Differential Notch Expression In BAVM And The Roles Of NCEx In EICH Rats And Hypoxia Injured HBVEC

Part IDifferential Notch signalling pathway gene expression of nidus and adjacent brain tissure parts in human brain arteriovenous malformationsOBJECTIVE:To investigate the expression of genes involved in Notch signalling pathway of different parts in human brain arteriovenousmalformations(BAVM) by using Notch signalling pathway microarray. METHODS:Five BAVM cases with stroke history were collected, whose sample of nidus (N) and adjacent brain tissure (B) were obtained in surgery. Signal intensity of the genes were examed and calculated by using Oligo GE Array Human Notch Signaling Pathway Microarray OHS-059. The gene whose ratio N/B larger than1.5or smaller than0.67was counted as markedly changed, two of which were verified by realtime PCR.RESULTES:Twenty seven of113genes involved in Notch signalling pathway are found differential expressed. Among those, four genes were up-regulated, twenty three genes were down-regulated. Most important genes include:Notch signalling pathway ligands DLL1/DLL3, key genes of cleavage ADAM10/ADAM17, Notch signalling pathway target gene HES5, Notch related Sonic Hedgehog pathway gene GLI1, Wnt signalling pathway gene FZD1, oncogene LM02. Data from verifying realtime PCR on two genes DLL1and HES5consisted with data from gene chip. Some important genes involved in Notch signalling pathway did not show differential expression at the two parts of BAVM.CONCLUSIONS:Many of the genes involved in Notch signalling pathway were differential expressed at nidus and adjacent brain tissue parts in BAVM, indicating the relationship between Notch signalling pathway and BAVM occurrence and developing. Notch signallling pathway gene expression pattern might be different at adjacent brain tissure. Differential expressed genes could contribute new ways to BAVM research. Part IISpecific expression of a New Sodium-calcium Exchanger-NCEx and other related factors in experimental intracerebral hemorrhage model of ratsOBJECTIVE:This study is to investigate early changes and typical time courses of NCEx and intracellular calcium concentration and other factors after experimental intracerebral hemorrhage(EICH) such as human glucose metablosism related protein-1(GMRP-1), Notch signalling pathway genes Notch4and ADAM10. The role of above factors in pathophysiological changes after EICH and their relationship were preliminarily studied.METHODS:EICH model was established as usual. After that, four parts of experiments were performed.1. Realtime PCR and Western blot were used to test the mRNA and protein level of NCEx in hemorrhagic side and contralateral side caudate nucleus, as well as right side of control group, at6h, dayl, day3, day5, day7after EICH establishment, respectively; Immunohistochemistry was used to analyze the staining and morphologic changes of NCEx in hemorrhagic side and contralateral side caudate nucleus, as well as right side of control group, at6h, day3, day7after EICH, respectively.2. Western blot was used to test the protein level of GMRP-1in hemorrhagic side and contralateral side caudate nucleus, as well as right side of control group, at6h, dayl, day3, day5, day7after EICH establishment, respectively.3. Fura-2/AM loaded, dual wavelength spectrophotofluorometry was used to test the intracellular calcium concentration in hemorrhagic side and contralateral side caudate nucleus, as well as right side of control group, at6h, dayl, day3, day5, day7after EICH, respectively.4. Realtime PCR was used to test the mRNA level of ADAM10and Notch4in hemorrhagic side and contralateral side caudate nucleus, as well as right side of control group, at6h, day3, day7after EICH, respectively.RESULTES:1. NCEx mRNA existence could be examed normally in caudate nucleus of rats. After EICH establishment, NCEx mRNA level decreased at6h and down to the lowest at day3, then back to the normal level at day7. The mRNA level of NCEx in hemorrhagic side was, especially3times at day3, lower than contralateral side. The data from western blot test accorded with the data above. Immunohistochemistry study showed lower and sparse density of ipsilateral side rather than contralateral side.2. GMRP-1protein existence could be examed normally in caudate nucleus of rats. After EICH, GMRP-1protein level decreased at6h and down to the lowest at dayl to day5, then back to the normal level at day7. The protein level of GMRP-1in hemorrhagic side was, except for day7, lower than contralateral side, especially four times at dayl.3. The tested intracellular calcium concentration of caudate nucleus was (277.1±22.0)nmol/L normally, which elevated to the highest level of (887.5±52.2) nmol/L at day3followed with a decrease at day5and day7. The correlation ratio of NCEx and calcium concentration was0.66(P<0.05).4. ADAM10and Notch4mRNA existence could be examed normally in caudate nucleus of rats. After EICH, either mRNA level decreased to the lowest at6h, then back to normal level at day3and to above at day7. CONCLUSIONS:Intracellular calcium concentration modulator NCEx, cell apoptosis/proliferation controller GMRP-1, cell differentiation and remodeling modulator Notch4and ADAM10, showed specific expression pattern after EICH. NCEx level correlated with calcium concentration. The dynamic expression pattern of NCEx, GMRP-1and Notch signalling pathway genes, might play an important role in early changes of cell disfunction and apoptosis after EICH and cell/tissure regeneration, proliferation and remodeling later. Part ⅢThe influence of hypoxia on the expression of NCEx in Human brain vascular endochelial cellsOBJECTIVE:To perform the preliminary survey of relationship between hypoxia, NCEX, vascular endothelial cell disfunction by investigating NCEx expression and intracellular calcium concentraion changes in Human brain vascular endothelial cells(HBVEC) cultured under hypoxia condition.METHODS:HBVECs cryopreservated were resuscitated and cultured, then randomized into two groups of normal culture and hypoxia culture, which was performed as treating cells with normal medium and anoxic incubator. Western blot was used to exam NCEx protein level; Fura-2/AM loaded, dual wavelength spectrophotofluorometry was used to exam the intracellular calcium concentration.RESULTES:HBVECs resuscitation and passage were successfully performed. Floating and dead HBVECs were rarely observed after cultured under hypoxia condition. NCEx protein existence could be examed in normally cultured. A remarkable downregulation of NCEx protein level was detected, which was0.16times of normal level, after cultured under hypoxia condition for24h. The intracellular calcium concentration elevated to2.2times above normal level.CONCLUSIONS:An HBVECs model of hypoxia injury could be performed as treating cells with normal medium and anoxic incubator for24h. Hypoxia could influence and downregulate NCEx mRNA, while elevating intracellular calcium concentration. These two variable factors might correlated with each other.