【作者】 韩冬；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 神经病学， 2009， 博士

【摘要】 缺血性脑卒中是最常见的脑血管疾病,由于脑血流减少以及缺血后再灌注造成神经细胞的损伤,脑缺血发生后多种因素和途径在神经细胞死亡过程中发挥重要作用。星形胶质细胞与神经元有着密切的关系,在缺血发生后对神经元具有一定的保护作用。在星形胶质细胞中大量存在着缝隙连接,它由两个在相互对应的细胞膜上的半通道组成,每个半通道由六个连接蛋白亚单位组成。缝隙连接通过参与细胞间物质交换的代谢偶联和电信号传递的电偶联,进行细胞间信息传递(gap junction intercellular communication,GJIC),从而在神经细胞的生长、分化、生理功能的调节中发挥作用。缝隙连接蛋白由许多成员组成,其中Cx43在神经系统中分布尤为广泛。近年来的研究表明,在脑缺血发生后,缝隙连接、半通道及连接蛋白对神经细胞起到了有害或有益的作用,并且它们与凋亡之间又存在着一定的关系,目前国内对这方面研究少见报道,尤其是半通道和连接蛋白以及与凋亡信号通路的关系研究甚少。因此,我们希望通过特异性抑制缝隙连接蛋白表达的方法来研究Cx43蛋白、半通道、缝隙连接与凋亡以及相关的凋亡信号转导通路之间的关系。本实验通过原代培养Wistar大鼠大脑皮层星形胶质细胞,运用连续传代法建立了星形胶质细胞的体外纯化培养体系;化学合成针对大鼠Cx43基因的小干扰RNA及其阴性对照,通过脂质体转染星形胶质细胞后,采用Western blot和染料传输实验来检测干扰效果。采用缺氧孵箱法成功建立了星形胶质细胞缺氧/再复氧模型,通过流式细胞术观察siRNA干扰对细胞凋亡的影响,通过免疫组化和Western blot观察其对Caspase-3以及对ERK信号转导通路的影响。结果表明:siRNA干扰能够明显抑制缝隙连接蛋白表达及其胞间通讯功能;抑制缝隙连接蛋白,星形胶质细胞缺氧/再复氧后细胞凋亡明显减轻;抑制缝隙连接蛋白,星形胶质细胞缺氧/再复氧后p-ERK蛋白表达明显减低。表明抑制缝隙连接蛋白,能够减轻凋亡发生,起到细胞保护作用,而且影响了ERK信号转导通路。本实验将缝隙连接蛋白作为干预靶点,应用RNA干扰技术,深入探讨连接蛋白对凋亡的影响及其可能机制,丰富了脑梗死后凋亡传导途径的理论,为临床治疗脑梗死提供了理论和实验基础。更多还原

【Abstract】 Cerebrovascular disease (CVD) refers to brain lesions caused by various cerebrovascular diseases. CVD is one of the three diseases which threaten human health and is one of the main reasons leading to human’s death and disabilities. Ischemic stroke is the most common in CVD, and is mainly related to the reduction of cerebral blood flow and its ischemic injury. There are evidences that there is a close relationship between astrocytes and neurons, which is important for the survival and protection of neurons after ischemia to some extent. Astrocytes form functional syncytium by gap junctions, realize the synchronization among the information transmission, metabolic substrates changes and ionic balance. Not only can gap junctions adjust the cell functions by low selective permeability of the second messagers such as calcium, cAMP and IP3 and energy substances, but also can they allow harmful substances transfer between cells and cause the transmission of death signals. After the cerebral ischemia occurs, gap junctions are still open making metabolites and harmful substances communication between astrocytes and resulting in more cell damages. In addition, connexin and semi-channels have certain effects on cell survival. Under this background, our experiments establish the hypoxia/ reoxygenation model which is based on the successfully primary culture of the rat cortex astrocytes, discuss the effects of the inhibitive Cx43 on apoptosis of astrocytes and its related transduction signals by applying RNAi technology, and we hope to provide theoretical and experimental foundation for clinical researches on ischemic cerebrovascular diseases. Objective: To prepare primary culture model of rat cortical astrocytes, to identify purity, to transfect astrocytes by siRNA and to test interference effect. To establish astrocytes hypoxia/reoxygen model, to observe the influence of inhibitive Cx43 to astrocytes apoptosis and ERK transduction pathway. Methods: Astrocytes in primary culture were prepared from the cortex of 1-day-old newborn Wistar rats to establish astrocytes purification system in vitro. The cells were cultured in DMEM with 20% newborn calf serum. The cells were cultured in a 95% air and 5% CO2 humidified incubator at 37℃. After 3-4 replatation procedure, the cultured cells morphous were observed and GFAP immunological cells were identified chemically. Small interfering RNA and its negative RNA aim to rat Cx43 gene were synthesized chemically, transfected astrocytes by liposomes, detected Cx43 expression and gap junction intracellular communication by Western blot and dye coupling method. Then the interfering effects of siRNA to Cx43 were observed. The toxic effects of siRNA transfection to cells were detected by MTT. When the cells reached 80% confluent, the cultures were transferred into oxygen-poor incubator of O27%/CO25%/N288% for 12 hours and reoxygen were undertaken for respectively 6 hours, 12 hours, 24 hours, 48 hours and 72 hours. Flow cytometry was applied to detect the change of apoptosis rate of cell hypoxia/reoxygen at different time. Western blot was applied to observe the change of caspase-3 and ERK expression at 12 hours after hypoxia and different time of reoxygen, and then the most obvious time of expression was chose to be the proving time for next interfering experiments. At proving time, the influences of inhibitive Cx43 to astrocytes apoptosis rate, caspase-3 and p-ERK1/2 were observed by flow cytometry, immunocytochemical method and western blot. Results: 1. The primary culture cell bodies were large and flat, cytoplasms were rich, cytoplasmic processes were distinct, the branches were slender and intensive, crossed to each other and had uniform appearance. GFAP cell purity was 97.63%. According to Western blot, Cx43 expression began to inhibit 12 hours after siRNA transfection and was inhibited the most 48 hours later. According to dye coupling method, siRNA transfection group was negative, while the control group was positive. MTT results showed that, the cell activities of siRNA group and liposome group were much lower than that of untransfection group (p<0.05), what’s more, the activities of different groups increased gradually along with time. 2. The morphous of cultured astrocytes were normal 12 hours after hypoxia, but the morphous changed along with the beginning of reoxygen. The cell bodies changed to be round and protrude cell surface, and vacuoles appeared in part cytoplasm 6 hours after reoxygen. The gaps between fusion cell layers became larger, cell protuberances decreased and the network among cells were destroyed 12 hours after reoxygen. Part cell bodies became round and cell protuberances decreased or broke 24 hours after reoxygen. The destruction became worse 48 hours after reoxygen. At 12 hours after hypoxia and reoxygen, the apoptosis rate increased gradually and the apoptosis rate was highest at 48 hours after reoxygen, what’s more, siRNA group was obviously lower than control group(p<0.05). The expression of caspase-3 increased after reoxygen and was the most obvious at the 48-hour, siRNA group was obviously lower than control group(p<0.05). The expression of p-ERK1/2 increased after reoxygen and was the most obvious at the 6-hour, siRNA group was obviously lower than control group(p<0.05). TUNEL results showed that the apoptosis rate of siRNA group was obviously lower than that of control group(p<0.05). Conclusions: 1. The astrocytes purification system was established successfully. 2. siRNA transfection could inhibit Cx43 expression and GJIC effectively and RNAi technique is an effective method to research the functions of Cx43. 3. The cytotoxicity of siRNA transfection was from liposome. 4. The astrocytes hypoxia/reoxygen model in vitro was established successfully. 5. Cx43 inhibited by siRNA could distinctly lessen the apoptosis after astrocytes hypoxia/reoxygen. 6. Cx43 inhibited by siRNA could distinctly lessen the increasing expression of p-ERK1/2 after astrocytes hypoxia/ reoxygen and affected its transduction pathway.更多还原