穹隆海马伞切断与卵巢切除大鼠大脑皮质和海马CA1区酪氨酸激酶A与淀粉样β蛋白前体β位点裂解酶共存观察

Coexistence of tyrosine kinase A and beta-site beta-amyloid precursor protein cleaving enzyme in cerebral cortex and hippocampus CA1 region of rats after fimbria-fornix transection and ovariectomy

目的:观察穹隆海马伞切断加卵巢切除大鼠大脑皮质区、海马CA1区酪氨酸激酶A与淀粉样β蛋白前体β位点裂解酶共存表达的变化。方法:实验于2003-03/12在青岛大学医学院附属医院脑病防治重点实验室完成。选择成年健康雌性Wistar大鼠14只,随机分为穹隆-海马伞切断加卵巢切除组和对照组,每组7只。穹隆海马伞切断参照Klein等的方法,动物麻醉固定于脑立体定位仪后,参照Paxinos等大鼠脑立体定位图谱于前囟后2.2～2.5mm,中线外1.0mm处凿开颅骨,切开硬脑膜,自制双刃刀完全切断海马伞外侧缘。双侧卵巢切除参照Singh等的方法,麻醉动物后,打开腹腔,在肾脏下方的脂肪内取出卵巢,结扎并切除。对照组除不切断穹隆-海马伞和不切除卵巢外,其余步骤同穹隆-海马伞切断加卵巢切除组。免疫荧光双标细胞化学染色,激光共聚焦显微镜下观察大鼠大脑皮质区、海马CA1区酪氨酸激酶A、淀粉样β蛋白前体β位点裂解酶阳性细胞表达及其共存表达的变化。结果:实验过程中,无动物死亡,全部进入结果分析。穹隆-海马伞切断加卵巢切除组大鼠大脑皮质区、海马CA1区酪氨酸激酶A绿色荧光细胞数与对照组比较显著减少[皮质区(11.85±8.11),(22.14±8.68),P<0.05];[海马CA1区(11.28±3.04),(17.28±5.85),P<0.05]。淀粉样β蛋白前体蛋白β位点裂解酶红色荧光细胞数与对照组比较显著增多[皮质区(21.14±6.20),(5.57±3.10),P<0.01];[海马CA1区(12.43±6.24),(3.42±2.76),P<0.01]。海马CA1区呈现黄色荧光的双标细胞数与对照组比较显著增多[(5.71±3.64),(0.57±1.51),P<0.01],大脑皮质区呈现黄色荧光的双标细胞数与对照组比较,差异无显著性意义[(5.71±3.14),(2.85±3.07),P>0.05]。结论:穹隆海马伞切断加卵巢切除大鼠大脑皮质区、海马CA1区出现淀粉样β蛋白前体β位点裂解酶表达增多、酪氨酸激酶A表达减少,海马CA1区共存细胞表达增多现象,说明神经生长因子系统与淀粉样β蛋白前体系统可能存在内在的相互联系。

AIM： To observe the change of coexist expressions of tyrosine kinase A （TrkA） and beta-site beta-amyloid precursor protein （APP） cleaving enzyme （BACE） in the cerebral cortex and hippocampus CAt region of the rat＇s brain after fimbria-fornix transection （FFT） and ovariectomy. METHODS： The experiment was carried out in the Key Laboratory of Preventing and Treating Encephalopathy, Medical School Hospital of Qingdao University from March to December 2003. Fourteen healthy female Wistar rats were randomized into 2 groups with 7 in each： FFT plus ovariectomy group （FFTO group） and control group, In accordance with the Klein＇s method, the anesthetized rats were fixed on the brain solid positioner, and the skull opening was performed at the coordinates of the bregma-posterior 2.2-2.5 mm and lateral 1.0 mm according to the Paxinos＇ methods, After exciding the dura, the lateral border of hippocampus fimbria was cut off with the self-made double blade. The bilateral ovariectomy was conducted referring to Singh＇ s method： The abdomens of rats were opened under anesthesia, and the bilateral ovaries were taken out of the fat under the kidney, and then deligated and resected, The control rats were treated identically as those of FFTO group except FFT and ovariectomy, The changes of positive cells＇ expression and coexist expression of TrkA and BACE in hippocampus CA1 region and cortex were observed with laser confooal microscope after immunofluorescent double labeling cytochemical staining. RESULTS： All the rats were involved in the result analysis without death. In the cerebral cortex and hippocampus CA1 region of the rats＇ brain, the amounts of the immunofluorescent green cells of TrkA remarkably decreased [（Cortex： （11.85±8.11）, （22.14±8.68）, P 〈 0.05; Hippocampus CAh （11.28±3.04）, （17.28±5.85）, P 〈 0.05）, while the amounts of the immunofluorescent red cells of BACE remarkably increased in FFTO group compared with the control group [cortex： （21.14±6.20）, （5.57±3.10）, P 〈 0.01; Hippocampus CA1： （12.43±6.24）, （3.42±2.76）, P 〈 0.01]. The amounts of immunofluoreseent double labeling yellow cells in the hippoeampus CA1 region of FFTO group was obviously more than those in the control group [（5.71±3.64）, （0.57±1.51）, P 〈 0.01], However, there was no significant difference in the amounts of immunofluorescent double labeling yellow ceils of cerebral cortex between the two groups [（5.71±3.14）, （2.85±3.07）, P 〉 0.05]. CONCLUSION： The BACE expressions increase but the TrkA expressions decrease in the hippocampus CA1 region and cerebral cortex of rats after FFT and ovarieetomy. The increased expression of coexist cells in the hippocampus CA1 region means there is possibly an internal relationship between the system of nerve growth factor and the system of beta-APP.