大鼠颅脑损伤后Caspase-9的表达变化及其法医学意义

The expression of Caspase-9 in rats after traumatic brain injury and its forensic significance

目的探讨大鼠颅脑损伤后Caspase-9随时间变化的规律及其法医学意义。方法将60只雄性SD大鼠随机分为正常对照组、假损伤组以及损伤后1、6、12h及1、2、3、5、7、10d和14d共12组。采用免疫组织化学SP方法、ELISA和图像分析技术观测大鼠脑外伤后不同时间段平均光密度值、阳性面积比及血清中Caspase-9含量,并对所测数据进行统计学分析。结果 ELISA检测结果,正常对照组血清Caspase-9含量为(8.75±2.37)ng/mL,伤后12h组血清中Caspase-9含量明显高于对正常对照组,至伤后1d组血清中Caspase-9含量达到高峰,为(45.23±3.31)ng/mL,此后血清中Caspase-9含量逐渐下降,至伤后7d组血清含量降至正常范围。Caspase-9免疫组织化学染色结果,脑损伤后1～6h,损伤区周围及海马可见个别Caspase-9细胞,着色较浅,损伤后12h,出现较多的Caspase-9阳性产物,损伤至1d时阳性细胞进一步增多,达到高峰,染色明显加深,伤后2～5d有很明显的Caspase-9阳性细胞,染色较深,伤后7～14d,阳性细胞明显减少。同比之下,假损伤组阳性反应物的平均光密度值及阳性面积比均高于正常组,但差异无统计学意义(P>0.05),而在损伤1～12h之间,损伤组阳性反应物的平均光密度值及阳性面积比逐渐升高,在1d时达到高峰,分别为(0.424±0.017)OD和(19.31±1.96)%,且与正常对照组及假损伤组相比较,差异均具有统计学意义(P<0.05),之后随着时间的延长,阳性反应物的平均光密度值及阳性面积比逐渐下降。结论大鼠脑外伤后Caspase-9的表达呈现出升高-高峰期-逐渐下降期的规律性变化,可能为法医学损伤时间推断和颅脑损伤鉴定提供一种新的指标。

Objective To analyze the rule of Caspase-9 changes with time after rats craniocerebral injury and its significance of forensic medicine.Methods 60 male SD rats were randomly divided into normal control group,Shan injury group and the injury 1,6,12 h,1,2,3,5,7,10 d and 14 d of 12 groups.Observing rats after trauma,the average light density value different period,and the masculine area ratio and serum Caspase-9 content were examined by immuno-histochemical SP method,ELISA and image analysis technology,and the data were analyzed by statistical analysis.Results ELISA test results showed that serum Caspase-9 content of normal control group is（8.75±2.37） ng/mL,Caspase-9 in serum with the group of 12 h after injury were significantly higher than the normal control group.And the content of Caspase-9 in serum reached a peak（45.23±3.31） ng/mLafter 1 d.Then Caspase-9 content in serum gradually declined,until to the normal range after the 7 d.Caspase-9 immunohistochemical results after 1~6 h,Caspase-9 cells,appeared individually in the damaged area and around the hippocampus,coloring was shallow.And after 12 h,it appeared more Caspase-9 positive product,the injury after 1 d positive cells further increased,peak dying obviously deepened,and the injury after 2~5 d there was clear Caspase-9 positive cells,dying deeply.7 to 14 d after injury,positive cells obviously reduced.1 h and 12 h after injury,the average optical density value and ratio of positive area after injury were rising gradually,and reached a peak in 1 d,respectively,it were 0.424±0.017 OD and 19.31±1.96%,all of it were higher than the normal control group and the shan injury（P0.05）.Then it declined gradually with the prolongation of time.Conclusion Caspase-9 showed increasing-the peak-the gradual decline in the regularity of period changes after traumatic brain injury in rats,and it may provide a new target for forensic wound age estimation and identification of traumatic brain injury.