1.Studies On The Relationship Between Expression Of Hypoxia Inducible Factor 1 And Hypoxia/reoxygenation Injury On PC12 Cells 2.Effect Of Snake Venom Thrombin-like Nzyme From Agristrodom Acutus In Fujian China On Hemostatic Action And It's Mechanism

Hypoxia induced injury on neuron cells, however reoxygenation maked the cells damage even worsen There are many reasons in hypoxia/reoxygenation injury .but the exact mechanism should be studied further.Hypoxia inducible factor l(HIF-l) is composed of HIF-1 alpha subunit and HIF-1 beta subunit. HIF-1 alpha is specific regulated oxygen subunit, which determined activity of HIF-1. The transcription factor mediates hypoxic induction of genes encoding certain vascular endothelial growth factor , erythropoietin (Epo) and tyroxine hydroxylase , which is important regulator to gene expression of vascular growth, blood steam accommodation, energy metabolism.We used subculturing PC 12 cells to replace for primary neuron cells culture. Cobalt chloride could mimic hypoxia , removal of cobalt chloride could imitate reoxygenation. We studied relationship between HIF-1 and hypoxia/reoxygenation injury and the influence of antisense RNA on expression of FflF-la protein .It is very important to biological treatment of angiogenesis diseases such as hypoxic/ ischemic cerebral diseases and cerebral ischemia/ reperfusion.PC 12 cells were treated with cobalt chloride (125uM) for 0,1, 2,4,8,12,16 hours. We measured leakage of Lactic dehydrogenase(LDH) in cell culture medium,and collected cells to observe ultraconstructure with electron microscope .During hypoxic exposure,the leakage of LDH was gradually increased , peaked at 8h ,then decreased. The same results were supported by electron telescope. At the same time, we used RT-PCR to observe the expression of HIF-1 a rnRNA. We found HIF-1 a mRNA was expressedin normoxic condition. After hypoxia exposure,it increased time-dependentedly , peaked at 4h, then decreased.In order to explore relationship between HIF-1 and hypoxia/reoxygenation injury on neuron cells further, we observed leakage of LDH, alteration of ultraconstructure, expression of rnRNA. The leakage of LDH and alteration of ultraconstructure were remarkable at 8h reoxygenation. On the contrary, the expression of HIF-1 a mRNA decreased to the lowest level at 4h reoxygenation. The results suggest that HIF-1 a exert certain protective effect on hypoxia/ reoxygenation injury on PC 12 cells, but the exact mechanism is being studied.Based on aboval researches, we observed the expression of HIF-la protein with laser sym-focus fluorescence microscope. After different hypoxia exposure, the presence and strength of fluorescence were time-dependented, peaked at 8h, then weakened. However it was decreased to the baseline at 8h reoxygenation, the same as the results of RT-PCR.To study the influence of antisense RNA on the expression of HIF-1 a protein ,we devised sense , antisense RNA probes. PC 12 cells were randomly divided into three experimental groups(control, sense RNA, antisense RNA). After incubate with cobalt chloride for 0,1, 2,4,8,12,16 hours, PC 12 cells were collected,fixed, blocked with first antibody of HIF-1 a and secondary antibody. The expression of HIF-1 a protein was significant both in cyto-plasm and nucleus, and was inhibited at 4h,8h reoxygenation after treated with the same amount of antisense RNA. This inhibition was remarkable mainly in nucleus .These results suggest that HIF-1 exert certain protective effects on hypoxia/reoxygenation injury on PC 12 cells. Antisense RNA of HIF-1 a can inhibit the expression of HIF-1 a protein after hypoxiaexposure.