人尿激肽原酶对大鼠急性缺血性卒中后血管新生作用的研究

发布时间：2018-03-07 19:02

本文选题：急性缺血性卒中　切入点：血管新生　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：背景：脑卒中已经在全球范围内成为导致死亡以及长期残疾的首要病因之一,因而寻找并发展新型有效的治疗缺血性卒中的方法显得至关重要。近年来,多项研究显示缺血性卒中发生后病灶部位的血管新生不仅能够提高局部的血供,同时也促进内源性的神经再生并且有助于卒中后的神经功能恢复,因此,血管新生已经成为缺血性卒中治疗的研究热点。人尿激肽原酶(即注射用尤瑞克林),作为国内近年来开发的Ⅰ类新药,临床试验已经证实其能够减少急性缺血性卒中患者的神经功能损伤,并提高远期预后；但关于人尿激肽原酶是否能够促进病灶部位的血管新生,目前的研究还比较少。68Ga-PRGD2 PET/CT作为一项能够特异性识别新生血管表面的整合素αvβ3受体、从而评估机体血管新生状况的无创的分子靶向显影技术,是目前新生血管显像研究的重要内容,并已在肿瘤、心肌梗死等疾病中得到应用,但将其应用于评估卒中后缺血灶血管新生的研究尚且较少。目的：1、评估68Ga-PRGD2 PET显影技术应用于大鼠大脑中动脉卒中(MCAO)模型中检测血管新生状况的可行性；2、结合分子影像学及生物学指标综合评估人尿激肽原酶(注射用尤瑞克林)对大鼠MCAO模型是否具有血管新生作用。方法：通过线栓法建立SD大鼠大脑中动脉卒中(MCAO)模型,将建模成功的大鼠分为①尤瑞克林给药组：通过静脉注射3.5×10-3PNA/(kg·d),疗程分为7天和14天；②对照组：通过腹腔注射等量生理盐水,疗程同样分为7天和14天。另选择健康SD大鼠组成假手术组,通过腹腔注射等量生理盐水,疗程分为7天和14天。对尤瑞克林给药组和对照组大鼠进行以下方面的评价：①建模后第1、3、7、14天行Longa评分评估神经功能恢复情况;②在建模后第7、14天分别完成18F-FDG MicroPET和68Ga-PRGD2 MicroPET检查；③在建模后第7、14天分别取给药组、对照组大鼠患侧大脑皮层病灶周边组织通过Western Blot法检测整合素β3水平；④在建模后第7天、14天分别取给药组、对照组和假手术组大鼠右侧大脑皮层病灶周边组织通过ELISA法检测VEGF-A水平。结果：1、建模后第1、3、7天给药组及对照组大鼠Longa评分均无显著性差异,建模后第14天给药组Longa评分较对照14天组降低,提示给药组可能有神经功能恢复更好的趋势,但差异不具有统计学意义(P=0.06)；2、18F-FDG MicroPET显像提示给药和对照7天组大鼠患侧大脑均可见代谢明显减低区,两组间放射性摄取无显著性差异(P0.05),给药14天组大鼠放射性摄取比对照14天组有升高的趋势,但两组间无显著性差异(P=0.051),给药14天组与7天组相比放射性摄取无显著性差异(P0.05)；3、68Ga-PRGD2 MicroPET显像提示给药7天组、给药14天组大鼠梗死区放射性摄取均较对照7天组、对照14天组升高(P0.05),给药14天组摄取值与给药7天组相比无显著性差异(P0.05)；4、Western Blot检测结果显示给药7天组患侧脑组织整合素β3水平高于对照7天组(P0.05),给药14天组与对照14天组整合素β3水平无显著性差异(P0.05)；5、ELISA检测显示给药7天及14天组大鼠脑组织VEGF-A水平均高于对照7天及14天组(P0.05),给药14天组大鼠脑组织VEGF-A水平较给药7天组无显著性差异(P0.05)。结论：1、人尿激肽原酶(注射用尤瑞克林)对MCAO大鼠具有促进血管新生的作用,并可能对改善急性缺血性卒中后的神经功能恢复发挥一定的作用：2、68Ga-PRGD2 MicroPET可以显示血管新生空间分布,用于实验动物缺血性卒中后血管新生的评估具有一定价值,需要扩大研究以进一步证实。
[Abstract]:Background: stroke has become one of the leading causes of death and long-term disability worldwide, and therefore look for method for the development of new and effective treatment of ischemic stroke is crucial. In recent years, many studies have shown that ischemic stroke occurred after lesion of angiogenesis can not only improve the local blood supply, but also promote the recovery of endogenous. The nerve regeneration and contribute to the neurological function after stroke. Therefore, angiogenesis has become a hot topic in the treatment of ischemic stroke. Urinary kallikerein (Urinary Kallidinogenase for Injection), as a new drug developed in recent years in China, clinical trials have confirmed that it can reduce the neurological damage in patients with acute ischemic stroke, and improve long term prognosis; but about human urinary kallikrein whether lesion can promote angiogenesis, the present study also compared .68Ga-PRGD2 PET/CT as a specific recognition of neovascular surface integrin alpha v beta 3 receptor, to evaluate the angiogenesis status of body without a molecular target to the developing technology, is the important content of angiogenesis imaging research, and has been applied in tumor, myocardial infarction and other diseases, but its application the new evaluation on vascular ischemia after stroke research even less. Objective: To evaluate 68Ga-PRGD2 1, PET imaging technology applied in rats with middle cerebral artery stroke (MCAO) the feasibility of detecting angiogenesis in the model; 2, combined with molecular imaging and comprehensive assessment of biological indicators of human urinary kallikrein (Urinary Kallidinogenase for Injection) to whether the rat model of MCAO with angiogenesis. Methods: a SD rat model of middle cerebral artery stroke by suture method (MCAO) model, the successful model rats were divided into the Yuri Franklin administration Group by intravenous injection of 3.5 * 10-3PNA/ (kg - D). The course is divided into 7 days and 14 days; the control group by intraperitoneal injection of saline, treatment is also divided into 7 days and 14 days. The other healthy SD rats were selected consisting of sham operation group by intraperitoneal injection of saline, treatment for 7 days and 14 days. Yuri Klein on treatment group and evaluate the rats in control group: 1,3,7,14 day Longa score to assess the recovery of nerve function after the modeling; modeling in 7,14 days after the completion of 18F-FDG MicroPET and 68Ga-PRGD2 MicroPET were examined in the model; after 7,14 days respectively the medicine group, the control group rat ipsilateral cerebral cortex tissue surrounding the lesions detected by Western Blot method 3 integrin beta level; the seventh day 14 days after modeling, respectively treatment group, control group and sham operation group rats with right cerebral cortex lesions surrounding tissue by E Detection of VEGF-A level by LISA. Results: 1. The modeling of 1,3,7 days after treatment group and control group Longa rats were no significant difference between the model fourteenth days after the treatment group Longa score than the control group decreased 14 days, suggesting that the drug group may have better neurological recovery trend, but the difference is not statistical significance (P=0.06); 2,18F-FDG MicroPET imaging suggest that treatment and control group of 7 day rat ipsilateral brain showed significantly lower metabolism, uptake between the two groups had no significant difference (P0.05), administered 14 days group uptake in rats than in the control group of 14 days has increased, but the two groups there is no significant difference between (P=0.051), administered 14 days group and 7 days group compared uptake had no significant difference (P0.05); 3,68Ga-PRGD2 MicroPET imaging that administered 7 days group, administered 14 days group infarction rats uptake were lower than those in the control group of 7 days, 14 days in control group increased (P0 .05), administered 14 days group uptake value compared with medication for 7 days showed no significant difference (P0.05); 4, Western Blot showed that the administration of 7 days group ipsilateral brain tissue integrin beta 3 was higher than the control group for 7 days (P0.05), administered 14 days group and the control group of 14 days integrin beta 3 level no significant difference (P0.05); 5, ELISA showed that the administration of 7 days and 14 days group VEGF-A level in brain tissue of rats was higher than that of the control group 7 days and 14 days (P0.05), administered 14 days group VEGF-A level in brain tissue of rats were administered for 7 days were no significant the difference (P0.05). Conclusion: 1, human urinary kallidinogenase (Urinary Kallidinogenase for Injection) can promote angiogenesis of MCAO rats, and may improve the neurological function after acute ischemic stroke recovery play a role: 2,68Ga-PRGD2 MicroPET can show the distribution of angiogenesis in space, is used to evaluate the angiogenesis of ischemic animal experiment after stroke There is a certain value that needs to be expanded to be further confirmed.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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