摘要
本研究主要分为两部分。
第一部分:脑损伤后脑内Homer与Shank基因的改变及意义
脑损伤主要包括创伤性脑损伤(Traumatic brain injury,TBI)和缺血性脑损伤,残、死率高[1-2]。脑损伤的发生、发展可能与谷氨酸神经毒性、钙离子超载、炎症反应和血-脑脊液屏障破坏等因素有关,但其确切的分子病理机制尚不清楚,这是治疗效果差的根本原因[3-4]。我们曾在体外培养大鼠脑皮层神经元机械性损伤模型上,发现一种即早基因( immediately early gene,IEG) Homer及其下游分子Shank,能调控多种细胞分子,通过多种信号通路广泛参与脑损伤发生、发展的核心过程[5]。本课题拟在以往工作基础上,通过在体动物的多种脑损伤模型,如侧向瞬时旋转致弥漫性轴索损伤(diffuse axonal injury,DAI)模型、Marmarou加速致弥漫性脑损伤(diffuse brain injury,DBI)模型及缺血性脑损伤模型,检测Homer和Shank在损伤脑组织中的表达及其意义,为探索脑损伤的发生机制及其相关治疗提供理论依据。
实验一大鼠DAI后,脑内Homer的表达改变及意义
目的:研究大鼠DAI后,脑内Homer蛋白各亚型表达变化规律及意义。方法:选择成年雄性SD大鼠120只,随机分为正常对照组、假手术组与DAI后30min、1h、3h、6h、12h、24h、48h及72h共10组,每组12只;应用侧向瞬时旋转致DAI,取大鼠脑干区主要核团、皮层和海马组织进行检测;采用免疫组化染色法行免疫组化评分检测;Western blot法行蛋白测定;实时荧光定量RT-PCR法行mRNA测定。结果:①免疫组织化学法结果显示,正常对照组和假手术组中神经元Homer1a染色呈阴性,DAI组神经元Homer1a染色呈阳性,阳性染色呈颗粒状分布于胞浆、胞膜及突起结构;伤后30 minHomer1a蛋白出现表达,一直持续至伤后72 h,与正常对照组和假手术组Homer1a蛋白免疫评分相比,DAI后24h出现表达高峰(P<0.01);Homer1b/c在各组神经元中均有一定程度表达,但表达量无明显变化;②与正常对照组和假手术组相比较,在DAI后30min,Homer1a蛋白及其mRNA开始表达,1h ~72 h表达量明显增加,在24h达到高峰(P<0.01),但72h仍维持较高水平(P<0.05);③Homer1b/c在正常对照组、假手术组和DAI组神经元中均有一定程度表达,无论蛋白还是mRNA表达水平,其表达量均无明显变化(P > 0.05);④DAI后,在30min~24h,脑干Homer1a蛋白及其mRNA表达量均大于皮层和海马(P<0.05),但在48~72h无统计学差异(P>0.05);其在皮层和海马之间无差异(P>0.05);Homer1b/c蛋白及其mRNA表达量在上述三个部位也无差异(P>0.05)。结论:DAI刺激可诱导Homer1a基因快速表达,可能与DAI后,大量谷氨酸释放,细胞兴奋性增加有关,而Homer1b/c表达不受神经元兴奋性活动调节。结合以往文献认为,DAI后,动态表达的Homer1a与持续表达的Homer1b/c共同调节mGluR1a的结构和功能,其机制可能是Homer1a与Homer1b/c竞争结合mGluR1a,减少细胞内Ca2+超载,调节mGluR1a在突触部位的分布及受体信号传递效率,防止mGluR1a过度兴奋。神经元兴奋性增高时,Homer1a表达增加,这是一种自然的、选择性地调节mGluR1a与Homer1b/c结合的负反馈机制。而脑组织不同部位Homer1a表达量存在差异可能与侧向旋转DAI致伤模型造成中线部分受损最严重有关,提示根据Homer1a表达量多少可判断脑损伤严重程度。
实验二大鼠DBI后,脑内Homer的表达改变及意义
目的:研究大鼠DBI后,脑内Homer蛋白各亚型表达变化规律及意义。方法:选择成年雄性SD大鼠120只,随机分为正常对照组、假手术组DBI后30min、1h、3h、6h、12h、24h、48h及72h共10组,每组12只;采用Marmarou加速致DBI模型,其它检测内容和方法同实验一。结果:①免疫组织化学法结果显示,正常对照组和假手术组中神经元Homer1a染色呈阴性,DBI组神经元Homer1a染色呈阳性,阳性染色呈颗粒状分布于胞浆、胞膜及突起结构;伤后30 min Homer1a蛋白开始表达,一直持续至伤后72 h,与正常对照组和假手术组Homer1a蛋白免疫评分相比,DBI后24h出现表达高峰(P<0.01),同时,Homer1a在损伤胶质细胞中也表达;Homer1b/c在各组神经元中均有一定程度表达,但表达量无明显变化;②与正常对照组和假手术组相比,DBI后3h和24h,Homer1a蛋白及其mRNA表达量出现2个峰值(P<0.01),72h时仍维持较高水平(P<0.05);③Homer1b/c结果同实验一结果;④DBI后30min~24h,Homer1a蛋白及其mRNA表达量在皮层和海马均大于脑干的相应值(P<0.05),而在48~72h两者无差异(P>0.05);实验中,Homer1a蛋白及其mRNA表达量在皮层和海马之间无差异改变(P>0.05),而且,Homer1b/ c蛋白及其mRNA表达量在脑干、皮层和海马之间也无差异改变(P>0.05)。结论:DBI后Homer1a表达有两个高峰,第一个高峰的出现可能与早基因蛋白Homer1a竞争性与Homer1b/c结合代谢性谷氨酸受体,降低其在突触部位的聚集量,减少细胞内Ca2+超载有关;第二个高峰的出现可能与Homer1a还可能参与促进受损神经元和胶质细胞病理性凋亡有关,这种Homer1a蛋白上调可能对受损脑组织有保护作用。另外,与实验一DAI后的结果不同,Homer1a表达量在海马和皮层明显增高,这可能与模型不同、海马和皮层受损更严重、发生机制不同有关。
实验三大鼠缺血再灌注脑损伤后,脑内Homer的表达改变及意义
目的:研究大鼠缺血再灌注脑损伤后,脑内Homer蛋白各亚型表达变化规律及意义。方法:选择成年雄性SD大鼠120只,随机分为正常对照组、假手术组与缺血再灌注脑损伤后30min、1h、3h、6h、12h、24h、48h及72h共10组,每组12只;采用大脑中动脉阻塞致缺血再灌注损伤模型(MCAO),其它检测内容和方法同实验一。结果:①免疫组织化学法结果显示,正常对照组和假手术组中神经元Homer1a染色呈阴性,损伤组神经元Homer1a染色呈阳性,阳性染色呈颗粒状分布于胞浆、胞膜及突起结构;缺血再灌注损伤后30 min Homer1a蛋白开始表达,一直持续至伤后72 h,与正常对照组和假手术组Homer1a蛋白免疫评分相比,损伤后24h出现表达高峰(P<0.01),同时,Homer1a在损伤胶质细胞中也表达;Homer1b/c在各组神经元中均有一定程度表达,但表达量无明显变化;②与正常对照组和假手术组相比,缺血再灌注脑损伤后,Homer1a蛋白及其mRNA表达量出现30min(P<0.01)、3h(P<0.01)和24h(P<0.01)3个高峰,72h时仍维持较高水平(P<0.05);③Homer1b/c结果同实验一结果。结论:首先,缺血再灌注损伤后Homer1a表达有三个高峰,伤后30min出现的第一个高峰,可能与缺血损伤和再灌注损伤“叠加”,在损伤早期共同刺激及早基因Homer1a大量表达,与Homer1b/c竞争结合mGluRs,降低其在突触部位的聚集量,减少细胞内Ca2+超载有关,DBI后无此表达高峰。其余两个表达高峰则与Homer1a调节mGluRs分布,及促进神经元和胶质细胞病理性凋亡有关;其次,缺血再灌注脑损伤后Homer1a表达时空变化规律与上述两个实验结果不同,可能与发生机制不同,缺血性脑损伤病理生理过程牵涉更多、更复杂信号通路有关。
实验四大鼠脑损伤后,脑内Shank各亚型的表达改变及意义
Shank是一种多结构域的骨架蛋白,可介导多种蛋白连接而成功能复合体,包括3个亚型,Shank1,Shank2和Shank3,在神经系统有广泛分布,对于维持神经元突触的形态和功能有重要作用[6-7]。文献报道,Shank蛋白通过调控Homer蛋白与mGluRs的锚定与信号传导过程,参与了突触可塑性、学习和记忆等的病理生理机制。但是脑损伤后不同亚型Shank蛋白的表达时空变化规律,以及与脑损伤的关系等都未见报道。
目的:本课题拟在以往工作基础上,通过上述3种在体动物脑损伤模型,初步检测脑损伤后不同亚型Shank蛋白的表达时空变化规律以及与脑损伤的关系,力求寻找出关键性Shank分子,为探索脑损伤的发病机制及其相关治疗提供了实验依据。方法:取上述3种模型致损伤后的大鼠皮层组织进行检测。采用免疫组化染色法和Western blot法行蛋白定位定量测定,实时荧光定量RT-PCR法行mRNA测定。结果:①免疫组织化学法结果显示,在3种模型中,脑损伤前后,Shank的3个亚型- Shank1、Shank2和Shank3都阳性表达,它们的阳性染色都呈颗粒状分布于胞浆、胞膜及突起结构。②DAI后,Shank的蛋白定量和mRNA定量检测结果一致。Shank1,在所有组别都高表达,无变化趋势,损伤前后表达量一致(P>0.05);Shank2,正常组和假手术组都有表达,DAI后30 min其表达量开始增加,6h达到高峰(P<0.01),随后下降,72h又出现表达高峰(P<0.01);Shank3,正常组和假手术组都有表达,DAI后,30min到6h一直高表达(P<0.01),然后开始下降,至72h仍维持较高水平(P<0.05)。③DBI后,Shank的蛋白和mRNA定量检测结果一致。Shank2,正常组和假手术组都有表达,DBI后30 min其表达量开始增加,在3h到6h期间达到高峰(P<0.01),而DBI前后Shank1和Shank3亚型表达变化结果,和DAI前后此两个亚型结果基本一致。④缺血再灌注损伤后,Shank的蛋白和mRNA定量检测结果一致。Shank1,正常组和假手术组都有表达,损伤后30min其表达量开始增加,出现2个高峰,3h(P<0.01)和12h(P<0.01),随后下降,至72h仍维持较高水平(P<0.05);Shank2,在所有组别都高表达,无变化趋势,损伤前后表达量一致(P>0.05);Shank3,正常组和假手术组都有表达,损伤后30min其表达量开始增加,3h达到最高峰(P<0.01),然后开始下降,至12h开始下降明显(P>0.05),至72h时表达量和正常组表达量一致(P>0.05)。结论:我们首次在脑损伤后,对脑皮层组织中Shank的表达变化及意义进行研究。首先,Shank各亚型在损伤前后都有表达,但表达量有明显变化,证明Shank可能在维持和调节神经元兴奋性方面发挥重要的功能,而且各亚型蛋白通过结合不同的信号分子,参与不同的信号通路共同调节神经元功能;其次,DAI和DBI后,损伤刺激可诱导Shank2和Shank3基因快速表达,可能与损伤后,大量谷氨酸释放,Shank和动态表达的Homer1a相结合,改变信号传导信息,共同影响细胞兴奋性,而Shank1表达不受神经元兴奋性活动调节,这些变化提示动态表达的Shank2和Shank3可能与持续表达的Shank1共同调节Homer、mGluRs以及其他信号分子的结构和功能,影响细胞内Ca2+超载,共同调节神经元的兴奋性;再次,缺血性损伤后Shank各亚型表达变化和创伤性脑损伤后Shank亚型表达变化差异很大,提示3种类型脑损伤病理生理过程可能不一致,复杂多变的信号通路在各种脑损伤的发病机制中发挥不同的作用;最后,脑内Shank各亚型在不同类型脑损伤后有不同表达变化,提示其可能成为潜在的靶向诊治脑损伤的目标。
第二部分:Homer在脑胶质瘤中表达、功能及作用机制的研究
脑胶质瘤是中枢神经系统最常见的恶性肿瘤,其发生率约占颅内肿瘤的45%,恶性脑胶质瘤患者术后中位生存期尚不足一年[8-9]。脑胶质瘤治疗困难的根源与其所具有的恶性生物学特性密切相关。研究表明,脑胶质瘤表现有过度增殖、对抗凋亡以及旺盛的血管形成等恶性表型[10-11]。揭示决定脑胶质瘤恶性生物学行为的关键基因对于攻克人类这一顽疾有着重要的科学意义和临床应用价值。结合文献和我们预实验结果发现,Homer在人脑胶质瘤的细胞凋亡和血管形成过程中扮演重要角色,提示Homer与人类肿瘤的形成和发展有着密切关系。目前,未见有关Homer与脑胶质瘤发生与发展关系的研究,Homer在脑胶质瘤恶性增殖、抗凋亡以及血管形成中的作用很不清楚。基于此,本课题从以下三个方面进行了研究。
实验一Homer1在人脑胶质瘤中的表达及意义
目的:研究Homer1在人脑胶质瘤中的表达及意义。方法:采用免疫组织化学化方法、Western-blot和实时荧光定量RT-PCR检测Homer1蛋白在3株人脑胶质瘤细胞系(U251、U87和SHG-44)、60例人脑胶质瘤组织和5例正常人脑组织中的表达水平。结果:①免疫组化结果显示:SHG-44和U87细胞中Homer1a染色呈强阳性,在U251细胞中弱阳性染色,肿瘤细胞的阳性染色都呈颗粒状分布于胞核、胞浆、胞膜及突起结构。3株细胞系中Homer1b/c染色呈阴性;Homer1a蛋白在人脑胶质瘤及正常人脑组织中的表达阳性率及免疫反应评分(IRS)分别为61.8%、4.35±3.99和1.2%、0.09±0.35(P均<0.01);Homer1a蛋白表达阳性率和IRS与脑胶质瘤Ⅰ~Ⅳ级病理级别间呈“U”型关系,其中Ⅰ和Ⅱ、Ⅲ级间差异非常显著(P<0.01),Ⅳ和Ⅱ、Ⅲ级间差异非常显著(P<0.01),Ⅰ和Ⅳ级、Ⅱ和Ⅲ级间差异显著(P<0.05),良、恶性脑胶质瘤之间Homer1a蛋白表达阳性率差异显著(P<0.05);Homer1b/c在人脑胶质瘤标本中不表达。②Homer1a蛋白及其mRNA在SHG-44和U87细胞中高表达,在U251细胞中低表达。Homer1b/c蛋白及其mRNA在3株细胞系中均无表达;Homer1a蛋白表达水平与Homer1 mRNA表达水平无明显差异(P>0.05);其他结果,mRNA表达情况和蛋白表达情况一致。结论:以上结果提示Homer1a蛋白和mRNA在人脑胶质瘤中有表达,并与脑胶质瘤的发生和发展密切相关;Homer1b/c不参与人脑胶质瘤发生和发展。
实验二人Homer1基因真核干涉表达的构建和鉴定
目的:构建人Homer1基因真核干涉表达载体,并转染人脑胶质母细胞瘤U87细胞。方法:根据人Homer1a基因序列,设计、合成了针对人Homer1的特异性RNAi片断,并克隆入pGCsi载体,构建了Homer1a shRNA真核干涉表达载体pGCsi-H1。通过脂质体介导的基因转染方法将将干涉载体pGCsi-H1和空载体pGCsi质粒以及阴性对照质粒pGCsi-NC分别转染入人脑胶质母细胞瘤U87细胞,暂时分别命名为U87-H1、U87-P和U87-NC。结果:实时荧光定量RT-PCR检测证实U87-H1细胞Homer1 mRNA表达受到明显抑制, Western blot检测证实U87-H1细胞Homer1a蛋白表达也受到明显抑制。结论:Homer1 shRNA成功抑制了Homer1 mRNA和蛋白表达,这为进一步研究Homer1a在胶质瘤中的生物学意义和其作用机制奠定了实验基础。
实验三重组人pEGFP-N1-Homer1-GFP基因表达质粒的构建
目的:克隆人Homer1基因的全长cDNA,并构建其真核表达载体。方法:从人脑组织中提取总RNA,通过RT-PCR扩增Homer1基因的全系列cDNA。将Homer1基因插入真核表达载体pEGFP-N1中EGFP基因的5‘端,获得与EGFP融合表达的真核表达质粒pEGFP-N1-Homer1-GFP。结果:经双酶切及测序鉴定证实人Homer1基因的真核表达载体构建成功。免疫印迹法证实人Homer1基因的表达。结论:成功克隆了人Homer1编码区序列,并构建了其真核表达载体,为胶质瘤等中枢神经系统恶性肿瘤的治疗奠定基础。
This study includes two parts.
First part: The expression and significance of Homer and Shank gene in brain injury
Brain injury, which mainly included traumatic brain injury (TBI) and ischemia brain injury, has long been a major cause of disability and death to society. The pathphyciological progression of TBI maybe includes neural toxicity of glutamate, overloading of Ca2+, inflammation and obstruction of blood-cerebrospinal fluid barrier and so on, however, the assured developmental mechanism concerning the path physiology of TBI has been unclear. Our previous study indicated that a immediately early gene(IEG), Homer, can regulates many cell-molecule and cell signal pathways, and participate in the pivotal process of the developmental mechanism of TBI in mechanically injured model of cortical neurons of rat in vitro. In this study, we examined the expression and significance of Homer after brain injury in neurons in vivo (3 kinds of brain injury models were produced: diffuse axonal injury (DAI) was produced by accelerated lateral head rotation model; diffuse brain injury (DBI) was manufactured by acceletation-deceletation model of DBI by Marmarou et al; and ischemia-reperfusion injury was made by middle cerebral artery occlusion model). The present study may provide experimental basis for the mechanism and correlated therapy of brain injury.
PartⅠExpression and significance of Homer after DAI in vivo
Objective: To study the variational regulation of expression and significance of Homer after DAI in vivo. Methods: 120 young-adult male Sprague-Dawley rats were randomly assigned into normal control (n=10), sham-operated control (n=10), and DAI group (n=80). The DAI group was subdivided so that different time points (30min, 1h, 3h, 6h, 12h, 24h, 48h and 72h) post-injury could be studied. Accelerated lateral head rotation was used to produce DAI in rats in this study. And the rats’brainstem, cortex and hippocampus were detected for Homer by immunohistochemistry, Western blot and quantitative real-time RT-PCR after DAI. Results:①Immunohistochemical analysis showed that there was negative-expression of Homer1a in normal control and sham-operated control group. And Homer1a was increased significantly from 30 min to 72 hours after DAI, peaked at 24h (P<0.01). The positive particles were distributed in the neuronal cytoplasm, cyto-membrane and dendrites. No change of Homer-1b/c expression was found before and after injury of neurons;②Compared to the normal control and sham-operated control samples, the levels of Homer1a mRNA and protein expressed in samples obtained from DAI rats at 30min, and were strongly up-regulated from 1h to 72h after DAI, peaked at 24 h (P < 0.01), and remained at a high level until 72 h (P<0.05);③Homer1b/c could be observed and was constitutively expressed at similar levels in neurons of all three groups. There was no significant statistical difference in the intensity of Homer1b/c protein band among normal control, sham-operated control and DAI group (P>0.05);④In all three groups, the expression of Homer1a was significantly higher in brainstem than in cortex and hippocampus (P<0.05), during the early part of the post-injury period (30min-24 h). However, the significance of this difference was lost during the latter part of the post-injury period (48 h-72 h, P>0.05). Meanwhile, there was no difference of the expression of Homer1a between cortex and hippocampus (P>0.05). The expression of Homer1b/c was nearly the same in these anatomic sites (P>0.05). Conclusions: It was verified for the first time that DAI could induce Homer1a gene expression, which may be due to the excitement of neurons as a result of glutamic acid release after injury. Expression of Homer1b/c was not modulated by the neuronal exciting activity. Dynamically expressed Homer1a and constitutively expressed Homer1b/c might further modulate the distribution and function of mGluR1a. Potential mechanism might be that Homer1a, which competes with Homer1b/c to couple to mGluR1a, regulates the distribution of receptor at synapse and the efficiency of signal transduction, prevents neurons from excess excitement. Homer1a functions as a natural dominant negative to selectively regulate association of mGluR1a with Homer1b/c after neuronal excitement increased. In addition, the results which there was a significantly difference of the expression of Homer1a in different anatomic sites was indicated that in the DAI, the injured degree of axons in brainstem were more severe than that in cortex and hippocampus, because it more approached the midline of brain.
Part II Expression and significance of Homer after DBI in vivo
Objective: To study the variational regulation of expression and significance of Homer after DBI in vivo. Methods: 120 young-adult male Sprague-Dawley rats were randomly assigned into normal control (n=10), sham-operated control (n=10), and DBI group (n=80). The DBI group was subdivided so that different time points (30min, 1h, 3h, 6h, 12h, 24h, 48h and 72h) post-injury could be studied. DBI in rats was manufactured by acceletation-deceletation model of DBI by Marmarou et al in this study. The content and method of examine were the same as that of in partⅠ. Results:①the results of immummunohistochemical analysis were the same as that of in partⅠ. And Homer1a expressed in glia cell after DBI, also.②Compared with normal control and sham-operated control samples, there are two peak values of mRNA and protein of Homer1a following DBI, increased in 3h (P<0.01) and in 24h (P<0.01), then decreased in 72h(P<0.05)with high value.③The results of expression of Homer1b/c were the same as that of in partⅠ.④In all three groups, the expression of Homer1a was significantly higher in cortex and hippocampus than in brainstem (P<0.05), during the early part of the post-injury period (30min-24 h). However, the significance of this difference was lost during the latter part of the post-injury period (48 h-72 h, P>0.05). Meanwhile, there was no difference of the expression of Homer1a between cortex and hippocampus (P>0.05). Moreover, the expression of Homer1b/c was similar in the different anatomic sites (P>0.05). Conclusions: The expression of Homer1a reached two peak values, the mechanism of the first peak value maybe related with Homer1a competes with Homer1b/c to couple to mGluR1a, reduced the level of mGluR1a at synapse and Ca2+ influx; And the mechanism of the second higher value might connecte with Homer1a can induces apoptosis of neurons and glia, these indicated that overexpression of Homer1a might protect injured brain. Moreover, the expression of Homer1a was higher in neurons of cortex and hippocampus than in brainstem, which is different from the previous results of partⅠ, these phenomena might connect with injured models was different, the injured degree of neurons in cortex and hippocampus was severe and the mechanism of TBI was diverse.
Part III Expression and significance of Homer after ischemic- reperfusion injury in vivo
Objective: To study the variational regulation of expression and significance of Homer associated with ischemic-reperfusion injury in vivo. Methods: 120 young-adult male Sprague-Dawley rats were randomly assigned into normal control (n=10), sham-operated control (n=10), and ischemic-reperfusion injury group (n=80). The ischemic-reperfusion injury group was subdivided so that different time points (30min, 1h, 3h, 6h, 12h, 24h, 48h and 72h) post-injury could be studied. The ischemia-reperfusion injury of rat was made by middle cerebral artery occlusion model. The content and method of examine were the same as that of in partⅠ. Results:①The results of immunohistochemical analysis were the same as that of in partⅡ.②C ompared normal control and sham-operated control samples, there are three peak values of mRNA and protein of Homer1a following ischemia-reperfusion injury, increased in 30min (P<0.01), in 3h (P<0.01) and in 24h (P<0.01), and decreased in 72h(P<0.05)with high value.③T he results of expression of Homer1b/c were the same as that of in partⅠ. Conclusions: First, the expression of Homer1a reached three peak values, and peaked at 30min firstly, which maybe relate with both the ischemia injury and reperfusion injury stimulate jointly Homer1a expression, and overexpression Homer1a competes with Homer1b/c to couple to mGluR1a“timely”, induced the injured degree of neuron, there was no the first peak value after DBI. The mechanism of other two peak value of Homer1a expression connected with Homer1a maybe regulates the distribution of mGluRs and induces pathological apoptosis of neurons and glia. Second, the characteristic spatio-temporal of expression of Homer1a after ischemia- reperfusion injury was found, which might because of the mechanism of ischemia-reperfusion injury was different from that of DAI and DBI, and maybe added and more complex cell signal pathways were involved in the pathophysiology of ischemia-reperfusion injury.
PartⅣExpression and significance of isoforms of Shank after brain injury in vivo
Shank, a scaffold protein in the post-synaptic density (PSD), which including 3 subtypes, Shank1, Shank2 and Shank3, has multi-functional regions, link and interact with many different classes of proteins, distribute extensively in the nervous system, is believed to play an important role in the synaptic construction and function. Some previous studies suggest that Shank may be involved in synaptic plasticity and, by extension, learning and memory through modulate the signal transduction of Homer and mGluRs. However, the space-time transformation law of isoforms of Shank protein associate with brain injury in vivo, and the relationship between Shank and brain injury are still unknown.
Objective: In this study, we examined the expression and significance of Homer after brain injury in rat in vivo (3 kinds of brain injury models above-mentioned). We examine the expression of isoforms of Shank, and find the“key”Shank molecule. This study may provide experimental basis for the mechanism and correlated therapy of brain injury. Methods: The rats’cortex was detected for Shank by immunohistochemistry, Western blot and quantitative real-time RT-PCR after brain injury (3 kind’s models of brain injury) which mentioned in above study. Results:①Immunohistochemical analysis showed that no change of isoforms of Shank-Shank1, Shank2, Shank3 expression were found before and after brain injury of neurons; The positive particles were distributed in the neuronal cytoplasm, cyto-membrane and dendrites.②The results of protein expression and mRNA levels of Shank which examined by Western-blot and quantitative real-time RT-PCR were consistent after DAI. Shank1 could be observed and was constitutively expressed at similar levels in neurons of all three groups (P>0.05); Shank2 could be observed in neurons of all three groups, however, compared to normal control and sham-operated control samples, the levels of Shank2 mRNA and protein expressed in samples obtained from DAI rats at 30min, and were strongly up-regulated from 1h to 72h after DAI, and peaked at 6 h and 72 h (P < 0.01); Shank3 could be observed in neurons of all three groups, strongly rose from 30min to 6h after DAI (P < 0.01), then decreased, but remained the high level until 72 h (P < 0.05).③the results of protein expression and mRNA levels of Shank which examined by Western-blot and quantitative real-time RT-PCR were consistent after DBI. Except that Shank2 reached the peak from 3 h to 6 h (P < 0.01), other results were coincident with the expression of isoforms of Shank after DAI.④The results of protein expression and mRNA levels of Shank which examined by Western-blot and quantitative real-time RT-PCR were consistent after ischemia- reperfusion injury. Shank1 could be observed in neurons of all three groups, and compared to normal control and sham-operated control samples, the levels of Shank2 mRNA and protein expressed rose at 30min after injury, and reached to the peak at 3 h and 12 h (P < 0.01), then decreased, but remained the high level until 72 h (P < 0.05); Shank2 could be observed and was constitutively expressed at similar levels in neurons of all three groups (P>0.05); Shank3 could be observed in neurons of all three groups, and the levels of Shank3 increased from 30min after ischemia-reperfusion injury (P<0.05), peaked at 3h, then decreased markedly from 12 h to 72 h (P>0.05); The variational trends of isoforms of Shank were different obviously among ischemia- reperfusion injury, DAI and DBI. Conclusions: We firstly study the expression and function of isoforms of Shank after brain injury in vivo. Firstly, isoforms of Shank all expressed in brain cortex before and after brain injury and the expression levels of Shank changed remarkable proved that Shank maybe play important role in keep and management of the excitement of neurons, and every isoforms of Shank binded with different signal molecules, participated in different signal pathways and regulated the function of neurons together; Secondly, injury induce Shank2 and Shank3 gene expression associated with DAI and DBI, which may be due to the lots of release of glutamic acid after injury, and Shank combined with dynamically expressed Homer1a to affect the efficiency of signal transduction and the excitement of neurons, meanwhile, expression of Shank1 was not modulated by the neuronal exciting activity, these indicated that dynamically expressed Shank2 and Shank3 and constitutively expressed Shank1 might further modulate the distribution and function of Homer, mGluRs and other signal molecules; Thirdly, there were various different between the expression of isoforms of Shank after ischemia-reperfusion injury and that of associated with traumatic brain injury, which suggested suggested that different kinds of brain injury has various developmental mechanism respective, and complex signal pathway play different effects in pathphyciological progression of brain injury; In conclusion, differently variational regulation expression of isoforms of Shank in different types of brain injury prompted that Shank may be become a potential target of therapy and diagnosis of brain injury.
Second part: The expression, function and mechanism of Homer gene in human brain glioma
Brain glioma is the most common malignancy of central nervous system, which occupies almost half of all intracranial tumors. The postoperative median survival is less than one year for patients with malignant brain glioma. The main reason for difficulties on treatment is associated closely with the malignant biology phenotype of brain glioma, which including excessive proliferation, antiapoptosis and abundant angiogenesis. So, it has important science significance and clinical application value to identify the key genes involved in the malignant biology behavior of this malignancy for overcoming it. Combined literature and our pre-experiment’results, we found Homer play important role in antiapoptosis and angiogenesis of brain glioma, which suggested there is close relationship between Homer and human tumor. At present, the research on the relationship between Homer and the pathological mechanism of brain glioma is lack, and the role of Homer in malignant proliferation, antiapoptosis and angiogenesis of brain glioma is not completely clear. For this, the current study was performed from three aspects as follows.
PartⅠExpression and significance of Homer in human brain glioma
Objective: To investigate the expression and significance of Homer in human brain glioma. Methods: Three human brain glioma cell lines (U251, U87 and SHG-44), 60 cases of human brain glioma tissue and 5 cases of normal human brain tissue were investigated by immunohistochemistry method for protein expression of Homer. Results:①Homer1a protein was highly expressed in U87 and SHG-44 cells, and low expression of Homer1a protein in U251 cells, meanwhile, there was no expression of Homer1b/c in 3 human brain glioma cell lines. It was 61.8%, 4.35±3.99 and 1.2%, 0.09±0.35 respectively that the positive expression percent and immunoreactivity score (IRS) of Homer1a in brain glioma tissues and normal brain tissues, there were especially significant difference (P<0.01 for both) respectively in the positive expression percent and IRS of Homer1a between them. There were‘U’ relationship between the positive expression percent and IRS of Homer1a and type pathological grades of glioma, there were specially significant difference (P<0.01 for both) between gradeⅠand gradeⅡ,Ⅲ, and between gradeⅣand gradeⅡ,Ⅲ, also, there were significant difference (P<0.05 for both) between gradeⅠand gradeⅣ, and between gradeⅡand gradeⅢ. In addition, there was specially significant difference in the positive expression percent and IRS of Homer1a between benign and malignant brain glioma. Moreover, there were no significant difference (P>0.05 for all) respectively in the positive expression percent and IRS of Homer1b/c between benign and malignant brain glioma and among different pathological grades of glioma.②The results of Western blot and quantitative real-time RT-PCR were the same as that of immunohistochemistry. Conclusions: The results suggested that, Homer1a not Homer1b/c protein be expressed in brain glioma, and which may associate closely with initiation, progress, malignant proliferation, antiapoptosis and angiogenesis of brain glioma.
PartⅡConstruction and identification of human Homer1 shRNA
Objective: To construct the eukaryotic expression vector pGCsi-H1 of Homer1 shRNA and transfected human brain glioblastoma U87 cells. Methods: According to Homer1 cDNA sequence, the specific RNAi fragments targeting Homer1 were designed and synthesized, which were cloned into pGCsi vector, and the eukaryotic expression vector pGCsi-H1 of Homer1 shRNA was constructed. The pGCsi-H1 vector, blank pGCsi vector and pGCsi-NC were transient transfected respectively into human brain glioblastoma U87 cells by lipofectin medium, and temporarily called them: U87-H1、U87-P and U87-NC. Results: Quantitative real-time RT-PCR results showed that mRNA expression of Homer1 was inhibited markedly, while the results of Western blot indicated that protein expression of Homer1a was also suppressed significantly in U87-H1 cells. Conclusions: The Homer1 shRNA can suppress significantly expression of mRNA and proteins of Homer1, which offers a basis for study of biological significance of glioma with lack of Homer1a.
PartⅢConstruction of recombinant plasmid pEGFP-N1- Homer1-GFP in human
Objective: To clone human Homer1 cDNA and construct eukaryotic expression vector carrying human Homer1 gene. Methods: The cDNA encoding the human brain glioma Homer1 was isolated by using RT-PCR method with total RNA extracted from human brain glioma. Homer1 gene was cloned into eukaryotic expression vector pEGFP-N1 of EGFP reported gene encoding green fluorescence protein in the form of fusion protein. The expression vector of recombinant plasmid pEGFP-N1-Homer1-GFP is successfully constructed. Results: Automatic DNA sequence analysis demonstrated that the sequence of the recombinant plasmid pEGFP-N1-Homer1-GFP was in accordance with that published in GenBank. Conclusions: pEGFP-N1-Homer1-GFPis constructed successfully, which may provide a novel and important means for gene therapy of glioma.
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