用定量RT-PCR方法检测脑梗死半影区内凋亡相关基因的表达

目的 建立脑梗死后脑组织凋亡相关基因表达定量检测的精确、可靠的方法.方法 采用SYBR Green荧光定量RT-PCR法,结合免疫组织化学,测定自发性高血压大鼠脑梗死后不同时相点几种凋亡相关基因的表达水平,包括：caspase 3/CPP32、rIAP、caspase 2等,同时比较其与半影区凋亡细胞数的相关性.结果 在脑梗死半影区,凋亡细胞数量自3 h开始逐渐升高,24 h达到高峰,然后保持较高水平.而caspase 3/CPP32自8 h才升高,12 h达到高峰,3 d时便迅速回落到正常水平,而rIAP,在8 h升高一倍,然后迅速降至正常,caspase 2表达水平在脑梗死后与假手术组比较无明显改变.结论 caspase 3/CPP32是影响神经元凋亡的重要因素,但非唯一的和必不可少的,在脑缺血后,caspase 3/CPP32等的激活或许与神经元凋亡的加重有关.

Quantitative RT-PCR in detecting the apoptosis associated gene expression in penumbra area of cerebral infarction in rats

Objective To set up a comparatively precise method to measure quantitatively the expression of the apoptosis associated genes in penumbra area after permanent middle cerebral artery occlusion (MCAO) in spontaneous hypertensive rats (SHRs).Methods The fluoresce SYBR Green quantitative RT-PCR with the immunohistochemistry were used to detect the expressions of apoptosis associated genes, such as caspase-3/CPP32, rIAP and caspase-2, and to compare their correlation to counts of cerebral apoptosis cells.Results In the penumbra area, the number of apoptosis cells gradually increased as early as 3 h following the cerebral infarction and reached its peak at 24 h after MCAO, then retained at the higher level until 7 d. But caspase-3/CPP32 mRNA expression increased at 8 h after MCAO, and reached its peak at 12 h and rapidly returned to normal level at 3 d. Another apoptosis associated gene rIAP mRNA expression elevated about twice at 8 h point and returned to normal very quickly. The caspase-2 mRNA expression in penumbra area had no obvious changes comparing with control group through all time points after MCAO.Conclusions The increased expression of caspase-3/CPP32 is one of the most important but not the only necessary factor in initiating the apoptosis process after MCAO, which might be related to aggravation of neuron apoptosis.