【作者】 王伟；

【导师】 姚鑫；

【作者基本信息】 天津医科大学 ， 外科学， 2013， 硕士

【摘要】 目的：利用活体成像技术研究牛磺酸在重型颅脑创伤裸鼠体内实时代谢分布变化情况,并观察脑创伤后脑组织TauT、GFAP表达,探讨牛磺酸对重型颅脑创伤的治疗机制。方法：本课题分为活体成像和免疫组化两部分,①活体成像部分：取6-8周龄BALB/C裸鼠32只,随机数字表法分为正常(normal)组、假手术(sham)组、脑创伤染料(TBI-Cy7)组和牛磺酸治疗(TBI-Tau)组。后两组用液压打击仪制作左侧脑创伤模型,sham组在左侧相同位置只开骨窗,不予打击。TBI-Tau组、sham组伤后立即尾静脉注射Cy7-Tau, normal组不做处理,直接注射Cy7-Tau, TBI-Cy7组注射游离Cy7,剂量均为5mg/Kg。给药后1、2、4、8、12、24h行活体成像；②免疫组化部分：选用6-8周龄BALB/C小鼠48只,随机数字表法分为假手术(sham)组、脑创伤(TBI)组和牛磺酸治疗(TAU)和,造模同上,TAU组、sham组伤后立即尾静脉注射Tau (5mg/Kg), TBI组注射无菌生理盐水(5mg/Kg)。给药后12h、24h每组各处死8只,4%多聚甲醛灌注后冰冻连续切片,行HE染色、GFAP-DAPI、TauT荧光免疫组化染色。结果：1.活体成像：给药后Tau在正常裸鼠体内呈全身弥漫分布,主要通过肾脏代谢,12h大部分均可排出体外,仅剩胆囊存较高浓度Tau; sham组手术切口皮缘24h仍有较高浓度Tau富集；TBI-Tau组给药后24h脑创伤区域、皮肤切口处有Tau特异富集,Tau在离体脑组织的分布范围与脑创伤区域一致；Cy7在裸鼠体内弥漫分布于皮下,无特定器官富集趋势；2.HE染色：sham组神经细胞形态、结构正常,未见明显脑创伤；TBI组脑组织充血、水肿,灰质和白质弥漫性出血灶,脑皮质分层结构消失,灰白质分界不清,并伴有大量多形核炎性细胞聚集；Tau组脑组织轻度充血、水肿,实质内有局灶性出血,并伴有多形核炎性细胞聚集。3. TauT荧光免疫组化结果：TBI后12h,各组均有TauT表达于伤侧大脑皮质和海马的血管内皮细胞,偶见个别神经细胞,与Sham组比较,TBI组12h的皮质和海马中TauT显著降低(p<0.05),TAU组的TauT均显著高于TBI组(p<0.05),伤后24h,与sham组相比,TBI组脑皮质无显著变化,海马显著降低,TAU组TauT表达较sham组显著增高,荧光位于血管内皮细胞及少数神经细胞,数量增多,荧光强度增强。4. GFAP荧光免疫组化结果：TBI后12小时,sham组中,星形胶质细胞内GFAP阳性反应较低,细胞胞体较小,突起纤细。反应性星形细胞主要局限于TBI组、TAU组脑创伤侧胼胝体及海马,表现为细胞GFAP阳性反应增加,染色加深,突起增多。伤后24小时,TBI组、TAU组星形细胞胞体肥大,GFAP阳性反应细胞明显增加,在海马组织中尤其明显。无论胼胝体还是海马组织,TBI组的GFAP在12h、24h均显著较sham组升高(p<0.05),而Tau组与TBI组无显著区别(p>0.05)。结论：1.活体成像技术可实时监测Tau在裸鼠体内的分布、代谢,Tau主要通过肾脏代谢；TBI后Tau特异性富集于脑创伤区域、皮肤切口处,离体脑组织Tau的分布范围与脑创伤区域一致；2.重型颅脑创伤后小鼠脑组织形态学发生明显改变,皮层、海马区神经细胞结构受损严重,予牛磺酸治疗后24h可见神经细胞形态损伤有所减轻,神经细胞超微结构趋于正常,受损轻微,从形态学方面表现出了较好的治疗作用；3.TBI后TauT表达于伤侧大脑皮质及海马的血管内皮细胞、个别神经元,TBI对脑创伤后TauT的表达存在明显抑制作用,外源性Tau可促进TBI后12h、24h创伤侧脑组织皮质和海马中TauT的表达;4.TBI后12h、24h内胼胝体和海马中GFAP表达显著升高,反应性星形细胞局限表达于脑创伤区及伤侧海马,Tau不能降低TBI后急性期的胶质细胞增生。更多还原

【Abstract】 Objective To conduct an in vivo optical imaging analysis of the metabolism and biodistribution of taurine in nude mouse with severe traumatic brain injury, to observe the expression of TauT and GF AP after brain trauma,Methods This study includes two experimental parts, one is in vivo optical imaging, the other is immunohistochemistry.32nude mice were randomly divided into normal group, sham operation group, brain trauma group, taurine treatment group. TBI models were made by fluid-percussion. All liquid used was injected via caudal vein immediately after TBI. In vivo optical imaging system was used to observe the distribution and metabolism of taurine at1,2,4,8,12and24h after injection. Then, the nude mice were executed, fluorescence Imaging of the isolated organ was conducted. The second part,48BALB/C mice were randomly divided into normal group, sham operation group and taurine treatment group. The building of TBI model and injection of taurine were the same with the in vivo optical imaging experiment.8mice of each group were executed at12h and24h after TBI. Morphology change of brain was observed by HE staining. The expression of TauT and GF AP was analysed by immumohistochemical staining.Results1.In group normal, Tau diffused in the whole body after injection, and the majority of Tau was excreted through kidney within12h, only the gallbladder with high concentration Tau. Tau in group sham still accumulated at skin incision margin24h after TBI. High intensity of Tau still accumulated at brain trauma area24h after TBI in group TBI-Tau. In group TBI-Cy7, Cy7diffused in subcutaneouse, without concentration in any organ.2. In group sham, there was no obvious damage in cellular morphology and structure. The ultrastructure of neuron and glia of group TBI-Cy7were severely destroyed. In group TBI-Tau, mild hyperemia, edema observed in traumatic brain tissue, with focal hemorrhage, and accompanied by polymorphonuclear inflammatory cells.3.12h after TBI, TauT expressed in vascular endothelial cell and several nerve cells in cerebral cortex and hippocampus of traumatic brain tissue. Compare with group sham, the TauT of group TBI was lower both in cerebral cortex and hippocampus (p<0.05). In group TAU, the TauT was remarkably higher than group TBI (p<0.05).24h after TBI, compare with group sham, there was no evident difference in cerebral cortex, and the TauT in hippocampus was remarkably lower in group TBI.4.12h after TBI, reactive astrocytes mainly limited in corpus callosum and hippocampus of brain trauma side of group TBI and TAU,24h after TBI, in group TBI and TAU, GFAP positive cells increased significantly, especially in the hippocampus. Both at12h and24h, the GFAP in group TBI and TAU was remarkably higher than group sham, and there were no significant differences between them.Conclusion1. The in vivo optical imaging technology can accurately monitor the distribution and metabolism of Tau in nude mouse. The majority of Tau was excreted through kidney. After TBI, Tau could accumulate specifically at brain trauma area and skin incision margin. The distribution of Tau in in vitro brain tissue was consistent with brain trauma area.2. Tau could effectively alleviate the damage of neural cells24h after TBI, and the ultrastructure of neuron and glia returned to normal, which showed effective therapeutic effect of Tau from morphology aspect.3. TauT expressed in vascular endothelial cell and several neural cells in cerebral cortex and hippocampus of brain trauma area. TBI could obviously inhibit expression of TauT, and exogenous Tau could promote TauT expression in cerebral cortex and hippocampus of brain trauma area.4.12h after TBI, the expression of GFAP was significantly increased in corpus callosum and hippocampus of brain trauma area, Tau could’t reduce the proliferation of glial cells in the acute phase of TBI.更多还原