EPO can obviously reduce the neurocyte apoptosis of the cerebral ischemia-reperfusion, augment the expression of Bcl-2 and depress the expression of Bax and Caspase-3. Combined treatment can have synergetic effect of inhibiting apoptosis partly by means of activating PI-3K/Akt signaling pathway. EPO has significant effect in respect of inhibiting neurocyte apoptosis.
Part Four Research of the effect of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin on the neural regeneration of the cerebral ischemia-reperfusion in Rats
Objective: To observe the effect of G-CSF or EPO only or combined treatment on the neural proliferation, differentiation and the expression of neurotrophic factors of the cerebral ischemia-reperfusion in rats.
Method: The proliferative cells were marked by Brdu. After each corresponding time of reperfusion, the Brdu positive cells of cerebral ischemia in rats of each group were detected by single fluorescent staining. The Brdu/NeuN and Brdu/GFAP both positive cells were detected by double-label immunofluorescence assays. The expression of neurotrophic factors including BDNFmRNA and NGFmRNA are detected by means of RT-PCR.
Results:
1. In I/R group sporadic BrdU positive cells were observed after reperfusion for 1d, the quantity of the BrdU positive cells peaked after reperfusion for 7d and began to decline after reperfusion for 14d. There was no significant difference in the BrdU positive cell quantity of the three treatment groups after reperfusion for 1d and 3d when compared with each other or compared to that of the I/R group. After reperfusion for 7d and 14d, the BrdU positive cell quantity of the three treatment groups respectively exceeded that of the I/R group significantly (P<0.05) and the BrdU positive cell quantity of G-CSF+EPO group significantly exceeded that of the G-CSF group or the EPO group respectively (P<0.05). After reperfusion for 7d and 14d, the BrdU positive cell quantity of the G-CSF group was obviously increased compared to that of the EPO group (P<0.05 respectively).
2. The Brdu/NeuN and Brdu/GFAP both positive cells were observed in the I/R group. The Brdu/NeuN and Brdu/GFAP both positive cell quantity of the three treatment groups respectively exceeded that of the I/R group significantly (P<0.05); and the Brdu/NeuN and Brdu/GFAP both positive cell quantity of G-CSF+EPO group significantly exceeded that of the G-CSF group or the EPO group respectively (P<0.05). The Brdu/NeuN and Brdu/GFAP both positive cell quantity of the G-CSF group was obviously increased compared to that of the EPO group (P<0.05).
3. The expression of BDNFmRNA in the I/R group was augmented obviously. The expression of BDNFmRNA of the three treatment groups respectively was augmented significantly compared to that of I/R group (P<0.05 respectively). But there was no significant differance in the expression of BDNFmRNA of the three treatment groups when compared with each other.
4. The expression of NGFmRNA in the I/R group was augmented obviously. The expression of NGFmRNA of the three treatment groups respectively was augmented significantly compared to that of I/R group (P<0.05 respectively). The expression of NGFmRNA of the G-CSF+EPO group was augmented significantly compared to that of the G-CSF group or the EPO group. The expression of NGFmRNA of the G-CSF group was augmented significantly compared to that of the EPO group (P<0.05)
Conclusions: Therapy of G-CSF combined with EPO can obviously promote the neural proliferation and the differentiation to neurons and glial cell of the cerebral ischemia-reperfusion in Rats, augment the expression of neurotrophic factors including BDNFmRNA and NGFmRNA. G-CSF has significant effect in respect of promoting neural regeneration.
引文
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