颅脑损伤后脑糖代谢及神经特异性标志物相关研究

发布时间：2018-04-29 23:02

  本文选题：颅脑创伤 + 糖代谢　； 参考：《重庆医科大学》2014年博士论文

【摘要】：第一部分 大鼠脑损伤后急性期脑葡萄糖代谢和缺氧诱导因子、葡萄糖转运蛋白表达变化时序规律的研究 目的：通过检测大鼠颅脑损伤模型急性期血糖、脑损伤区局部细胞外液(Extracellular Fluid,ECF)糖代谢指标的变化以及损伤区局部皮层脑组织缺氧诱导因子-1α(Hypoxia-inducible Factor-1, HIF-1α)、葡萄糖转运蛋白1(glucose transporter-1,GLUT-1)和葡萄糖转运蛋白3(glucosetransporter3,GLUT-3)表达的变化，分析这些变化的时序性规律，以探讨颅脑损伤后血糖控制的最佳时机。 方法：成年雄性SD大鼠35只，随机分成7组，每组包含正常对照、颅脑创伤(traumatic brain injury,TBI)后不同时间点六组，每组5只。在伤后15m、30m、45m、75m、90m、3h、6h、12h、24h、48h、72h不同时间点测定血糖值并收集脑损伤区局部脑细胞间(ECF)透析液，监测透析液葡萄糖含量([Glu]d)和乳酸含量([Lac]d)的变化，并在伤后3h、6h、12h、24h、48h、72h六个时间点断头取脑，采用RT-PCR和Western-blot法检测局部皮层脑组织HIF-1α、GLUT-1、GLUT-3三者mRNA和蛋白的表达。 结果：1.颅脑创伤组伤后血糖水平逐渐上升，6h上升明显，24h达到峰值，48h开始下降，72h仍高于正常水平，对照组血糖水平变化不明显。 2.颅脑创伤组脑细胞间透析液葡萄糖水平明显降低，15～30min时间段达低谷，明显低于对照组(P0.05)，其后逐渐回升，至75～90min时间段接近对照组；对照组脑细胞间透析液葡萄糖水平变化不明显。 3.颅脑创伤组脑细胞间透析液乳酸水平明显升高，伤后15～30min时间段达最高峰，可达基础水平的2~3倍，明显高于对照组(P0.05)，其后逐渐降低，至75~90min时间段接近对照组，而对照组脑细胞间透析液乳酸水平变化不明显。 4.颅脑创伤组脑细胞间液葡萄糖（[Glu]d）、乳酸（[Lac]d）变化和对照组相比，变化具有统计学意义，P0.05。[Glu]d下降和[Lac]d上升呈相反趋势变化。 5.颅脑创伤组脑损伤区皮层脑组织伤后3h可见有HIF-1α阳性表达，后逐渐增强，48h后达高峰，和对照组比较具有显著差异（P0.05），随后表达逐渐减少；创伤组脑损伤区皮层脑组织GLUT-1表达伤后12h开始降低，持续低至伤后72小时，和对照组比较具有显著差异（P＜0.05）；创伤组脑损伤区皮层脑组织GLUT-3表达伤后12h开始有增加，48h表达最明显，72h开始下降，但仍高于正常水平，和对照组比较有显著差异（P＜0.05）。对照组对应区皮层脑组织GLUT-1、GLUT-3表达在各时间段变化不明显，偶见HIF-1α阳性表达。 结论：颅脑创伤后血糖、脑损伤区皮层脑细胞间液糖、乳酸及HIF-1α、GLUT-1、GLUT-3的变化在时间上具有一定特点：损伤区皮层脑细胞糖代谢最先发生变化，随后HIF-1α开始表达，血糖升高，最后，GLUT-1、GLUT-3逐渐出现表达变化。这种变化可能是机体对局部缺氧代偿和葡萄糖转运、利用障碍的结果，作用机制复杂。伤后6～12小时是脑创伤后局部脑细胞糖代谢相关基因、蛋白发生变化的关键时期，，可能为控制性降糖预防继发性脑损伤发生的最佳时间段。 第二部分 高灵敏葡萄糖生物传感器的制备及其在颅脑损伤患者脑脊液糖含量检测中的运用 目的：制备基于纳米材料的高灵敏葡萄糖生物传感器，探讨高灵敏葡萄糖生物传感器在脑脊液葡萄糖水平监测中运用的可行性。 方法：采用高灵敏葡萄糖生物传感器对5例颅脑损伤患者所采集的脑脊液中糖含量进行检测，同时运用实验室葡萄糖氧化酶法检测，对两种方法的检测范围、准确性、灵敏度、精密度以及抗干扰能力进行比较。 结果： 1.高灵敏葡萄糖生物传感器响应电流值随着葡萄糖从1.2×10-6到1.6×10-3M的加入而呈线性增加，相关系数为0.998（n=20）。检测限为4.0×10-7M,信噪比为3，高灵敏葡萄糖生物传感器检测范围广，灵敏度高。 2.组内和组间精密度的相对标准偏差分别为3.8%和5.1%,高灵敏葡萄糖生物传感器检测具有良好的准确性和重现性。 3.在含0.2mM葡萄糖的PBS中加入一定浓度干扰物质（抗坏血酸，尿酸，对乙酰氨基酚以及L-半胱氨酸），对葡萄糖的检测无干扰，高灵敏葡萄糖生物传感器具有良好的抗干扰能力。 4.检测结果的准确性与临床检验方法（GLU-GOD-PAP方法）进行对比，样本稀释10倍，所得结果的相对偏差为-6.45%到8.69%之间，高灵敏葡萄糖生物传感器检测精密度高。 结论： 运用高灵敏葡萄糖生物传感器对颅脑损伤患者脑脊液葡萄糖水平检测响应快，灵敏度、精密度高，检测结果准确，抗干扰能力强，和传统葡萄糖氧化酶法检测相比更灵敏、快捷。可进一步用于颅脑外伤后患者脑脊液葡萄糖水平的动态监测。 第三部分 颅脑损伤后动态监测颅内压、灌注压及血脑屏障指数的临床意义 目的：通过检测、计算颅脑外伤后血脑屏障指数，并动态监测其变化，探讨血脑屏障指数与颅脑损伤严重程度及病情演变之间是否存在相关性。 方法：对30例中、重型颅脑损伤患者的血脑屏障指数进行动态监测结合GCS(glasgow coma scale，GCS)评分、颅内压及灌注压，分析血脑屏障指数和颅脑损伤严重程度和病情演变之间的相关性。 结果：所有患者血脑屏障指数均高于文献中的正常范围。血脑屏障指数和颅脑损伤的严重程度（GCS评分）呈正相关，和高颅压及低灌注压累及时间（pressure times Time dose,PTD）呈正相关，和病情演变具有一致性。 结论：颅脑损伤后血脑屏障通透性发生改变，影响其功能。血脑屏障指数的动态监测结合颅内压、灌注压可以客观反映病情的演变，血脑屏障指数可以作为临床监测指标之一。 第四部分 颅内压及脑脊液生物标记物S100β和NSE联合监测在颅脑损伤中的临床意义 目的：通过动态监测颅脑损伤后脑脊液S100β(S100β protein)和NSE(neuron-specific enolase,NSE)水平，分析二者和高颅压及脑低灌注压的相关性，探讨监测脑脊液S100β和NSE水平在评估颅脑损伤的严重程度及病情进展中的意义。 方法：对重庆医科大学附属第二医院和重庆市江津区中心医院2013年1月至2013年10月符合入选标准的颅脑损伤患者进行前瞻性的研究。所有符合入选标准的病例从脑室引流管收集脑脊液，检测S100β和NSE水平，每天2次，取平均值，一共7天。记录每小时ICP、CCP，取超过临界值压力的均值，将压力均值和24小时内ICP20mmHg，CPP60mmHg累计时间相乘，看作高颅压、低灌注的累及时间（pressure times Time dose,PTD）PTD20、PTD60。比较脑脊液S100β和NSE水平和PTD20、PTD60。分析脑脊液S100β、NSE的水平和ICP及CCP的动态变化的关系。 结果：36例病人被纳入。中型组(GCS9～12)20例，重型组(GCS4～8)16例。采集脑脊液标本，对这些标本进行了检测，结果所有患者S100β和NSE水平均增高，和颅脑损伤严重程度（GCS评分）呈正相关。同时S100β水平增高和颅内压20mmHg、灌注压60mmHg累及时间关系密切（PTD20:r=0.35,P 0.001;PTD60:r=0.24,P 0.001）；NSE水平增高和颅内压20mmHg累及时间PTD20呈弱相关(r=0.17, P=0.01)，和灌注压60mmHg累及时间PTD60关系密切(r=0.20, P0.01)。脑脊液S100β和NSE水平的变化和颅脑损伤严重程度及病情演变具有一致性。 结论：颅脑损伤后脑脊液S100β和NSE水平的增高程度反映了颅脑损伤严重程度，脑脊液S100β和NSE水平的动态变化反映了病情的演变。对二者脑脊液水平的变化结合颅内压、灌注压进行动态监测可以客观反映病情的进展，可以在出现明显的继发性脑损伤临床症状之前预测继发性脑损伤的发生。对中、重型颅脑损伤患者实行S100β、NSE联合ICP、CCP的动态监测具有重要临床意义。
[Abstract]:Part one
Timing of brain glucose metabolism and expression of hypoxia inducible factor and glucose transporter in rats with acute brain injury
Objective: to detect the changes of glucose metabolism in the local extracellular fluid (Extracellular Fluid, ECF) and the hypoxia inducible factor -1 alpha (Hypoxia-inducible Factor-1, HIF-1 alpha), glucose transporter 1 (glucose transporter-1, GLUT-1) and grape (glucose transporter-1, GLUT-1) in the local cortical tissue of the injured area by detecting the acute blood glucose in the rat brain injury model. The changes of glucosetransporter3 (GLUT-3) expression were analyzed, and the timing rules of these changes were analyzed to explore the best timing of blood glucose control after craniocerebral injury. 3.
Methods: 35 adult male SD rats were randomly divided into 7 groups, each group contained normal control, traumatic brain injury (TBI), six groups at different time points, 5 in each group. After the injury, 15m, 30m, 45m, 75m, 90m, 3h, 6h, etc. were determined at different time points and collected local brain cells in the brain injury area and monitored dialysate. The changes of glucose content ([Glu]d) and lactic acid content ([Lac]d) were observed at six time points of 3H, 6h, 12h, 24h, 48h and 72h after injury, and HIF-1 alpha, GLUT-1, and protein expression were detected by RT-PCR and Western-blot methods.
Results: 1. after craniocerebral trauma group, blood glucose level increased gradually, 6h increased obviously, 24h reached peak value, 48h began to decrease, 72h was still higher than normal level, and the blood sugar level in control group was not obvious.
2. the level of glucose in the dialysate between brain cells in the brain trauma group decreased significantly, and the time period of 15 ~ 30min was lower than that of the control group (P0.05), and then gradually recovered to the control group from 75 to 90min, and the glucose level of the dialysate between the cerebral cells of the control group was not obvious.
3. the level of lactic acid in the dialysate between brain cells in the traumatic brain injury group was significantly higher, the peak of the 15 ~ 30min period after injury reached the peak, up to 2~3 times of the basic level, obviously higher than that of the control group (P0.05), and then gradually decreased to the 75~90min time period to close to the control group, but the change of lactic acid level of the dialysate between the brain cells of the control group was not obvious.
4. the changes of glucose ([Glu]d) and lactic acid ([Lac]d) in the brain cells of the traumatic brain injury group were statistically significant compared with the control group. The decrease of P0.05.[Glu]d and the rise of [Lac]d were in the opposite direction.
5. the positive expression of HIF-1 alpha was found in 3h after brain injury in brain injury area, and then increased gradually, and then reached the peak after 48h. There was a significant difference between the control group and the control group (P0.05), and then the expression gradually decreased. The 12h began to decrease after GLUT-1 expression in the cortex brain tissue of the traumatic brain injury group and was lower to 72 hours after the injury, compared with the control group. There was significant difference (P < 0.05). After the GLUT-3 expression of the cortical brain tissue in the trauma group, the 12h began to increase, the expression of 48h was the most obvious, the 72h began to decline, but it was still higher than the normal level, and there was a significant difference between the control group and the control group (P < 0.05). The expression of GLUT-1 in the corresponding cortex and brain group of the control group was not obvious in each time period, occasionally HI. The expression of F-1 alpha was positive.
Conclusion: after craniocerebral trauma, the changes of sugar, lactate and HIF-1 alpha, GLUT-1, GLUT-3 in cerebral cortex and cerebral cells in the brain injury area have some characteristics in time: the glucose metabolism in the cortex brain cells of the injured area changes first, then the HIF-1 alpha begins to express, the blood sugar rises, and finally, GLUT-1, GLUT-3 changes. This change may be The mechanism of the body to local anoxic compensation and glucose transport and the use of obstacles is complicated. 6~12 hours after injury is the key period of the related genes of glucose metabolism in local brain cells after traumatic brain injury and the best time for controlling the occurrence of secondary brain injury by controlling hypoglycemic control.
The second part
Preparation of high sensitive glucose biosensor and its application in detecting CSF sugar content in patients with craniocerebral injury
Objective: to prepare a highly sensitive glucose biosensor based on nanomaterials, and to explore the feasibility of using highly sensitive glucose biosensor to monitor the glucose level in cerebrospinal fluid.
Methods: the glucose content in cerebrospinal fluid of 5 patients with craniocerebral injury was detected by high sensitive glucose biosensor, and the glucose oxidase method was used in the laboratory. The detection range, accuracy, sensitivity, precision and anti-interference ability of the two methods were compared.
Result锛

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