| 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.
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