Rh-EPO干预早产儿脑白质损伤模型鼠的作用及机制研究

发布时间：2018-04-21 00:40

本文选题：脑室周围白质损伤 + 促红细胞生成素　；参考：《东南大学》2015年博士论文

【摘要】：目的：脑室周围白质损伤(periventricular white matter damage,PWMD)是早产儿脑损伤的主要神经病理学形式,已成为早产儿脑瘫、智力障碍等神经系统后遗症的主要原因之一,给家庭和社会带来沉重负担。目前,早产儿脑白质损伤的发病机制尚未完全明确,缺氧缺血是早产儿PWMD发生的两个主要病因之一,而脑室周围血管发育不完善是早产儿发生PWMD的重要解剖因素,目前针对早产儿PWMD尚无有效的干预措施。所以寻找治疗或预防早产儿脑白质损伤的方法、改善早产儿生存质量已是目前迫切需要解决的问题之一。重组人促红细胞生成素(recombinant human erythropoietin,rh-EPO)多年来广泛用于早产儿贫血的治疗且取得较好的效果,随着深入的研究发现EPO不仅具有造血功能,而且具有良好的神经保护作用。研究表明外周静脉或腹腔内注射rh-EPO能通过血脑屏障直接在脑内发挥神经保护作用。目前国内外对rh-EPO与脑损伤的关系及机制研究主要集中于rh-EPO对脑卒中及脑外伤等的神经保护作用,与早产儿PWMD的关系的研究仍少,尤其是其中的机制目前不明确。因此,本研究通过建立缺氧缺血性PWMD新生鼠模型,并给缺氧缺血后的新生鼠腹腔内注射一定剂量的rh-EPO,观察rh-EPO对缺氧缺血的PWMD新生大鼠脑保护功能,并探讨可能机制。该研究将为临床治疗早产儿PWMD提供一定的实验信息及依据,为今后进一步研究早产儿PWMD的防治提供新的思路和途径。方法：本实验采用生后3日龄的新生大鼠,除假手术(Sham)组外,其余各组大鼠乙醚麻醉后取颈部正中切口,游离右侧颈总动脉并结扎,术毕放回原饲养环境中恢复2-3小时后置于恒温密闭容器中,以2L·min-1的速度输入6%02、94%N2混合气体,持续4小时。Sham组大鼠手术仅游离右侧颈总动脉但不结扎,不进行缺氧通气。药物干预组(HI-EPO)大鼠于缺氧后1小时内给予腹腔注射(5U/g)的rh-EPO。缺氧缺血(hypoxia-ischemia, HI)组和Sham组大鼠给予腹腔注射相同剂量的无菌生理盐水。第一部分：整个实验过程中观察大鼠的生长发育状况,术后11天(P14)天断头取脑行HE染色观察脑组织病理改变,生后(postnatal day,P)30天动物行为检测(悬吊实验、旷场实验、拒俘反应实验、圆筒实验)和P90天学习记忆检测(三臂迷宫实验)。每小组完成实验保证6只。第二部分：分别于术后2天(P5)、术后5天(P7)、术后7天(P10)和术后11天(P14)断头取脑,一部分标本用于免疫组化检测EPO受体(Eythropoietin receptor, EPOR)和携氧蛋白神经珠蛋白(Neuroglobin,Ngb),一部分标本用于Western Blotting方法检测EPOR蛋白,另一部分用于Real-time PCR方法检测Ngb。每小组完成实验保证6只。第三部分：分别于术后2天(P5)、术后5天(P7)、术后7天(P10)和术后11天(P14)断头取脑,一部分标本用于免疫组化检测血管生成细胞内皮祖细胞(Endothelial progenitor cells,EPCs),一部分标本用于Western Blotting法检测EPCs,另一部分用Real-time PCR方法检测血管内皮生长因子(Vascular endothelial growth factor,VEGF)、血管生成素-1(Angiopoietin-1, Ang-1)、新生微动脉标志EphrinB2和微静脉标志EphB4。每小组完成实验保证6只。结果：1.Sham组幼鼠死亡率为2.50%；HI组幼鼠死亡率为16.12%：HI-EPO组幼鼠死亡率为8.23%；三组间死亡率差异存在统计学意义(χ2=8.32,P0.05)。术前,各组大鼠体重差别无统计学意义(F=0.448,P=0.647),自术后2天(P5)起HI组大鼠体重增长幅度低于Sham组,两组间差异存在统计学意义(P0.05)：HI-EPO组大鼠在手术后初期体重增长情况不良,从术后7天始体重增长加快,体重高于HI组但略低于Sham组,而且与Sham组和HI组间体重差异均存在统计学差异(P0.05)2. Sham组鼠双侧脑室大小相同,细胞排列整齐,形态大小正常。HI组鼠右侧脑室较对侧明显增大,脑室形状不规则,脑室周围组织疏松,细胞变性、水肿,部分可见内囊部位空腔形成：HI-EPO组鼠脑室周围白质病变减轻。3.悬吊实验、旷场实验、拒俘实验及三臂迷宫评分结果均显示三组间存在统计学差异,进一步两两分析,HI组评分明显低于Sham组(P0.05),HI-EPO组评分改善高于HI组(P0.05),且与Sham组间差异无统计学意义(P0.05)。圆筒实验评分结果显示HI组偏好右前足的频率与Sham组间存在明显统计学差异(P0.01),HI-EPO组则有改善,但未恢复到正常,与前两组间均存在统计学差异(P0.05)。4. Sham组大鼠EPOR表达在一较低水平；大鼠缺氧缺血后脑内EPOR在术后2天(P5)即明显增多,术后4天(P7)则逐渐开始下降,但一直到术后11天(P14)仍保持在一较高水平,术后各时间点与Sham组比较有统计学意义(P0.05)；HI-EPO组大鼠EPOR在术后2天(P5)较HI组及Sham组均增高且达到一高峰,与HI组及Sham比较增高有统计学意义(P0.05),但很快开始下降,在术后4天(P7)就降至HI组水平,术后7天(PI0)和术后11天(P14)进一步下降,与Sham组比较无统计学意义(P0.05),与HI组比较存在统计学意义(P0.05)。5.Sham组大鼠Ngb随着日龄的增大而略微增加；大鼠HI后脑内Ngb在术后2天(P5)即开始增加,术后7天(PI0)及11天(P14)未表现出进一步增多,术后各时间点与Sham组比较有统计学意义(P0.05)；HI-EPO组大鼠Ngb在术后2天(P5)至术后7天(PI0)较HI组增高,且两组之间比较有统计学意义(P0.05),术后11天(P14)Ngb则下降,与HI组比较无统计学意义,但与Sham组比较仍有统计学意义(P0.05)6. Sham组CD34+细胞随着日龄的增加而增加；HI后CD34+细胞较相同日龄Sham组大鼠初始增多(P5和P7),且两组间差异存在统计学意义(P0.05),但术后7天起(PI0)开始下降,并少于相同日龄Sham组大鼠,两组间差异存在统计学意义(P0.05)；HI-EPO组大鼠CD34+细胞变化趋势同缺氧缺血组,且较相同日龄缺氧缺血组鼠增多,各时间点两组间差异存在统计学意义(P0.05),同时与Sham组间差异也存在统计学意义(P0.05)。7.Sham组大鼠VEGF、Ang-1表达随着日龄的增加而增加：大鼠HI后脑内VEGF水平在术后2天(P5)即开始增加,至术后7天(PI0)达到高峰,然后下降,各时间点VEGF表达量均较Sham组增多；HI-EPO组大鼠术后2天(P5)VEGF、Ang-1表达量即较缺氧缺血组大鼠开始增加,术后7天(PI0)达高峰,术后11天(P14)又开始下降但仍高于缺血缺氧组,各个时间点与缺血缺氧组间差异存在统计学意义(P0.05)8.Sham组大鼠EphrinB2及EphB4表达均随着日龄的增加而增加,各时间点EphrinB2/EphB4比例接近0.5；大鼠缺氧缺血后脑内EphrinB2及EphB4在术后2天(P5)未见增加,EphrinB2/EphB4为0.49,与Sham组比较差异无统计学意义(P0.05),但自术后4天(P7)EphrinB2及EphB4迅速增加,与Sham组比较差异有统计学意义(P0.05),术后7天(PI0)开始则下降,但EphrinB2表达较EphB4多,EphrinB2/EphB4为0.56;HI-EPO组大鼠术后2天(P5)EphrinB2及EphB4表达较HI组大鼠明显增加(P0.05),术后4天(P7)达高峰然后开始下降,但仍高于缺血缺氧组,且EphrinB2表达较EphB4多。结论：1.缺氧缺血后脑组织有内源性修复反应,表现为脑室周围白质EPOR表达增加,携氧蛋白Ngb呈现持续上升表达,并有血管新生反应。2. Rh-EPO对PWMD起到了保护作用,rh-EPO干预缺氧缺血脑白质损伤鼠可以明显减轻脑室周围白质区域的损伤,可以改善大鼠的远期神经行为学能力,促进脑白质内EPOR表达进一步上调,给EPO提供受体发挥效应。3. Rh-EPO与EPOR结合后,一方面可以通过促进Ngb为脑组织提供更多的氧,另一方面可以通过促进血管新生反应为脑组织提供更多脑血流发挥干预效应。
[Abstract]:Objective: periventricular white matter damage (PWMD) is the main neuropathological form of brain injury in preterm infants. It has become one of the main causes of neurological sequelae in premature infants, such as cerebral palsy and mental retardation, and brings heavy burden to the family and society. At present, the pathogenesis of brain white matter injury in premature infants has not yet been found. It is clear that hypoxic ischemia is one of the two main causes of PWMD in preterm infants, and the poor development of peripheral blood vessels around the ventricle is an important anatomical factor for the occurrence of PWMD in preterm infants. There is no effective intervention for premature infant PWMD, so it is necessary to find a method to treat or prevent the brain white matter injury in preterm infants and improve the quality of survival in premature infants. It is one of the urgent problems that need to be solved. Recombinant human erythropoietin (recombinant human erythropoietin, rh-EPO) has been widely used for the treatment of anemia in preterm infants for many years and has achieved good results. With further research, it has been found that EPO not only has hematopoiesis, but also has good neuroprotective effect. Intravenous or intraperitoneal injection of rh-EPO can play a neuroprotective role through the blood-brain barrier directly in the brain. Research on the relationship and mechanism of rh-EPO and brain injury at home and abroad is mainly focused on the neuroprotective effect of rh-EPO on stroke and brain injury, and the study of the relationship with premature infant PWMD is still less, especially the mechanism of which is unclear. Therefore, in this study, a hypoxic ischemic PWMD rat model was established and a dose of rh-EPO was injected into the abdominal cavity of neonatal rats after hypoxic ischemia. The protective function of rh-EPO on hypoxic-ischemic PWMD neonatal rats was observed and the possible mechanism was discussed. This study will provide some experimental information and basis for the clinical treatment of PWMD in the treatment of premature infants. In order to further study the prevention and control of PWMD in preterm infants in the future, a new way of thinking and method is provided. In this experiment, the newborn rats of 3 days of age after birth, except for the sham operation (Sham) group, were taken from the middle incision of the neck after ether anesthesia, free right common carotid artery and ligated, and then returned to the original feeding environment for 2-3 hours after the operation. In a warm closed vessel, 6%02,94%N2 mixed gas was input at the speed of 2L min-1. For 4 hours, the right common carotid artery was free from the right common carotid artery in group.Sham rats, but no anoxic ventilation was carried out. The drug intervention group (HI-EPO) rats were given the rh-EPO. hypoxic-ischemic (hypoxia-ischemia, HI) group and Sham group within 1 hours after hypoxia. Rats were given the same dose of aseptic saline in the abdominal cavity. Part 1: the growth and development of rats were observed during the whole experiment. The brain tissue was observed by HE staining on the 11 day (P14) days (P14) days, and the 30 days after birth (postnatal day, P), the animal behavior test (suspension test, open field experiment, refusing reaction experiment, cylinder experiment) And P90 day learning and memory test (three arm labyrinth test). Each group completed the experiment to guarantee 6. Second parts: 2 days after operation (P5), 5 days after operation (P7), 7 days after operation (P10) and 11 days after operation (P14), and some specimens were used for immunohistochemical detection of EPO receptor (Eythropoietin receptor, EPOR) and oxygen carrying protein Neuroglobin (Neuroglo). Bin, Ngb), some specimens were used to detect EPOR protein by Western Blotting method. The other part was used for the Real-time PCR method to detect Ngb. in each group. 6. Third parts: 2 days after operation (P5), 5 days after operation (P7), 7 days after operation (P10) and 11 days after operation (P14), some specimens were used for immunohistochemistry to detect angiogenesis. Endothelial progenitor cells (EPCs), some specimens are used to detect EPCs in Western Blotting method, and the other part uses Real-time PCR method to detect vascular endothelial growth factor (Vascular endothelial growth), angiopoietin, and microarterioles and venules. The result: the mortality of young rats in group 1.Sham was 2.50%, and the mortality of young rats in group HI was 8.23% in group HI and in group 16.12%:HI-EPO, the mortality difference between groups was statistically significant (F=0.448, P=0.647) before operation (F=0.448, P=0.647), 2 days after operation (P). 5) the weight increase of the HI group was lower than that of the Sham group, and the difference between the two groups was statistically significant (P0.05) the body weight growth in the group:HI-EPO rats was poor after the operation, and the weight increased from 7 days after the operation, and the weight was higher than that of the HI group, but it was slightly lower than the Sham group, and there was a statistical difference between the group of Sham and the HI group (P0.05) 2. Sh. The size of the bilateral ventricle of the AM rats was the same, the cells arranged neatly, the right ventricle of the right ventricle increased obviously in the right ventricle of the normal.HI group, the shape of the ventricle was irregular, the periventricular tissue was loose, the cell degeneration, the edema and the formation of the cavity in the inner capsule: the peripheral white matter in the HI-EPO group was reduced by the.3. suspension experiment, the open field experiment, and the refusal of the capture. The results of the three arm labyrinth score showed that there was a statistical difference between the three groups. The score of group HI was significantly lower than that of group Sham (P0.05), and the improvement of group HI-EPO was higher than that of group HI (P0.05), and there was no statistical difference between the group of Sham (P0.05). The results of the test score of the cylinder experiment showed that the frequency of the HI group preference right front foot was clear between the Sham groups. The significant difference (P0.01), HI-EPO group improved, but did not recover to normal, and there were statistical differences between the first two groups (P0.05).4. Sham group EPOR expression at a lower level, EPOR in the brain after hypoxia ischemia in rats after 2 days (P5), the 4 days (P7) gradually began to decline, but until the 11 day after the operation (P14) still guaranteed. At a high level, there was a significant difference between the Sham group and the Sham group after the operation. The EPOR in group HI-EPO increased and reached a peak at 2 days after the operation (P5), which was higher than that in the HI group and the Sham group. It was significantly higher than the HI group and Sham (P0.05). But it began to descend quickly and decreased to the level of the HI group at the 4 day (P7) after the operation, and 7 days after the operation. After 11 days (P14), there was no significant difference from group Sham (P0.05), and there was a statistically significant difference between group HI and group HI (P0.05) in group.5.Sham, Ngb was slightly increased with the increase of day age, and Ngb in the brain after HI began to increase at the beginning of the operation (P5), and the 7 days (PI0) and 11 days (P14) after the operation did not increase further, and all the time after the operation. The point was statistically significant compared with that in the Sham group (P0.05), and the Ngb in group HI-EPO was higher than that in the HI group at 2 days after the operation (P5) and 7 days after the operation (PI0), and there was a significant difference between the two groups (P0.05), and the 11 days (P14) Ngb decreased, but there was no statistical significance compared with the HI group, but there was still a significant difference between the 6. group and the 6. group. The increase of day age increased; after HI, CD34+ cells increased initially in group Sham (P5 and P7), and there was a significant difference between the two groups (P0.05), but it began to decline (PI0) from the 7 day after the operation, and was less than that of the same age group of Sham rats. The difference between the two groups was significant (P0.05); the CD34+ cell trend of the HI-EPO group was deficient. Oxygen ischemia group, and more than the same age of hypoxia ischemia group increased, there was a statistically significant difference between the two groups at each time point (P0.05), and there was a statistically significant difference between the Sham group (P0.05) and the VEGF in group.7.Sham, Ang-1 expression increased with the increase of day age: the level of VEGF in the brain of the rat HI began to increase at the 2 day after the operation (P5), to the operation. The following 7 days (PI0) reached the peak, then decreased, and the expression of VEGF in each time point increased more than that in the Sham group; the expression of Ang-1 in the HI-EPO group was increased at 2 days (P5), and the expression of Ang-1 was increased in the hypoxic ischemia group, the peak of the 7 day (PI0) after the operation, and the 11 day after the operation (P14), but still higher than the ischemic anoxia group, and the difference between each time point and the ischemic anoxia group. There was statistical significance (P0.05) in group 8.Sham, the expression of EphrinB2 and EphB4 increased with the increase of day age, and the proportion of EphrinB2/EphB4 in each time point was close to 0.5. The EphrinB2 and EphB4 in the brain after hypoxic ischemia in rats did not increase at 2 days (P5), EphrinB2/EphB4 was 0.49, but there was no statistical difference from the Sham group (P0.05), but 4 after the operation. P7 EphrinB2 and EphB4 increased rapidly, compared with the Sham group, the difference was statistically significant (P0.05), and the 7 days after the operation (PI0) began to decrease, but the expression of EphrinB2 was more than EphB4 and EphrinB2/EphB4 was 0.56, and the 2 days after operation (P5) was significantly increased in the HI-EPO group. The expression of EphrinB2 was higher than that of ischemic hypoxia group, and the expression of EphrinB2 was more than that of EphB4. Conclusion: after hypoxic ischemia, the brain tissue has endogenous repair response, which shows an increase in the expression of white matter EPOR around the ventricle, the elevated expression of oxygen carrying protein Ngb, and a neovascularization reaction.2. Rh-EPO to the protection of PWMD, rh-EPO interfered with the white matter injury of hypoxic and ischemic brain. Rats can significantly reduce the damage of the white matter area around the ventricle, improve the long-term neurobehavioral ability of the rat, promote the further up of EPOR expression in the white matter, and provide EPO with the receptor effect of.3. Rh-EPO and EPOR, on one hand, by promoting Ngb to provide more oxygen to the brain tissue, on the other hand it can promote blood by promoting blood. The tube regeneration response provided more cerebral blood flow to the brain tissue and played an intervention role.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R-332;R722.6

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