摘要
目的:研究在缺血性脑水肿病理过程中,水通道蛋白家族(aquaporins, AQPs)表达变化的时空规律及其与脑水肿形成的关系,初步探讨AQPs在缺血性脑水肿过程中的作用及其表达调控机制。
方法:成年健康雄性Wistar大鼠392只,随机分为假手术对照组和缺血组,用大脑中动脉栓塞法制作脑缺血动物模型,假手术对照组除不插线栓外,其余操作与缺血组相同。每组在造模成功后1 h、3 h、6 h、12 h、24 h取出大鼠脑组织,分别采用HE染色、透射电镜对相应部位进行病理观察,干湿重法计算脑组织含水量变化,免疫组化(Immunohistochemistry,IHC)、免疫荧光(Immunofluorence)观察各组相应时间点脑组织AQPs的表达变化,免疫印迹、Real-time PCR、测定各组相应时间点水肿脑组织AQPs含量的变化。同时运用免疫印迹检测水肿脑组织ERK(Extracellular signal regulated kinase,ERK)含量的变化。
结果:在假手术对照组,脑组织形态、脑含水量、AQPs及其mRNA的分布及表达、ERK1/2总量以及磷酸化ERK1/2( phosphorylated ERK1/2, pERK1/2)量在各时间点均无明显变化。脑缺血后,光镜和电镜观察可见脑组织出现缺血性改变:细胞及血管周围间隙明显扩大,部分细胞坏死,胞核浓染或崩解,胶质细胞增生。缺血1小时后,脑含水量明显升高,并随缺血时间的延长而逐渐增加,在缺血后24小时达到峰值。免疫组化和免疫荧光结果显示,AQPs在海马、脉络丛、脑膜、视上核、室旁核、丘脑,大脑皮质等部位的星形胶质细胞上呈阳性表达;AQP3,AQP5,AQP8和AQP9还分布于丘脑和皮层下神经元。脑水肿组织中AQP4、AQP9及其mRNA的含量随脑缺血时间的延长而增加,在缺血后24小时表达最强。而AQP3, AQP5, AQP8及其mRNA在缺血后迅速增加,至6h到高峰,后缓慢降低,至24h仍高于对照组。脑水肿的形成过程中,AQPs mRNA和蛋白的表达呈高度正相关(P<0.05),AQP4、AQP9的表达与脑含水量的变化亦呈显著正相关(P<0.05)。脑缺血后,ERK1/2总量以及pERK1/2量在各时间点均无明显变化。
结论:脑缺血后,伴随脑水肿的加重,AQP4、AQP9及其基因表达上调,提示脑缺血后,AQP4,AQP9表达上调是缺血性脑水肿形成的重要因素;在缺血早期,AQP3, AQP5, AQP8可能与脑细胞(包括神经元和星形胶质细胞)水肿密切相关,而在后期参与星形胶质细胞的细胞性水肿,在脑水肿的形成过程中起协同作用。脑缺血后,ERK1/2总量以及pERK1/2量在各时间点均无明显变化,提示ERK-MAPK通路与缺血性脑水肿AQPs的表达调控并无直接关系。
第二部分
渗透压改变对星形胶质细胞AQPs表达的影响及其表达调控
目的:探讨在不同渗透压作用下,星形胶质细胞生物学特性的改变,以及AQPs表达的规律及其表达调控机制。
方法:取生后2d内的Wistar大鼠的大脑皮层进行星形胶质细胞原代培养,随机分为对照组、低渗组和高渗组,每组分别作用1h、3h、6h、12h、24h,共5个时间点。对照组以正常培养液常规培养,低渗组和高渗组分别予以不同程度的低渗液和高渗液作用于细胞,以建立星形胶质细胞水肿和脱水的实验模型。通过HE染色和透射电镜观察渗透压改变对星形胶质细胞形态的影响,采用四甲基偶氮唑蓝(MTT)比色法、乳酸脱氢酶活性(LDH)测定和台盼蓝染色法评价细胞活力,并通过免疫细胞化学、免疫印迹、实时荧光定量聚合酶链反应(Real time-PCR)研究星形胶质细胞AQPs及其mRNA的表达变化,免疫印迹测定各组相应时间点星形胶质细胞MAPK家族ERK1/2含量的变化。
结果:对照组细胞在正常渗透压培养液培养条件下,细胞形态、细胞活性、AQPs及其mRNA的表达、ERK1/2含量在各时间点均无明显变化。低渗培养可导致细胞水肿,细胞活性下降,细胞体积变大,胞质区域相对增大,核浆比减小,并随培养液低渗程度的下调和低渗作用时间的延长而加重。在低渗液作用下,星形胶质细胞AQPs及其mRNA的表达水平随低渗程度的下调和低渗作用时间的延长而增强,除AQP5于6~12h到峰值外,其余AQP均于24h达高峰;在低渗液培养的各时间段,AQPs的蛋白表达与AQPs mRNA的表达水平呈正相关(P<0.05)。低渗液作用下,ERK1/2总量以及pERK1/2量在各时间点均无明显变化(P>0.05)。高渗液作用可导致细胞皱缩,细胞活性下降,折光性差,并随培养液高渗程度的上调和高渗液作用时间的延长而加重。在高渗液作用下,星形胶质细胞的AQP4、AQP9及其mRNA表达随高渗程度的增加而增强,AQP4、AQP9及其mRNA表达水平在12小时内持续增加,随后开始下降,至24小时后仍高于对照组(P<0.05);AQP3,AQP5和AQP8及其mRNA表达随高渗程度的增加而增强,于6h达高峰,随后开始下降,至24小时后仍高于对照组(P<0.05);AQPs的表达与AQPs mRNA的表达水平呈正相关(P<0.05)。在高渗液作用的各时间点,细胞ERK1/2总量均无明显改变(P>0.05),而pERK1/2在高渗液作用1h后表达上调,于6h达高峰,随后开始下降,至24小时后仍高于对照组(P<0.05)。pERK1/2表达变化趋势与AQP3、AQP5和AQP8表达变化一致。给予ERK阻断剂U0126处理后,ERK1/2总量、pERK1/2以及AQP3、AQP5和AQP8的表达均下调。
结论: AQPs的表达与渗透压直接相关。低渗液可导致星形胶质细胞水肿和生存能力下降, AQPs及其mRNA的表达上调作为一种病理性适应反应参与了星形胶质细胞水肿的形成。高渗液作用可导致星形胶质细胞皱缩和生存能力下降,在一定时间内,AQPs的表达上调对高渗性细胞脱水起到重要的代偿作用。ERK1/2是高渗条件下AQP3、AQP5和AQP8表达上调的重要信号物质,而高渗作用下的AQP4和AQP9以及低渗作用下的AQPs的表达调节,可能与ERK/ERK2以外的信号通路有关。
PART ONE
THE EXPRESSION REGULATION OF AQPS AND THEIR RELATIONSHIP WITH ISCHEMIC CEREBRAL EDEMA
Objective: To investigate the relationship between the formation of ischemic cerebral edema and the changes of AQPs expression during pathological course of ischemic cerebral edema in rats, and preliminarily to discuss the role of AQPs and the mechanism of AQPs expression regulation during ischemic cerebral edema.
Methods: A total of 392 healthy male adult Wistar rats were randomly divided into two groups, the ischemic group and the sham operation control group. MCAO (middle cerebral artery occluded) models were established by occluding unilateral middle cerebral artery (MCA) of the rats with the suture method. The operation procedure of the control group was as same as the ischemic group but without occluding unilateral MCA. The brains of the animals were taken out at interval times of 1h、3h、6h、12h and 24h, respectively. Subsequently, the morphological changes were observed with HE staining and transmission electron microscope (TEM). Brain water content (BWC) was measured by using wet-dry weighing method. The expression of AQPs in the edema brain tissue was detected with immunofluorence (IFC) and immunohistochemistry (IHC), respectively. The changes of AQPs and their mRNA expression in the edema brain tissue were detected with Western Blot analysis and real time reverse transcription polymerase chain reaction (Real-time PCR). The changes of ERK expression in the edema brain tissue were measured by Western Blot.
Results: After operation, the morphological structure, BWC, expression of AQPs and their mRNA, the content of ERK were not significantly changed in the control group. While in the ischemic group, HE staining and transmission electron microscope (TEM) showed that the brain tissue had ischemic changes. The space around the cells and capillaries became larger, some of the neurons showed necrosis, the nuclei were deeply stained or disappeared and the number of the neurogliocyte increased. The brain water content of ischemic brain tissue significantly increased from MCAO 1h, and gradually increased along with the ischemic time extendence, and reached its peak at 24h after MCAO. The results of IHC and IFC showed that the positive expression of AQPs was observed in the hippocampus, choroid plexes, thalamus, supraoptic nuclei, brain cortex and other regions. The expression of AQP4, AQP9 and their mRNA was up-regulated along with the ischemic time extendence and reached their peak at MCAO 24h. At the same time, the expression of AQP3, AQP5 and AQP8 increased immedially and reached their peaks at MCAO 6h, then decreased gradually, but still higher than the control group at MCAO 24h. The expression of AQPs protein was positively correlated with AQPs mRNA (P <0.05). The expression of AQP4 and AQP9 was correlated with BWC after MCAO (P<0.05). The contents of ERK and phosphorylated ERK were not significantly changed both in the control group and ischemic group.
Conclusions: The expression enhancement of AQP4 and AQP9 was correlated with the brain edema aggravation after MCAO, suggesting that the expression changes of AQP4 and AQP9 would have a close relationship with the formation of brain edema after MCAO. The up-regulation of AQP3, AQP5 and AQP8 may play a role in the neurocyte edema (neuron and astrocyte) in the early stage and cooperate with AQP4, AQP9 for astrocyte edema in the later stage. The contents of ERK and phosphorylated ERK were not significantly changed, suggesting ERK-MAPK do not play a role in the regulation of AQPs after MCAO.
PART TWO THE EFFECT OF OSMOLARITY CHANGES ON EXPRESSION OF AQUAPORINS IN ASTROCYTES AND THE PRELIMINARY STUDY OF ITS EXPRESSION REGULATION
Objective: To investigate the expression rules of aquaporins (AQPs) and the mechanism of their regulation in cultured rat astrocytes after exposure to different osmotic stress.
Methods: Rat cerebral cortical astrocytes from less than 2 days newborn Wistar rats were undergone the primary culture. The cells were divided into control group、hypotonic groups and hypertonic groups randomly. The cells of the control group were cultured in normal culture medium, the model of cell edema in the hypotonic group was established by exposed to hypotonic medium, and cell dehydration in the hypertonic group was established by exposed to hypertonic medium. Each group was examined at interval times of 1h, 3h, 6h, 12h and 24h, respectively. HE staining and transmission electron microscope were used to observe the cell morphological changes. The viability of cells in hypotonic or hypertonic medium was measured by MTT colorimetric method, LDH assay and trypan blue staining. The alteration of AQPs was studied by the methods of immunofluorence, immunocytochemistry and imaging analysis. The contents of AQPs and their mRNA expression of astrocytes were detected with Western Blot Analysis and real time reverse transcription polymerase chain reaction (Real-time PCR). The content changes of ERK of cells were measured by Western Blot.
Results: There were no significant change of the cell morphology, the cell viability, the expression of AQPs and their mRNA and the content changes of ERK in the control cells, which were cultured in normal culture medium. At the same time point, astrocytes cultured in hypotonic medium (268, 254, 240 mmol/L) showed typical features of cell edema, and the cell viability decreased, especially in lower osmolality group. The expression of AQPs and their mRNA were remarkably higher in hypotonic group than that in the control group(P<0.05), and increased by the osmolarity decreased. The expression of AQPs reached their peak at 24 except for AQP5 which reached its peak at 6~12h after astrocytes were exposed to hypotonic medium. The changes of AQPs expression showed a positive relationship with AQPs mRNA(P<0.05). The content changes of ERK were constant in astrocytes which exposed to hypotonic medium(P>0.05). Exposed to the hypertonic medium (320, 333, 345 mmol/L), cells shrinked, and the cell viability decreased with a time dependent manner. The expression of AQP4 and AQP9 were significantly changes compared with the cells in control group before 12h, and then decreased until 24h, at 24h time point it was mild higher than control cells(P<0.05), while the expression of AQP3, AQP5 and AQP8 reached their peak at 6h, then decreased. The content of ERK had no changes in higher osmolality group, but the phosphorylated ERK increased before 6h and then decreased until 24, at 24h time point it was mild higher than control cells. Preincubation of U0126, the inhibitor for ERK, could down-regulate the expression of ERK/ERK2, phosphorylate ERK/ERK2 and AQP3, AQP5 and AQP8.
Conclusions: The expression of AQPs had a direct correlationship with osmotic changes. Hyponotic medium could induce cell edema, inhibit viability of astrocytes and up-regulate the expression of AQPs and their mRNA, which is thought to be a maladaptation reaction. Hypertotic medium could induce cell shrink, inhibit viability of astrocytes, up-regulate the expression of AQPs and their mRNA, which may play an important compensation for the hypertonic dehydration in the early stage. The results mentioned above imply that ERK-MAPK participate in the enhancement expression of AQP3, AQP5 and AQP8 in the hypertonic condition. The expression of AQP4, AQP9 in the hypertonic condition and AQPs in hypotonic condition could be regulated by other signal path.
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