| Abstract:bjective:To observe the effects on cerebral ischemia-reperfusion(CIR) injury after transfection of heme oxygenase-1 gene in rat for discussing the therapy prospect of regulating the heme oxygenase gene to stroke in practice. Methods:Adenovirus Vector Production:HO-1 gene was cloned by using the reverse-transcription polymerase chain reaction (RT-PCR) method from the total RNA extracted from rat spleen cells.According to published GeneBank sequence of rat HO-1(J02722) the primer sequence was designed. Adenoviral vectors carrying a rat HO-1 gene were constructed by Adenovirus-bacterial recombination system (AdEasy).The viral titers of Ad-Ho-1 and Ad-GFP were 2.5×1011 pfu/ml and 2.0×1011 pfu/ml.Male Sprague Dawley (SD) rats (n=78) were randomly divided into four groups: sham group (SH),vehicle group (V), empty adenovirus vector group (Ad) and Ad-HO-1 transfection group(18 each group).Rats of V, Ad and Ad-HO-1 groups were respectively injected with 20μl saline,20μl saline containing 1μl of empty vector adenovirus or 1μl of recombinant HO-1 adenovirus through the right ventricle for three days before ischemia. Injection site was reference to Paxinos and Watson rat brain stereotactic atlas.Then,cerebral ischemia-reperfusion was established by middle cerebral artery occlusion (MCAO) according to a method Zea—Longa described.Under halothane anesthesia (induction 2%, maintenance:2% in an oxygen and nitrous oxide 30:70 mixture), a nylon monofilament was introduced into internal carotid artery to occlude the middle cerebral artery. Two hours later, the filament was withdrawn and the wound was sutured.Rectal temperature was maintained at physiological levels (36.5-37.5℃) using a thermal blanket. Regional cerebral blood flow (rCBF) was measured using a laser Doppler flow meter probe.Neurological deficits were evaluated at 24 hours after ischemia using the scoring system reported by Garcia. Then the brain tissue was removed and was sectioned into slices, infarct volume was evaluated with 2,3,5-triphenyltet-razolium hydrochloride (TTC) staining.Infarct volumes were expressed as percentage of the contralateral hemisphere.Two hours after ischemia, brain tissue was homogenized.The homogenates were using to detect neuronal caspase-3 activity.24h after ischemia, brain tissue sections were using to detect apoptotic cells by TUNEL stain, GFP expression was observed by fluorescence microscope, and calculated the transfection rate.The expressions of HO-1 in brain tissue were detected by Western blot. Results:Western blot show the change of heme oxygenase-1 level after transfection:In HO gene treated rats, heme oxygenase-1 expression was significantly increased compared with that in vehicle and Ad groups.The HO-1 expression in Ad group was no difference than those in and V group.Expression of green fluorescent protein show transfection rate:Fluorescent protein expression was seen in brain tissue in both Ad group and HO group with no significant difference,and the transfection rate was 34.5%±3.4%. The neurological behavior score:The neurological behavior score among 4 groups:18,9.571±0.254,9.857±0.149,12.75±0.211.The neurological behavior score of HO group were significantly higher than those in Ad group and V group (P<0.001).It is no significant difference between Ad group and V group.Infarction volume and neuronal apoptosis:a. Percentage of infarction volume among 4 groups:0%,60.36±6.750%,61.80±7.544%,27.31±2.913%.Compared with the SH group, volume of cerebral infarction in V group and Ad group increased. Compared with V group or Ad group, these indexes in HO group significant reduced (P< 0.01).Compared with V group or Ad group, these indexes in HO group significant reduced (P<0.01).Those indexes were no significant difference between Ad group and V group.b.Neuronal apoptosis:Rate of apoptosis positive staining cell and Caspase-3 activity in caudate putamen,CAl of hippocampal and cortex among 4 groups:SH group:<0.1%/0.800±0.170, <0.1%/0.441±0.193,<0.1%/0.674±0.186.V group:(53.39±1.742)%/8.889±0.352,(34.81±1.206)%/7.750±0.446,(57.67±2.591)%/7.144±0.424.Ad group: (52.27±1.051)%/8.273±0.359,(36.31±2.210)%/8.284±0.264,(59.33±2.951)%/ 7.744±0.398。HO group:(46.55±1.001)%/6.217±0.313,(27.31±1.382)%/ 6.428±0.342, (48.94±2.120)%/6.472±0.347.Compared with the SH group, the. neuronal apoptosis in V group and Ad group increased.Compared with V group or Ad group,these indexes in HO group significant reduced (P<0.01). Compared with V group or Ad group, these indexes in HO group significant reduced (P<0.01).Those indexes were no significant difference between Ad group and V group.Conclusion:Adenoviral vectors carrying a rat HO-1 gene were constructed by AdEasy Vector System; the stabled animal models were established by transient occlusion of MCA according to a method Zea—Longa described. Brain stereotactic intraventricular injection of HO-1 gene carried by adenovirus can be effectively transfected into the brain tissue and express stably in vivo.Ad-HO-lhas neuroprotective effect against cerebral ischemic reperfusion in rats.The neuroprotective role of them to CIR may contribute to the increased expression of HO-1 protein. High expression of HO-1 can play an early (2h after reperfusion)anti-apoptosis effect may translate into clinically meaningful infarct reduction at a later time point (24h after reperfusion).
|
PDF Full Text Request