【作者】 李楠；

【导师】 刘亢丁；

【作者基本信息】 吉林大学 ， 神经病学， 2015， 博士

【摘要】 目的：观察IVH后TGF-β1、MMP9及脑内铁代谢的变化，探讨其在IVH后脑室损伤及细胞凋亡中的作用。方法：（1）健康雄性Wistar大鼠84只，随机分为：正常组，盐水对照组术后1d、3d、7d、28d，脑室注血组术后1d、3d、7d、28d共9组，130ul自体尾动脉血二次注射法制作大鼠右侧侧脑室出血模型，造模后不同时间点，大体标本、扫描电镜及HE染色法评价脑室损伤情况；（2）Wistar大鼠108只，随机分为：正常组、盐水对照组术后1d、3d、7d、28d，脑室注血组术后1d、3d、7d、28d共9组，荧光实时定量PCR法检测TGF-β1mRNA水平，Western blot法检测MMP9表达变化；（3）Wistar大鼠25只，随机分为盐水对照组和脑室注血组术后1d、3d、7d、28d（n=5）。红菲绕啉法测定血清及脑组织非血红素铁含量；（4）Wistar大鼠162只，随机分为正常组、盐水组术后1d、3d、7d、28d，脑室注血组术后1d、3d、7d、28d共9组，荧光实时定量PCR、Western Blot及免疫荧光染色法检测脑室周围铁蛋白（Fn）、转铁蛋白（Tf）、转铁蛋白受体1（TfR1）、二价金属离子转运体1（DMT1）、铁调节蛋白2（IRP2）、血红素加氧酶1（HO1）表达变化；8-OHdG免疫荧光染色及Tunel法检测细胞凋亡情况；（5）Wistar大鼠18只，随机分为对照组、脑室出血后去铁胺治疗组和盐水对照组。电镜下观察脑室壁结构变化，Tunel法检测细胞凋亡情况。结果：（1）对照组脑室及其周围组织无明显变化，室管膜细胞排列整齐，组织结构清晰，脑室注血组大鼠术后1d大体及HE染色见右侧脑室内充满积血，脑室周围白质水肿，室管膜细胞排列紊乱，松散；术后3d侧脑室积血减少，室管膜部分断裂，可见脑室膜细胞增生；术后28d胶质细胞增生及室管膜断裂均较明显。电镜下对照组纤毛致密，排列规则有序，基膜完整；脑室注血组纤毛紊乱、断裂，基膜脱落逐渐加重。（2）术后1天TGF-β1mRNA水平即明显上升，显著高于盐水组（P<0.01），术后3天TGF-β1mRNA水平较术后1d明显下降（P<0.001），但仍高于对照组（P<0.05），术后7d TGF-β1mRNA水平再次上调，形成第二高峰，术后28dTGF-β1mRNA水平再次下降，但仍高于盐水对照组，差异具有统计学意义（P<0.05）；术后1d MMP9蛋白含量即开始上升，与盐水组比较差异具有显著性（P<0.05），随术后时间延长，MMP9蛋白表达持续增加，至术后28天达高峰，术后28天MMP9蛋白含量与盐水组比较差异显著（P<0.001）。(3)IVH后血清铁含量组间比较差异具有显著性（P<0.01），但各组与对照组相比均无差别。IVH术后1d脑铁含量即明显增加，术后7d表达量最高，之后持续高表达至术后28d。(4)Tf mRNA水平随术后时间延长有升高趋势，术后1d即显著高于对照组，术后3d达高峰，之后逐渐下降，28d时明显低于对照组。Tf术后1d表达较对照组增多，3d达高峰，7d开始下降，至28d表达量明显低于对照组（P<0.01）。TfR1术后3d表达量下降，之后表达上调，术后28d与术后7d相比无明显变化但均高于对照组。DMT1mRNA水平术后第1d明显低于对照组（P<0.01），随术后时间延长继续下降，术后7d表达量最低，术后28d较前恢复，与对照组比较无明显变化。DMT1术后1d表达与对照组比较无明显变化，之后随时间延长表达量下降，至术后7d最低，明显低于对照组，术后28d基本恢复正常，与对照组相比无明显差异。IRP2mRNA水平术后第1d较对照组下降，之后持续下降至术后7d达到最低水平，术后28d较前恢复，但仍明显低于对照组（P<0.001）。IRP2蛋白术后1d表达与对照组比较无明显变化，之后随时间延长表达量下降，术后3d表达量最低，术后28d基本恢复正常，与对照组相比无明显变化。术后1d HO1表达量较对照组明显上升，3d略有下降，但与术后1d相比无显著性差异；术后7d表达较3d明显下降，术后28d表达仍高于正常组。（5）8-OHdG在对照组阳性率较低，随术后时间延长，阳性率持续增加，术后7d与28d相比无明显差异，均明显高于正常。海马及脑室周围组织Tunel染色变化基本一致，对照组阳性率较低，术后1d阳性率即较对照组增高，之后阳性表达持续增加，至术后28d阳性率最高。（6）脑室出血后7d盐水治疗组电镜下脑室顶璧纤毛变细、断裂明显，基膜脱落，基膜下组织裸露；DFO治疗组纤毛断裂减轻，基膜脱落不明显；Tunel染色阳性率DFO治疗组较盐水组明显降低。结论：（1）IVH后脑室周围组织病理改变早期以水肿为主，后期增生逐渐明显；（2）IVH后脑室损伤、扩张可能与TGF-β1、MMP9表达失衡有关；（3）铁代谢紊乱在IVH后脑室损伤及细胞凋亡中发挥重要作用；（4）去铁胺治疗能减轻IVH后脑室壁损伤及细胞凋亡。更多还原

【Abstract】 Objective:To observe the change of TGF-β1、MMP9and iron metabolism in rats secondaryto intraventricular hemorrhage and to investigate the mechanism of the ventriculardamage and apoptosis after IVH.Methods:(1)84male Wistar rats were randomly divided into9groups:normal、shamsurgery（1d,3d,7d,28d after saline injection）and IVH groups(1d,3d,7d,28d afteroperation).Total130ul autologous tail arterial blood was injected into right ventriclefor two times to establish animal models. And sham groups animals were injected thesame amount of saline.Brain specimen, electron microscopy scanning and HE stainingwers used to evaluate ventricle damage at the different time points after sugery.(2)108male Wistar rats were randomly assigned to: normal、sham surger（y1d,3d,7d,28d after saline injection） and IVH groups（1d,3d,7d,28d after operation）. Wedetected the TGF-β1mRNA level with real-time PCR and the expression of MMP9with Western blot.(3)25male Wistar rats were randomly divided into sham surgery and IVH groups(1d,3d,7d,28d after operation).Bathophenanthroline was used to measure the serumand brain nonheme iron content.(4)162male Wistar rats were randomly assigned to normal、sham surgery andIVH groups (1d,3d,7d,28d after operation).Real-time PCR, Western Blot andimmunofluorescence staining were used to detect ferritin (Fn), transferrin (Tf),transferrin receptor1(TfR1), divalent metal ions transporter1(DMT1), ironregulatory protein2(IRP2), heme oxygenase1(HO1) expression in tissues aroundthe ventricle. immunofluorescence staining for8-OHdG and Tunel staining wereused for apoptosis detection.(5)18Wistar rats were randomly divided into normal、deferoxamine treatmentand placebo groups after IVH. We observed ventricles under electron microscope andapoptosis was detected by Tunel. Results:(1)Ventricular wall and its surrounding tissues of control groups were normalwith ependymal cells in alignment and structure organized. In IVH groups it’sshowed that the ventricle full of blood.We can also observed white matter edemaaround ventricle and disordered arrangement of ependymal cells1d after operation.3days after surgery lateral ventricle hematocele begin to scatter.ependymal fracture andependyma cells hyperplasia can also be found. glial cell hyperplasia and ependymalfracture were even more apparent at day28.Under the electron microscope,cilia wasdense、 regular and orderly and basement membrane was intact in controlgroups.While in IVH groups cilia was disorder and fractured and the basementmembrane was bare.(2)Postoperative1day TGF-β1mRNA rise obviously and significantly higherthan that of saline group (P <0.01).Then it significantly decreased (P <0.001)3dayslater, but still higher than the normal(P <0.05).7d after surgery TGF-β1mRNA riseagain to form the second peak and fell at days28.But it’s still higher than salinegroup.The differences between levels on28d and control groups were statisticallysignificant (P <0.05). MMP9protein rised from day1with a significant differencecompared to saline group (P <0.05).Then with the time gone its expression continueincrease and peaked at day28.(3)Serum iron concentration between groups were significantly differente afterthe IVH (P <0.01),while each group was undifferentiated compared to control group.1day postoperative brain iron content increased and peaked at7day. Then thequantity of iron in brain was high continuously even at day28.(4) Tf mRNA increased over time after operation. It’s significantly higher on day1than that of control group. Tf mRNA reach its peak at3day and then graduallydecline While obviously lower than that of the control group.Tf protein wasincreased at day1and peaked at day3.It declined from day7and the quantity wasobviously lower compared to the control group (P <0.01).TfR1decreased aftersurgery.It underwent the minimum at day3and then upregulated with time go on.There’sno difference between day7and day28but they were all higher than the control group.DMT1mRNA continued to fall with the time go on. It was significantly lowerthan the control group at day1(P <0.01) and got minimum at day7.It seemd to getrecovery at day28with no significant change compared to the control group.DMT1expression has no obvious change compared with controls at day1and thendecreased.Its value was minimum at day7and significantly lower than the controlgroup. It seemed to be normal at day28with no statistical difference compared withcontrol group.IRP2mRNA was also decreased after IVH.It fell to the lowest7d aftersurgery.The level of mRNA recover to some extent, but still significantly lower thanthe control group (P <0.001). A same trend of IRP protein was appeared. HO1increased significantly than the control group1day after IVH while fell slightly atday3with no significant difference compared to the level of day1.It was still higherthan normal group28days later.(5)8-OHdG positive cells in the control group were very little. Then the positiverate increased continuously. And there was no significant difference between day7and28.The trends of Tunel staining for hippocampal and ventricle surrounding tissueswere consistent. The low positive rate was very low in the control group thenincreased significantly and peaked at day28.（6）When observed under electron microscope7d after IVH and salinetreatment. It’s found that ventricular wall cilia was attenuated and fractured withbasal membrane peeling. Change in DFO treatment group was less obvious; Thepositive rate of tunel staining in DFO treatment group was significantly declineingcompared with the saline group.Conclusions:(1)The early pathological change of ventricle surrounding tissues after IVH isedema and hyperplasia was obvious gradually with time go on;(2)The ventricular injury caused by IVH may be associated with the imbalanceof TGF-β1and MMP9;(3)Iron metabolism disorder and iron overload plays an important role on theventriclar damage and apoptosis secondary to IVH;(4)Deferoxamine treatment can reduce the brain ventricular wall injury and apoptosis after IVH.更多还原